Tri-Manganese(III) Salen-Based Cryptands: A Metal Cooperative Antioxidant Strategy that Overcomes Ischemic Stroke Damage in Vivo

Article abstract of DOI:10.1021/jacs.0c03805

Oxidative stress is one of the hallmarks of ischemic stroke. Catalase-based (CAT) biomimetic complexes are emerging as promising therapeutic candidates that are expected to act as neuroprotectants for ischemic stroke by decreasing the damaging effects fro

Full text of DOI:10.1021/jacs.0c03805

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Article
Tri-Manganese(III) Salen-based Cryptands: A Metal Cooperative
Antioxidant Strategy that Overcomes Ischemic Stroke Damage In vivo
Yingying Ning, Yan Huo, Haozong Xue, Yujing Du, Yuhang Yao, Adam C. Sedgwick, Hengyu Lin, Cuicui
Li, Shang-Da Jiang, Bing-Wu Wang, Song Gao, Lei Kang, Jonathan L. Sessler, and Jun-Long Zhang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c03805 • Publication Date (Web): 10 May 2020
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Tri-Manganese(III) Salen-based Cryptands: A Metal
Cooperative Antioxidant Strategy that Overcomes Ischemic
Stroke Damage In vivo
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†

†

†
§
Yingying Ning, Yan Huo, Haozong Xue, Yujing Du, Yuhang Yao, Adam C. Sedgwick,
†

†
†
† ‡

Hengyu Lin, Cuicui Li, Shang-Da Jiang, Bing-Wu Wang, Song Gao, Lei Kang,* Jonathan L.
Sessler* and Jun-Long Zhang*
§
†
†Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Rare Earth Materials Chemistry and
Applications, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China
Department of Nuclear Medicine, Peking University First Hospital, Beijing 100034, P. R. China
§Department of Chemistry, The University of Texas at Austin, 105 East 24th Street-A5300, Austin, TX 78712-1224,
USA
‡School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, 510640 P. R.
China
KEYWORDS. Manganese; Catalase; Stroke; Metal Cooperativity; Synthetic Antioxidant Therapeutics.
ABSTRACT: Oxidative stress is one of the hallmarks of ischemic stroke. Catalase-based (CAT) biomimetic complexes are
emerging as promising therapeutic candidates that are expected to act as neuroprotectants for ischemic stroke by
decreasing the damaging effects from H O . Unfortunately, these molecules result in the unwanted production of the
2 2
harmful hydroxyl radical, HO. Here, we report a series of salen-based tri-manganese (Mn(III)) metallocryptands 1-3 that
function as catalase biomimetics. These cage-like molecules contain a unique “active site” with three Mn centers in close
2 2
proximity, an arrangement designed to facilitate metal cooperativity for the effective dismutation of H O with minimal
HO• production. In fact, significantly greater oxygen production is seen for 1-3 as compared to the monomeric Mn(Salen)
complex, 1c. The most promising system, 1, was studied in further detail and found to confer a greater therapeutic benefit
both in vitro and in vivo than the monomeric control system, 1c, as evident from inter alia studies involving a rat model of
ischemic stroke damage and supporting histological analyses. We thus believe that metallocryptand 1 and its analogues
represent a new and seemingly promising strategy for treating oxidative stress related disorders.
7
reactive hydroxyl radical (OH). As a result, it is believed
INTRODUCTION
2 2
that the targeted deactivation of H O is key to the design
Ischemic stroke is one of the leading causes of death
of an effective neuroprotective strategy that could reduce
1-2
worldwide.
The majority of all cases are due to the
8-14
or mitigate ischemic-based brain damage.
Inspired by the biological antioxidant enzyme catalase
(CAT) that serves to dismutate H into dioxygen and
water, efforts have been directed towards the development
of CAT-like biomimetics as a means of deactivating H
formation of blood clots, which restricts cerebral blood
flow to the central nervous system, decreasing the
availability of oxygen (hypoxia) and glucose to the brain.
This stroke-induced hypoxia generates neurotoxic reactive
2 2
O
2 2
O .
