Abstract
Despite the proceeds in the management of acute myocardial infarction
(AMI), the current therapeutic landscape still suffers from limited
success in the clinic. Exaggerated inflammatory immune response and
excessive oxidative stress are key pathological features aggravating
myocardium damage. Herein, catalytic immunomodulatory nanocomplexes as
anti‐AMI therapeutics to resolve reactive oxygen species
(ROS)‐proinflammatory neutrophils‐specific‐inflammation is engineered.
The nanocomplexes contain lyophilic S100A8/9 inhibitor ABR2575 in the
core of nanoemulsions, which effectively disrupts the
neutrophils‐S100A8/A9‐inflammation signaling pathway in the AMI
microenvironment. Additionally, ROS scavenger ultrasmall Cu[x]O
nanoparticles are incorporated into the nanoemulsions via coordinating
with SH groups of poly(ethylene glycol) (PEG)‐conjugated lipids, which
mimic multiple enzymes, dramatically alleviating the oxidative stress
damage to myocardial tissue. This combination strategy significantly
suppresses the infiltration of pro‐inflammatory monocytes, macrophages,
and neutrophils, as well as the secretion of inflammatory cytokines.
Additionally, it potentially triggers cardiac Tert activation, which
promotes myocardial function and decreases infarction size in
preclinical murine AMI models. This approach offers a new nanomedicine
for treating AMI, resulting in a dramatically enhanced therapeutic
outcome.
Keywords: immunotherapy, myocardial infarction, nanoemulsion,
neutrophilic inflammation, ROS scavenger
__________________________________________________________________
This study constructs a catalytic immunomodulatory nanoemulsion for
effective treatment of acute myocardial infarction (AMI) by targeting
ROS‐neutrophilic inflammation. This approach effectively disrupts
neutrophil‐S100A8/A9 signaling pathway, alleviates the levels of
multiple radicals, and activates cardiac Tert signaling pathway,
thereby remarkably enhancing myocardial functions, and reducing
infarction sizes of AMI mice.
graphic file with name ADVS-11-2402267-g005.jpg
1. Introduction
AMI remains one of the leading causes of death and frailty in the
modern world, despite significant progress in the field of AMI
management.^[ [42]^1 ^] About 40% of AMI patients still have long‐term
or even life‐lasting heart failure.^[ [43]^2 ^] Myocardial infarction
triggers the infiltration of massive heterogeneous subsets of
pro‐inflammatory immune cells, of which the frequency of infiltrating
neutrophils is positively associated with increased infarct size and
declined heart function, representing an independent prognostic factor
in AMI patients.^[ [44]^3 ^] Therefore, a broad spectrum of treatments
has been explored to target neutrophils in animal AMI models, including
neutrophils‐derived alarmins S100A8/A9 inhibitors (ABR2575),^[ [45]^4
^] metoprolol (β1‐adrenergic‐receptor antagonist),^[ [46]^5 ^] and
anti‐Ly6G antibody (neutrophil depletion).^[ [47]^6 ^] Although the
significant improvements of these strategies in cardiac function and
tissue injury are evident, their clinical translation has yet to be
unsuccessful. This is mainly caused by the inappropriate
pharmacokinetic distribution of these small molecular drugs with
suboptimal effectiveness and unsatisfied safety profiles.^[ [48]^7 ^]
Emerging advanced nanoplatforms with finely tuned physiochemical
features can markedly alter pharmacokinetics, biodistribution, and
targeted delivery to diseased tissue.^[ [49]^8 ^] The implementation of
nano‐engineering means to target AMI‐specific inflammatory immune cells
has rarely been reported.^[ [50]^9 ^]
Massively increased ROS immediately post‐myocardial infarction is
another key pathological feature in the AMI microenvironment, which in
turn enhances the infiltration of circulating immune cells, the
activation of pro‐inflammatory phenotype (M1‐type) macrophages, and the
production of pro‐inflammatory cytokines.^[ [51]^10 ^] Excessive ROS
further deteriorates the inflammatory microenvironment and damages the
myocardial cells.^[ [52]^11 ^] Meanwhile, the intensive level of ROS
also greatly diminishes the therapeutic effects of small drugs.
Therefore, eliminating ROS to suppress oxidative stress injury and
improve drug efficacy is critically important for AMI treatment.