oxygen
species
(ROS),
which
leads
to
the
Within this paradigm, Mn-based complexes have been
extensively explored with a porphyrin (AEOL10150), a Salen
3
dysfunction/death of neurons. The associated oxidative
stress is thought to play a key role in the pathogenesis of
ischemic-based brain injury. Among the various
deleterious species generated during ischemic stroke, is
2 2 2 2
hydrogen peroxide (H O ). On its own H O is recognized
as being a long-lived, cell permeable ROS with a relatively
low reactivity. However, in the presence of the redox active
metal ions, particularly copper (Cu(I)) and iron (Fe(II)),
(
EUK-134),
and a macrocyclic aza-ligand (M40403)
entering preclinical/clinical trials as synthetic catalytic
4-6
1
5
2 2
antioxidants for H O dismutation. However, without
exception these Mn-based complexes result in the
generation of the unwanted OH, which is expected to
1
6
limit their therapeutic utility. Production of OH occurs
in these instances via formation of an H -generated Mn-
peroxide intermediate that then undergoes homolytic
2 2
O
2 2
H O is converted via the Fenton reaction to the highly
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cleavage to produce OH (Figure 1a). Note - this process is
in direct competition with a heterolytic cleavage pathway
that results in the formation of the essentially non-toxic
–
17
hydroxide anion (HO ) (Figure 1a). An ideal therapeutic
CAT antioxidant biomimetic should not only promote
effective
2
H O
2
dismutation but also minimize the
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formation of HO•.
18-19
Nocera,20-21 Nam22-23
Previous efforts by Groves,
,
24-25
26-27
Gross
and others
have shown that the addition of
an axial ligand or the presence of a secondary coordination
sphere to Mn-porphyrin or -Salen cores, switches the ratio
of cleavage in favor of the heterolytic cleavage pathway
(Figure 1a). Unfortunately, various limitations, including a
combination of poor aqueous solubility, pH dependent
effects, and chemical stability, prevented the exploration of
these Salen derivatives in vivo. Alternative approaches
using di- and polynuclear Mn complexes have been shown
to exhibit enhanced catalase activities with the Mn-Mn
separation playing a key role in regulating activity. 28-31
Nevertheless, to date, minimal efforts have been devoted
to preparing and testing polynuclear Mn biomimetics in
vivo as a possible means of reducing HO formation. On
the other hand, more broadly, Mn(Salen)-based complexes
have attracted interest as potential antioxidant-based
Figure 1. Schematic illustration of a) homolytic and heterolytic
O-O bond cleavage of an edge bound Mn(III) peroxide complex,
b) the effect of metal cooperativity in binding and splitting H
and c) the strategy developed in the present study.
2 2
O ,
RESULTS AND DISCUSSION
Synthesis
and
Characterizations
of
Tri-
Manganese(III) Salen-based Cryptands.
Early on Akine, 33-35 Nabeshima,36 and others37 pioneered
3
2
therapeutics. We thus postulated that a tri-Mn(Salen)
cryptand complex might mimic in rudimentary fashion the
binuclear nature of the native CAT active site. As detailed
below, we have found that this approach, embodied in
complexes 1-3, provides systems that allow for effective
CAT activity while simultaneously minimalizing OH
production in vitro (Figure 1b). Specifically, 1-3 were found
exhibit a ca. 7-fold CAT enhancement relative to a
monomeric Mn(Salen) control, 1c. The most promising
system, complex 1, also gave rise to a significant reduction
in HO• formation (ca. 50-fold) in comparison to this same
control. In vivo studies, involving occlusion of the middle
cerebral artery followed by the administration of 1,
revealed a statistically significant reduction in brain
infarctions and prevention of further infarct growth in a rat
model. This work represents a new strategy for the design
of effective molecular antioxidants for the treatment of
oxidative stress related diseases.
the chemistry of Salen-based cryptands with an eye toward
improving guest recognition and selectivity for a range of
applications, including materials and catalysis. Within the
context of ongoing efforts to explore potential biological
38-40
applications of metallosalen complexes,
we have
41
42
recently reported biocompatible luminescent Zn and Al
tris(Salen) cryptands that are effective for live cell imaging.
We reasoned that such complexes could also provide a
platform for the cooperative binding of guest molecules as
the result of the metal centers being in close proximity. We
further expected cooperative binding of H
2 2
O between the
Mn cores would catalyze H dismutation, mimicking the
2 2
O
native CAT Mn core. The present study was undertaken in
an effort to test this hypothesis.
3
As shown in Scheme 1, three C symmetric tri-Mn(Salen)
cryptand complexes, 1-3, were synthesized to evaluate the
structural effects of the diamine moiety on the CAT
reactivity. Three diamines were chosen, namely o-
phenylenediamine,
1,2-diaminocyclohexane
and
ethylenediamine. They were incorporated into the 3-
41-42
dimensional hosts
tris(2-hydroxybenzaldehyde) at reflux with the chosen
diamine (1.5 equiv) and Mn(OAc) (1.5 equiv) in a mixed
CH CN/MeOH solution (1:1). This afforded complexes 1-3
by heating 3,3’,3’’-methanetriyl-
43
2
3
in good yields (≥40% in all cases). As a control, the
monomeric Mn(Salen) complex 1c was synthesized
44
following a known literature protocol. All isolated
complexes were characterized by high resolution mass
spectrometry, elemental analysis, and UV–Vis and IR
spectroscopies (Figure S1-7). The UV-Vis spectra of the
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trinuclear Mn(Salen) complexes 1-3 are characterized by
it is worth noting that Mn-based complexes are emerging
as attractive magnetic resonance imaging (MRI) relaxation
agents owing to their high-spin electronic configuration,
broad absorptions from 250-380 nm with an additional
peak at 400-480 nm (Figure S8), assigned to an overlap of
a ligand-centered transition and LMCT transition from the
4
9-50
long T1 and relatively lower toxicity.