Beyond transport function, certain types of nanoparticles intrinsically
possess ROS‐scavenging capability and have demonstrated their potential
in the treatment of distinct inflammatory diseases, including
nanoenzymes for AMI treatment,^[ [53]^12 ^] ultrasmall metal
nanoparticles (e.g., Cu[5.4]O) for treating acute lung/kidney/liver
injury,^[ [54]^13 ^] 2D MXene as therapeutics for the treatment of
neurodegenerative diseases and hypertension,^[ [55]^14 ^] black
phosphorus nanosheets for osteoarthritis treatment,^[ [56]^15 ^] carbon
dots for ischemic stroke treatment,^[ [57]^16 ^] and ceria nanoclusters
for depression treatment.^[ [58]^17 ^] However, the integration of
nano‐ROS scavenger with neutrophil‐targeting therapy has been scarcely
exploited for enhanced AMI immunotherapy.
In the present study, we developed catalytic immunomodulatory
nanoemulsion complexes for the treatment of AMI using a self‐assembly
agent, 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine conjugated
PEG‐thiol (DSPE‐PEG‐SH), where S100A8/A9 inhibitor ABR molecules were
encapsulated in the lyophilic core and Cu[x]O nanoparticles were
coordinated with thiol groups on the surface of nanoemulsions
(NE‐ABR‐Cu[x]O, Scheme [59] 1A). Upon arriving at the infracted area
(Scheme [60]1B), Cu[x]O nanoparticles effectively scavenged distinct
types of ROS, alleviating oxidative stress. Additionally, ABR blocked
the activation of exaggerated neutrophils infiltrated in the infarct
area by interrupting the S100A8/A9‐NLPR3‐IL‐1β inflammation signaling
pathway, thereby suppressing inflammatory immune responses. In
particular, the frequencies of pro‐inflammatory phenotypes of innate
immune cells, such as Ly6C‐high monocytes and neutrophils, dramatically
decreased in the damaged myocardial tissue, while anti‐inflammatory
phenotypic M2 macrophages were significantly increased. Beyond that,
RNA sequencing analysis uncovered that NE‐ABR‐Cu[x]O enhanced
cardioprotection was also potentially medicated by a new therapeutic
pathway of cardiac Tert activation. NE‐ABR‐Cu[x]O nanocomplexes
combination therapy markedly repaired the myocardial function and
decreased the infarction size with a long‐term protection effect
against murine AMI, offering great potential in clinical invention
implementation.
Scheme 1.
Scheme 1
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An illustrative scheme shows A) the construction of NE‐ABR‐Cu[x]O
nanocomplexes via a nanoemulsion method, and B) the mechanism of action
in alleviating the oxidative stress and regulating neutrophilic
inflammation for effective treatment of acute myocardial infarction.
2. Results and Discussion
2.1. Construction and Characterization of NE‐ABR‐Cu[x]O
To prepare NE‐ABR‐Cu[x]O complexes, Cu[x]O ultrasmall nanoparticles
were first obtained by a reduction reaction between CuCl[2] and
L‐ascorbic acid, which were suspended in an aqueous solution. The
dimethylformamide (DMF) oil phase that contained DSPE‐PEG‐SH monomer
and ABR inhibitor molecules was slowly mixed with the aqueous phase to
form nanoemulsions, where the Cu[x]O nanoparticles were coordinated
with SH groups of monomers. The resultant NE‐ABR‐Cu[x]O complexes were
harvested via dialysis after the removal of DMF. Transmission electron
microscopy (TEM) images revealed that Cu[x]O nanoparticles displayed an
ultrasmall size of 5 nm (Figure [62] 1A). X‐ray photoelectron
spectroscopy (XPS) was performed to assess the chemical valence states
of Cu in Cu[x]O. The survey spectrum clearly implied the characteristic
peaks of Cu 2p[1/2] at 953.0 eV and Cu 2p[3/2] at 933.0 eV
(Figure [63]S1, Supporting Information). Further analysis of the XPS
spectrum of Cu 2p revealed strong characteristic peaks of Cu/Cu^+ at
952.9 and 933.2 eV (Figure [64]1B).^[ [65]^18 ^] The typical peaks at
955.0 eV and 935.2 eV and their satellite peaks at 963.0 and 943.0 eV
suggest a significant proportion of Cu^2+. To differentiate Cu and
Cu^+, the Cu LMM (L‐inner level‐M‐inner level‐M‐inner level electron
transition) Auger spectrum was collected. The results showed an Auger
electron kinetic energy peak at 915.4 eV, which corresponded to the
typical peak of Cu^+ (Figure [66]1C; Figure [67]S1, Supporting
Information). Collectively, Cu[x]O exhibited the coexistence of two
copper species of Cu^2+ and Cu^+. Following assembly with ABR, the
obtained NE‐ABR‐Cu[x]O complexes were well dispersed and spherical in
morphology with a size of ≈100 nm (Figure [68]1D, left). the Cu[x]O
nanoparticles were clearly observed in the spheres, as indicated by the
white arrows (Figure [69]1D, right). Dynamic light scattering (DLS)
analysis suggested that the average hydrodynamic size of NE‐ABR‐Cu[x]O
was ≈140 nm (Figure [70]1E), which was slightly larger than the size
measured by TEM measurement caused by hydration layers. The loading
capacity of ABR in NE‐ABR‐Cu[x]O was estimated to be 14.13% by UV–vis
spectrophotometry analysis (Figure [71]S2, Supporting Information), and
the concentration of Cu in the obtained NE‐ABR‐Cu[x]O solution was
0.03 mg/L determined by inductively coupled plasma‐atomic emission
spectrometer (ICP‐AES) analysis (Figure [72]S3, Supporting
Information). These quantified values were used for the dose
calculation in the following experiments.