3
+
phenoxo oxygen to the coordinated Mn ion. The ESI-
mass spectrum of 1-3 (Figures S5-7) revealed peaks
at m/z 1197.0397, 1215.1797 and 1053.0391, attributed to [1-
In Vitro Catalase-based Studies of 1-3. Next, we
examined the ability of 1-3 and 1c to catalyze H
dismutation in PBS buffer (pH 7.4). Upon the addition of 1-
or 1c (10 μM) to a solution of H (10 mM), vigorous
evolution of O was observed. This is expected for a system
that promotes CAT-like H dismutation. As shown in
Figure 2a, a significant enhancement in the rate of H
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2 2
O
–
+
– +
– +
Cl ] , [2-Cl ] and [3-Cl ] , respectively, which are fully
consistent with the proposed structures. Detailed
synthetic procedures and corresponding characterization
data can be found in the Supporting Information.
3
2 2
O
2
2 2
O
2 2
O
Scheme 1. Synthetic Procedure Used to Access the
Manganese(III) Complexes of the Present Study.
dismutation was observed for 1-3 (87, 101, 69 μM/min)
relative to 1c (22 μM/min). As a result, an overall 6.8-fold
in oxygen production was observed for 1 (277 μM) relative
to 1c (41 μM) on a per mole basis. The corresponding
2
maximum O concentrations for 2 and 3 were 198 and 234
μM, respectively (Figure 2b). Importantly, the oxygen
production observed for 1 was greater than the simple use
of three molar equiv. of the complex 1c. This relative
increase on a per manganese equivalent basis was taken as
evidence that favorable metal-metal cooperative effects
contribute to the overall efficacy in the case of 1. In addition,
an obvious color change from brown to yellow was
observed for 1c, indicative of decomposition. No color
change was observed for 1-3 and ESI-MS analysis revealed
With complexes 1-3 and 1c in hand, the redox properties
were evaluated using cyclic voltammetry in DMF (cf.
Figure S9 and Table S1). These studies revealed two quasi-
reversible oxidation couples at approximately +1.1 and +1.3
V vs SCE for each complex, which were assigned to the
2 2
that complex 1 was stable after the H O dismutation
reaction (Figure S11), which were taken as an indication of
stability sufficient to allow for more detailed investigations.
These further studies focused on comparisons between 1
and 1c since the tris-Mn system 1 proved more effective at
–
PhO /PhO· and Mn(IV)/Mn(III) redox pairs, respectively,
45-46
in accord with literature reports.
The irreversible
reductive peaks seen around -1.6 V are believed to reflect
ligand centered reductions. The reversible peaks, seen
between - 0.5 V and 0.0 V, were assigned to Mn(III)/Mn(II)
conversion. Only a single reversible Mn(III)/Mn(II)
reduction was observed, a finding that is attributed to the
similar coordination environment of each Mn ion leading
to overlapping peaks. Similar observations have been made
previously in the case of similar metallocryptand
promoting overall H
Figure 2b).
2 2
O dismutation than either 2 or 3 (cf.
To determine the amount of OH produced during the
H
2
O
2
dismutation process, a known OH fluorescence
51
detection assay was used. In this protocol, the addition of
OH to p-phthalic acid results in production of the highly
fluorescent 2-hydroxyterephthalic acid (emitting between
3
2 2
70 – 550 nm). To our delight, the addition of 1 and H O
4
7-48
systems.