Figure 1.
Figure 1
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Characterization and radical‐scavenging capability of NE‐ABR‐Cu[x]O. A)
A TEM image of Cu[x]O nanoparticles and their size distribution by
measuring 100 nanoparticles (inset). B) Cu 2p XPS and C) Cu LMM spectra
of Cu[x]O nanoparticles. D) TEM images of NE‐ABR‐Cu[x]O. E) The
hydrodynamic diameter distribution curve of NE‐ABR‐Cu[x]O in PBS
(phosphate‐buffered saline) solution by DLS measurement. F–K) UV−vis
spectra of detection agents and quantitative analysis of free‐radical
scavenging ability of NE‐ABR‐Cu[x]O toward F) DPPH·, G) PTIO·, H) ·OH,
I) ·O‐ 2, J) ·ABTS^+, and (K) H[2]O[2]. All data are shown as the mean
± standard deviation (SD).
2.2. Multiple Radical‐Scavenging Capability of NE‐ABR‐Cu[x]O Nanocomplexes
To evaluate the antioxidant ability of NE‐ABR‐Cu[x]O nanocomplexes,
multiple representative radicals were included in scavenging
assessments, i.e., 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH·),
2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide radical (PTIO·),
hydroxyl radical (·OH), superoxide anion (·O‐ 2), 2,2′‐azino‐bis
(3‐ethylbenzothiazoline‐6‐sulfonic acid) radical cation (·ABTS^+) and
hydrogen peroxide (H[2]O[2]). NE‐ABR‐Cu[x]O nanocomplexes displayed
high efficiency in scavenging of all examined radicals in a
concentration‐dependent manner. For DPPH· (Figure [74]1F;
Figure [75]S4, Supporting Information), PTIO· (Figure [76]1G), ·OH
(Figure [77]1H), and ·O‐ 2 (Figure [78]1I), NE‐ABR‐Cu[x]O exhibited
potent ROS scavenging under a low concentration of 30 ng/ml (Cu
concentration), with inhibition percentages of ≈50%, 31%, 34% and 45%,
respectively. Particularly, remarkable inhibition rates of ≈84% and 94%
were found for ·ABTS^+ (Figure [79]1J) and H[2]O[2] (Figure [80]1K) at
the same low concentration of NE‐ABR‐Cu[x]O. By comparison, free Cu[x]O
nanoparticles exhibited similar inhibition rates for DPPH· (43%), PTIO·
(44%), and ·ABTS^+ (75%) at a significantly higher concentration of Cu
(100 ng/mL in free Cu[x]O versus 30 ng mL^−1 NE‐ABR‐Cu[x]O) in
Figure [81]S5 (Supporting Information). The substantially enhanced
radical‐scavenging capability of NE‐ABR‐Cu[x]O possibly contributed to
the free reductive SH groups in DSPE‐PEG‐SH and improved interaction
sites between the coordinated Cu[x]O nanoparticles and free radicals.
Collectively, NE‐ABR‐Cu[x]O nanocomplexes are potent anti‐antioxidants
with superior performance in scavenging a wide spectrum of radical
species.
2.3. Protective Effect of NE‐ABR‐Cu[x]O on Primary Cardiomyocytes Against
Mitochondrial and DNA Damage
To assess the influence of NE‐ABR‐Cu[x]O on oxidative stress injury,
the primary cardiomyocytes were isolated and cultured as previously
described,^[ [82]^19 ^] the purity of which was verified via the
staining of α‐actinin (Figure [83]S6, Supporting Information). For
comparison, we constructed nanoemulsions in the absence of therapeutics
or in the presence of only ABR or Cu[x]O, which were denoted as NE,
NE‐ABR, and NE‐Cu[x]O, respectively. These formulations were similar in
sizes (30–200 nm) (Figures [84]S7A–C and [85]S8A–C, Supporting
Information). Of note, the TEM image of NE‐Cu[x]O clearly demonstrated
that Cu[x]O nanoparticles were uniformly decorated on the surface of
nanoparticles (Figure [86]S8B, Supporting Information), implying
successful coordination of Cu[x]O and SH groups. The quantified content
of Cu in the stock solution was 0.03 µg mL^−1 measured by ICP analysis
(Figure [87]S9, Supporting Information). All nanocomplexes displayed a
similar negative surface charge with ζ potential values between −20 and
−30 mV (Figure [88]S10, Supporting Information). Fourier transform
infrared (FTIR) spectroscopy analysis (Figure [89]S11, Supporting
Information) presented the characteristic peak at 770 cm^−1 (C = C
bending band) in the groups of NE‐ABR and NE‐ABR‐Cu[x]O, which was
assigned to C = C groups in ABR molecules, verifying the successful
encapsulation of ABR. The typical stretching peaks of 1100, 1705, and
2920 cm^−1 were ascribed to C‐O, C = O, and C‐H bonds from
DSPE‐PEG‐SH/ABR in all nanocomplexes.
H[2]O[2] was added into the medium (500 µM) for 2 h to mimic the
AMI‐induced oxidative stress injury. H[2]O[2] significantly promoted
the production of intracellular ROS, as indicated by ROS probe
dichlorodihydrofluorescein diacetate (DCFH‐DA) that was converted into
highly green fluorescence DCF upon oxidation (Figure [90] 2A,B). The
excessive ROS led to mitophagy, as indicated by MitoTracker
(Figure [91]2A). The treatment of NE‐Cu[x]O or NE‐ABR‐Cu[x]O but not NE
or NE‐ABR remarkably scavenged intracellular ROS and alleviated
mitophagy. H[2]O[2] also significantly decreased the mitochondrial
membrane potential, as indicated by an increased JC‐1 monomer/JC‐1
aggregate ratio (green/red) (Figure [92]2C; Figure [93]S12, Supporting
Information), which indicated mitochondrial damage. Treatment with
NE‐Cu[x]O or NE‐ABR‐Cu[x]O significantly increased the mitochondrial
membrane potential compared to the PBS, NE, and NE‐ABR‐treated groups
(Figure [94]2D, all P < 0.001). The restoration of mitochondrial
function in cardiomyocytes was attributed to the potent effect of
eliminating intracellular ROS. By contrast, the treatment of NE or
NE‐ABR showed a limited effect on mitochondrial damage (both P > 0.05),
which was caused by the negligible role in ROS scavenging. In addition,
the exposure to H[2]O[2] induced the expression of γ‐H2AX in
cardiomyocytes (green, Figure [95]2E), suggesting DNA damage under
oxidative stress. Consistently, NE‐Cu[x]O and NE‐ABR‐Cu[x]O but not NE
or NE‐ABR significantly alleviated the H[2]O[2]‐induced DNA damage
(Figure [96]2F). The cell viability assay revealed that H[2]O[2]
significantly decreased the cell viability to ≈25% (Figure [97]2G),
which was dramatically rescued via the treatment of NE‐Cu[x]O or
NE‐ABR‐Cu[x]O (Figure [98]2G, both P < 0.01, compared to the
PBS‐treated group), rather than the NE and NE‐ABR (both P > 0.05). In
summary, these results confirmed the superior protective effects of
NE‐ABR‐Cu[x]O and NE‐Cu[x]O on primary cardiomyocytes against ROS
injury.
Figure 2.