. However, we cannot discount potential weak
to a solution of p-phthalic acid resulted in no change in the
fluorescence intensity whereas a significant enhancement
in the fluorescence intensity was observed for 1c under
otherwise identical conditions (Figure 2c). These
differences are ascribed to the promotion of heterolytic vs.
homolytic cleavage by 1 and 1c during the H O
2 2
dismutation process. Support for these observed
differences came from electron paramagnetic resonance
electrostatic couplings between each Mn center. Negligible
differences in the electrochemical feature were observed
between complexes 1 and 1c, leading us to suggest that each
Salen moiety within the cryptand structure 1 acts as a
discrete entity and does not influence the redox potentials
of the other Salen subunits. However, on passing from 1 to
3
, a decrease in the reduction potential (E1/2) for the
Mn(III)/Mn(II) couple is seen (i.e., from 0.16 V in 1 to ca. -
.04 V in 2 and 3). This is thought to reflect the electron-
(
EPR) studies of 1 and 1c. In these experiments, 5,5-
0
dimethyl-1-pyrroline N-oxide (DMPO) was employed as a
radical trapping agent. As shown in Figure 2d, the addition
of DMPO to a solution of 1c and H O led to a gradual
2 2
increase in the intensity of the signal at g = 2.0094. This
increase corresponded to the reaction between OH and
DMPO to form the DMPO-OH• radical. Under otherwise
donating ability of the diamine moieties in the order of o-
phenylenediamine > cyclohexanediamine > ethanediamine.
Ligand-based effects were also apparent in the relaxivities
(r
22.9 and 2.5 mM s , respectively, in 9:1 H
(Figure S10). While not a focus of the present study per se,
1
) of 1-3 and 1c, which were determined to be 10.5, 19.8,
-1 -1
2
O:DMSO, 37 °C
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identical conditions, the use of the tris-Mn complex 1
instead of 1c produced a relatively weak signal at the same
g value. The relative amounts of hydroxyl radical produced
by 1 and 1c were quantified by EPR signals and found to be
ca. 1:2; again, this is consistent with the notion that use of
1 as a CAT mimic produces minimal OH.
radical complexes display features at g~5.5 and ~2.0 with
Mn(IV) and Mn(IV)═O
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55-56
species displaying signals at ca.
g~5.0, 3.0, 1.8, which vary depending on the ligands. The
key signals in Figure 2e are thus assigned readily to Mn(IV),
phenol radical, and Mn(III) species, which were ascribed to
oxidation of the Mn(III) center, the Salen ligand, and
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2 2
possible H O -mediated reduction of free manganese(IV)
EPR experiments were also performed without DMPO
and the EPR spectra of 1 and 1c in the presence of H O
2 2
cations, respectively. The similarity in the g values seen for
the mono- and tri-Mn complexes supports the postulate
that the valence changes for Mn for both 1 and 1c are
. Considered in light of the
, these results provide
support for the core postulate underlying this study,
namely that cooperation between metal centers helps
were obtained at 10 K. As shown in Figure 2e, the EPR
spectra consisted of a number of Mn hyperfine lines (> 16)
55
similar when exposed to H
2
O
2
at ca. g ~ 2.0. This is a common observation for Mn(III)
5
2
disparate production of HO• and O
2
2 2
salen species. However, after the addition of H O , new
low field features were seen at ca. g ~ 4.5 and 3.0. At 60 s,
the EPR spectrum of 1c displayed EPR signals at g = 4.494,
3.053, and 1.896, with similar features being seen for 1
(4.456, 3.053, and 1.896, Figure 2e). Previous studies of Mn
salen complexes have shown that Mn(III) salen phenoxyl
prevent the formation of HO• while promoting H
2 2
O
dismutation.
Figure 2. a) Oxygen production over time (s) via catalytic H
buffer). b) Maximum concentration of oxygen (at 1000 s) produced via catalytic H
emission spectra of p-phthalic acid (5 mM) recorded 30 min after the addition of 1/1c (0.1 mM) followed by H
nm). d) X-band EPR spectra generated when either 1c (1.57 mM, left) or 1 (1.57 mM, right) were treated with 5.6 equiv of H
equiv of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) at 298 K in MeOH. Conditions: microwave frequency, 9.375 GHz; microwave power,
.5972 mW; modulation amplitude, 1 G; time constant, 59.6 ms; conversion time, 59.6 ms. e) X-band EPR spectra recorded at 10 K in
a frozen CH Cl -toluene glass after 1.57 mM 1c or 1 was treated with 1.0 equiv of H (7:3). Conditions: microwave frequency, 9.366
GHz; microwave power, 9.464 mW; modulation amplitude, 1 G; time constant, 29.3 ms; conversion time, 29.3 ms. f) Changes in the
UV-Vis spectra seen when 1 was treated with 3 equiv of H in 50 mM PBS buffer (pH = 6) at 5 °C.
2
O
2
(10 mM) dismutation mediated by 1-3 and 1c (10 μM) at pH 7.4 (PBS
dismutation process in a). c) Fluorescence
(1 mM) (λex = 330
and 34
2 2
O
2 2
O
2 2
O
0
2
2
2 2
O
2 2
O
To obtain insights into the mechanism of H
2
O
2
system (1) and the monomeric control 1c. The initial rate of
dismutation was found to be linearly correlated with
the concentration of both H and the Mn(Salen)
complex; this proved true for both 1 and 1c (Figure S12).
dismutation, we investigated the effect of both substrate
and catalyst concentration on the rate of reaction. For
these experiments, we focused on the most promising
2 2
H O
2 2
O
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Accordingly, we established a rate law of -d[H
][Mn(Salen)]. The H dismutation process was
also monitored by UV-Vis absorption spectroscopy. The
addition of H (3 equiv.) to a solution of 1 (10 μM, PBS,
pH = 6.0, 50 mM) at 5°C led to a change in UV-Vis
absorption spectral features similar to those previously
reported for Mn(IV) or Mn(V)═O Salen complexes (Figure
2 2
O ]/dt=
kcat[H
2
O
2
2 2
O
2 2
O
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0, 57-58
2f).
UV-Vis spectroscopic results, we believe a Mn(III) center
in 1 reacts with H to form an oxomanganese
intermediate Mn(IV)═O, which subsequently reacts with a
second H molecule to release O in direct analogy to the
Based on this precedent and the present EPR and
2 2
O
2
O
2
2
previously reported ping-pong mechanism for the
biological Mn CAT.5
9
Mn(IV)-oxo intermediates have been extensively shown
to
catalyze
the
oxidation
of
2,2'-azino-bis(3-
+
•
ethylbenzothiazoline-6-sulfonic acid (ABTS) to ABTS via
one-electron transfer and carry out the epoxidation of
60
styrene via oxygen atom transfer. In both reactions, we
found 1 exhibited a 7-10 fold greater reactivity than 1c
To investigate the potential of 1 to act as a synthetic
catalase mimetic, we evaluated its ability to scavenge
intracellular ROS using the ROS fluorescence probe
(
Figure S13). These results provide support for the
proposition that formation of Mn-oxo intermediates is
more facile under conditions of H dismutation in the
2 2
O
dichlorodihydrofluorescein
diacetate
(H2DCFDA).
case of 1 than it is for 1c. Again, this is rationalized in terms
of a cooperative effect between neighboring Mn(IV)═O
Treatment of HUVEC cells containing H2DCFDA with the
vascular endothelial growth factor (VEGF), an intracellular
moieties that could serve to stabilize various H
intermediates.
2 2
O -derived
61
ROS stimulator, resulted in a significant increase in the
fluorescence intensity ascribed to the fluorescein marker
2 2
Therefore, we believe a Mn(III) center first binds to H O
(
Figure 3). Incubation of HUVEC cells with 1c resulted in a
to release a water molecule with the concomitant
formation of an oxomanganese Mn(IV)═O intermediate.
This Mn(IV)═O intermediate is then believed to react with
reduced intracellular fluorescence signal. The effect was
even more dramatic when 1 was used in lieu of 1c. This was
taken as evidence that 1 has the ability to scavenge
intracellular ROS more effectively than 1c. This, in turn,
provides support for the suggestion that 1 could prove
useful as a CAT mimic in vitro. In addition, 1 displayed high
serum stability and proved inert to exchange in the
presence of 6 equiv of Zn(II) (Figure S14-15).
a second H
complex 1 is thought to support a metal cooperative effect
by stabilizing H between neighboring Mn(IV)═O
2 2
O molecule to release dioxygen. Importantly,
2 2
O
moieties resulting in an improved CAT activity, thus
providing an expected therapeutic benefit (Scheme 2).
Scheme 2. Proposed mechanism underlying the improved
catalase reactivity of triMn salen cryptand that is ascribed
to a cooperative effect involving neighboring Mn(IV)=O
moieties.
Figure 3. H2DCFDA fluorescence images (a) and relative
fluorescence intensities (b) of HUVEC cells co-incubated with
VEGF (2 ng/mL) and 1c or 1 (2 μM). Cells were treated with both
the VEGF HBSS solution and the indicated Mn complex and then
allowed to incubate in the dark for 2 h. A solution of H2DCFDA
1
00 μM was then added and the incubation continued for 30 min
prior to recording the images.
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Figure 4. a) Schematic of the stroke operation and injection procedure (intracerebroventricular injection, dose: 10 μL 2 mM 1 or 1c. b)
Biodistribution of 1 detected by MRI (7 T) after the surgery in a). c) PET images of rat brain following administration (tail vein injection)
of F-FDG to animals subject to the middle cerebral artery occlusion (MCAO) stroke model procedure and after treatment with 1 in
a). d) Coronal brain sections of rats (stained with triphenyltetrazolium chloride (TTC) solution) in the absence of MCAO (sham),
ischemia, DMSO, and treated with either 1c or 1. Brain slices are arranged in sequence. White areas represent the infarcted region after
MCAO. e) Calculated infarct volumes under the different conditions in d). ** P < 0. 01, *** P < 0.001.
18
In Vivo Studies. To test whether the ability to scavenge
ROS in vitro would translate into neuroprotective effects in
vivo, complex 1 was tested in an in vivo stroke model in
male Sprague-Dawley rats. This model was based on the
found that the neurological function of rats that were
injected with 1 was significantly greater compared to that
of the control group (Figure S16, P <0.01). Such findings
lead us to propose that treatment with 1 might have a role
to play in treating reperfusion injury.
endovascular suture middle cerebral artery occlusion
62
(
MCAO) method reported previously. In brief, a coated
Due to the good relaxivity seen for 1, we were able to use
MRI to determine qualitatively the biodistribution of the
complex within the brain in these animal studies. As shown
filament was inserted through the internal carotid artery
ICA) under anesthesia to achieve occlusion of the middle
(
cerebral artery (MCA), which was maintained for 90 min.
After the first 30 min of the occlusion event, either 1c or 1
were injected intracerebroventricularly (10 μL, 2 mM).
After an additional 60 minutes, reperfusion was performed
by withdrawing the thread and ceasing the anesthesia. This
stepwise process, designed to mimic the pathologic process
in clinic, is illustrated in Figure 4a. Neurological evaluation
was performed 24 hours after induction of ischemia and
scored on a 4-point scale: 0, no apparent deficit; 1,
contralateral forelimb flexion; 2, decreased grip of the
contralateral forelimb while tail pulled; 3, spontaneous
movement in all directions and contralateral circling only
if pulled by tail; 4, spontaneous contralateral circling. We
1
in Figure 4b, a strong T weighted signal enhancement was
observed at the injection point, indicating the successful
injection of the complex. Over a 24 h time period, the
signal gradually spread, which led to high contrast being
observed within the right side of brain. This finding was
taken as evidence of the ability of 1 to diffuse throughout
the brain.
As ischemic stroke results in the decreased availability of
1
8
glucose to the brain, F-FDG microPET (micro positron
63
emission tomography) was used to assess the difference
in glucose metabolism levels of the rat brains between the
control ischemic group and the group treated with 1 per
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the above protocol (Figure 4c). PET imaging revealed that
apoptosis, was also found to increase. In comparison, a
statistically significant decrease in the number of TUNEL-
positive cells per field was seen in the Mn(Salen)-treated
groups. This was true in the case of both the DAB and
fluorescence labelling experiments. Gratifyingly, the status
of the neuron cells after treatment with 1 was similar to the
sham group, whereas small amounts of neuronal apoptosis
was still observed for 1c. These results lead us to suggest
that reductions in ROS concentrations decrease the extent
of apoptosis in nerve cells and help account for the fact that
the glucose metabolism levels within the affected cerebral
hemisphere were significantly decreased as expected under
conditions of neuronal injury. In contrast the metabolic
activity of affected brains treated with 1 was near normal.
To test the effectiveness of 1 as a possible therapeutic
able to overcome ischemic-based brain injury in vivo,
triphenyltetrazolium chloride (TTC) staining was used to
quantify the infarct volume (taken as an indicator of brain
injury). As shown in Figure 4d, the sham group displayed
no evidence of infarction, while both the MCAO and the
untreated DMSO groups were characterized by large
infarct areas (white regions) with the measured percentage
being 27.51 ± 4.21 and 30.14 ± 5.03, respectively. However,
in comparison, animals treated with 1 and, to a lesser
extent 1c, displayed regions of damage that were
significantly reduced, as reflected in the corresponding
total infarct areas, i.e., 3.21 ± 1.01 and 11.12 ± 5.10 for 10 μL (2
mM) dosages of 1 and 1c, respectively. These effects were
found to be dose dependent, with the use of lower
quantities of these Mn(Salen) complexes leading to an
increase in the infarct regions. While not a proof, this
finding is consistent with the core suggestion underlying
this study, namely that the extent of recovery is correlated
with the antioxidant activity of each Mn-based complex
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1
proved more effective than 1c in overcoming the effects of
ischemic stroke in this rat model.
In order to determine whether the inhibition of cell
apoptosis by 1 was due to the regulation of apoptosis-
related
proteins,
caspase-3
and
Bax-2,
an
immunohistochemical staining method (IHC) was used to
determine the expression of these protein in neurons. As
shown in Figure 6, expression of caspase-3 increased for the
MCAO group. This is in accord with the expectation that
ischemic-based injury induces the expression of caspase-3
while promoting neuron apoptosis. However, the
expression level was reduced in a statistically significant
manner upon exposure to 1. A similar trend was seen for
Bax (P < 0.01). High Bax expression levels are found after
the MCAO procedure, while they decreased dramatically
upon treatment with 1. The disposition of caspase-3 and
Bax expression coincided with what was seen in the TUNEL
assays. On this basis, we infer that 1 may inhibit expression
of apoptosis related proteins, such as caspase-3 and Bax-2,
thereby suppressing neuronal apoptosis.
(
Figure S17). Most importantly, 1 demonstrated a
significantly greater therapeutic value (P < 0.01), which can
be seen visually in terms of the clear difference in the
infarct regions.
Hematoxylin and eosin (H&E) staining was employed to
determine the degree of neuronal damage and
degeneration in each group (Figure 5a). For the sham
group, the brain tissue was found to be compact, with the
shape and nucleus of neurons appearing to be normal (a
large number of Nissl bodies around the nucleus). However,
in the MCAO group, the infarct area was found to be fragile,
with a large area of severe damage reflecting necrosis of
neuronal cells. The damaged neuronal cells were reduced
in size and the nucleus was more heavily stained as
compared to the sham group as indicated by the black
arrow in Figure 5a. The group treated with 1c was found to
contain slightly disordered nerve fibers. In contrast,
minimal change in the morphology of neurons, including
no evident morphological change to the nucleus, was
observed in the case of 1.
To understand and elucidate the neuroprotective
mechanisms of 1, we evaluated the extent of neuronal
apoptosis using terminal deoxynucleotidyl transferase
biotin-dUTP nick end labelling (TUNEL, Figure 5b-c), and
fluorescence TUNEL methods (Figure 6d-e) to detect
64
fragmented DNA as described previously. As shown in
Figure 5, after the MCAO procedure, a TUNEL analysis
revealed an increase in brown staining of the TUNEL-
positive cells. In addition, the number of green
fluorescence labeled cells (yellow arrow), an indicator of
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reperfusion measured by immunohistochemical staining (IHC). *
P < 0.1, ** P < 0. 01, *** P < 0.001.
CONCLUSIONS
Inspired by the antioxidant properties of natural
catalases, three tri-Mn(Salen) cryptands 1-3 were prepared
as potential antioxidant metallodrugs. These catalase-
based biomimetics contain an “active site” with the Mn
centers in close proximity to each other. This structural
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arrangement was thought to allow for more effective H
2 2
O
dismutation as result of cooperative effects. In
a
operational terms, significantly greater oxygen production
was observed for 1-3 as compared to the monomeric
Mn(Salen) control 1c. Complex 1 proved more effective for
2 2
the overall dismutation of H O than either 2 or 3.
Therefore, it was subject to more detailed studies designed
to explore its potential as an antioxidant metallodrug.
Fluorescence and EPR spectroscopic analyses of 1 and 1c
revealed that 1, but not the control complex 1c, was
2 2
effective in promoting the dismutation of H O without
generating appreciable quantities of hydroxyl radicals. On
the basis of an in vivo stroke model, it was concluded that
the neurological function of rats was improved in a
statistically significant manner (P < 0.01; cf. Figure S16)
when treated with 1 relative to the control group following
1
8
ischemia. F-FDG microPET of the affected rat brains
revealed recovery of glucose metabolism levels following
neuronal injury and treatment with 1. Moreover, the infarct
volume was shown to be smaller in a statistically
significantly manner in comparison to the untreated
controls (total infarct area: 3.21±1.01 (1) vs 30.14 ± 5.03
control). Relative to the mono-Mn complex 1c, the tri-
Mn(Salen) system 1 provided a statistically significant
greater therapeutic effect (P value < 0.01), as judged by a
number of indicia. Fluorescence imaging assays and
immunohistochemical staining provide support for the
notion that the neuroprotective effect of 1 results from
reduction in the disease model-associated ROS
concentrations, which in turn leads to reduced apoptosis
of nerve cells and lower expression levels of apoptosis-
associated enzymes. This work thus highlights how multi-
centered manganese complexes, if appropriately designed,
might have a role to play in the treatment of diseases
related to oxidative stress. The ability to minimize
production of deleterious ROS, such as OH, is considered
to be a particularly desirable aspect of this potentially
generalizable approach.
Figure 5. a) Pathological examination of MCAO mice cerebral
sections by H&E staining. b) Microscopic analyses of cell death in
brain slices using TUNEL. TUNEL-positive cells are shown in
brown, and TUNEL-negative cells are shown in blue. Scale bar =
5
0 mm. c) Quantification of the TUNEL-positive cells in c). d)
Microscopic analysis of cell death in brain slices using fluorescent
TUNEL. TUNEL-positive cells give rise to green fluorescence,
while TUNEL-negative cells appear blue. Scale bar = 50 mm. e)
Quantification of the TUNEL-positive cells in e). * P < 0.1, ** P <
0
. 01, *** P < 0.001.
ASSOCIATED CONTENT
Supporting Information
Figure 6. Expression of a) caspase-3 and b) Bax positive cells per
region of interest in the cerebral cortex at 24 h following
Detailed synthesis, characterization and photophysical
1
data; H spectra, IR spectra, MS spectra. These materials are
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Amine-modified single-walled carbon nanotubes protect neurons from
injury in a rat stroke model. Nat. Nanotechnol. 2011, 6, 121-125.
0. Kim, C. K.; Kim, T.; Choi, I. Y.; Soh, M.; Kim, D.; Kim, Y.
J.; Jang, H.; Yang, H. S.; Kim, J. Y.; Park, H. K.; Park, S. P.; Park, S.;
Yu, T.; Yoon, B. W.; Lee, S. H.; Hyeon, T., Ceria nanoparticles that
can protect against ischemic stroke. Angew. Chem. Int. Ed. 2012, 51,
available free of charge via the Internet at
http://pubs.acs.org.
1
AUTHOR INFORMATION
Corresponding Author
1
1
1039-11043.
1. Kyle, S.; Saha, S., Nanotechnology for the detection and
*
*
*
Jun-Long Zhang. Email: zhangjunlong@pku.edu.cn
Lei Kang. Email: kanglei@bjmu.edu.cn
Jonathan L. Sessler. Email: sessler@cm.utexas.edu
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therapy of stroke. Adv. Healthc. Mater. 2014, 3, 1703-1720.
2. Davis, S. M.; Pennypacker, K. R., Targeting antioxidant
1
enzyme expression as a therapeutic strategy for ischemic stroke.
Neurochem. Int. 2017, 107, 23-32.
Author Contributions
1
3.
Liu, Y.; Ai, K.; Ji, X.; Askhatova, D.; Du, R.; Lu, L.; Shi, J.,
All authors have contributed to the design and implementation
of the studies reported here and have given approval to the final
version of the manuscript.
Comprehensive insights into the multi-antioxidative mechanisms of
melanin nanoparticles and their application to protect brain from injury
in ischemic stroke. J. Am. Chem. Soc. 2017, 139, 856-862.
14.
Jiang, X. C.; Xiang, J. J.; Wu, H. H.; Zhang, T. Y.; Zhang,
Notes
D. P.; Xu, Q. H.; Huang, X. L.; Kong, X. L.; Sun, J. H.; Hu, Y. L.; Li,
K.; Tabata, Y.; Shen, Y. Q.; Gao, J. Q., Neural stem cells transfected
with reactive oxygen species-responsive polyplexes for effective
treatment of ischemic stroke. Adv. Mater. 2019, 31, e1807591.
The authors declare no competing financial interest.
ACKNOWLEDGMENT
1
5.
Batinic-Haberle, I.; Reboucas, J. S.; Spasojevic, I.,
J.-L.Z. acknowledges financial support from the National Key
Basic Research Support Foundation of China (2015CB856301) and
National Scientific Foundation of China (NSFC) (21571007,
Superoxide dismutase mimics: Chemistry, pharmacology, and
therapeutic potential. Antioxid. Redox. Signal. 2010, 13, 877-918.
1
6.
Cheton, P. L.; Archibald, F. S., Manganese complexes and
21621061, 21778002, and 21861162008). We thank Dr. Yuanbo Cai,
the generation and scavenging of hydroxyl free radicals. Free Radical
Bio Med 1988, 5, 325-333.
Ms. Zi-Shu Yang for the help with in vitro data analyses and Mr.
Yuhui Fang, Zheng Liu and Xiaofan Wu for the help with EPR
measurements. L. K. acknowledges financial support from the
National Natural Science Foundation of China (81871385) and
PKU medicine-X Youth Program (PKU2020LCXQ007). The work
in Austin was supported by the National Institutes of Health (CA
1
7.
Baglia, R. A.; Zaragoza, J. P. T.; Goldberg, D. P.,
Biomimetic reactivity of oxygen-derived manganese and iron
porphyrinoid complexes. Chem. Rev. 2017, 117, 13320-13352.
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Groves, J. T.; Watanabe, Y., Heterolytic and homolytic 0-0
bond cleavage reactions of (acylperoxo)manganese(III) porphyrins.
Inorg. Chem. 1986, 25, 4808-4810.
68682 to JLS) and the Robert A. Welch Foundation (F-1018 to
J.L.S.).
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Jin, N.; Lahaye, D. E.; Groves, J. T., A "push−pull"
mechanism for heterolytic O−O bond cleavage in hydroperoxo
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