Figure 2
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NE‐ABR‐Cu[x]O maintains mitochondrial and DNA functions of
cardiomyocytes under oxidative stress. Representative confocal
microscopy images A,C,E) and quantitative analysis B,D,F) of
intracellular ROS A,B), mitochondrial membrane potential (JC‐1, C‐D),
and γ‐H2AX E,F) of primary cardiomyocytes treated with H[2]O[2] and NE,
NE‐ABR, NE‐Cu[x]O, NE‐ABR‐Cu[x]O or PBS. G) Viability of cardiomyocytes
under distinct treatments. The statistical difference between the
groups was analyzed with the Kruskal‐Wallis test followed by a Dunn's
multiple comparisons. Data are represented as mean ± SD. * and #
indicted P < 0.05, ** and ## indicted P < 0.01, *** and ### indicted P
< 0.001.
2.4. In Vivo Biosafety and Biodistribution Assessment of NE‐ABR‐Cu[x]O
To evaluate the biosafety of NE‐ABR‐Cu[x]O nanocomplexes, mice were
intraperitoneally injected with NE‐ABR‐Cu[x]O every two days over a
period of either 7 or 14 days. Blood tests and histological examination
of major organs were performed. The results demonstrated that there
were no significant differences in white blood cell (WBC), red blood
cell (RBC), platelet (PLT), and neutrophil (Neu) between treatment and
control groups (Figure [100]S13A–D, Supporting Information). The serum
biochemical indices, including creatinine (CREA), blood urea nitrogen
(BUN), aspartate aminotransferase (AST), and alanine transaminase
(ALT), in mice treated with NE‐ABR‐Cu[x]O nanocomplexes showed similar
levels to those in control mice (Figure [101]S13E–H, Supporting
Information). The histology analysis of the main organs, including the
lung, spleen, liver, heart, and kidney, revealed no apparent lesion
evidence of toxicity (Figure [102]S13I, Supporting Information).
To monitor the dynamic biodistribution of NE‐ABR‐Cu[x]O nanocomplexes
in vivo, NE‐ABR‐Cu[x]O was labeled with Cy5.5‐ maleimide via
maleimide‐thiol reactions, yielding NE‐ABR‐Cu[x]O‐Cy5.5. The mice were
sacrificed, and the main organs were harvested at 6 h, 48 h, or 7 days
after intraperitoneal injection for in vivo imaging systems (IVIS)
analysis. NE‐ABR‐Cu[x]O‐Cy5.5 nanocomplexes were found obviously
distributed in the heart and other vital organs, including the liver,
spleen, lung, kidney, and gastrointestinaltract at 6 h after injection
(Figure [103]S14A, Supporting Information). The distributions in all
the vital organs significantly declined at 48 h and Day 7
(Figure [104]S14B, Supporting Information). However, an opposite trend
was observed in the kidney, implying the renal retention and clearance
of NE‐ABR‐Cu[x]O‐Cy5.5 that might be degraded into nanoparticles less
than 10 nm.
Together, these results suggest that NE‐ABR‐Cu[x]O nanocomplexes can
effectively reach the therapeutic site in the heart, are systematically
and renally clearable, and are well tolerated without obvious systemic
toxicity. NE‐ABR‐Cu[x]O nanocomplexes are expected to be a safe therapy
for AMI.
2.5. Myocardial Function Restoration of NE‐ABR‐Cu[x]O in AMI Mouse Model
To assess the in vivo therapeutic effect of NE‐ABR‐Cu[x]O, AMI was
induced by the suture and ligation of the left anterior descending
artery (LAD) at a site of 3 mm from its origin post‐surgery in mice.
The sham mice underwent the same surgery, except that LAD was not tied.
Mice with AMI were treated with NE, NE‐Cu[x]O, NE‐ABR, NE‐ABR‐Cu[x]O,
or PBS immediately and one day after the AMI operation. The
transthoracic echocardiography was performed before and two days after
the AMI operation, and then the mice were sacrificed for triphenyl
tetrazolium chloride (TCC) staining. As shown in Figure [105] 3A,B, the
transthoracic echocardiography indicated that all nanoformulations
mentioned above had no effect on the cardiac function of healthy mice
before AMI operation, implying excellent biosafety on myocardial cells.
At day 2 post‐AMI, untreated mice showed an apparent decrease in left
ventricular ejection fraction (LVEF) and fraction shortening (FS),
indicating myocardial dysfunctions. However, the treatment of
NE‐Cu[x]O, NE‐ABR, or NE‐ABR‐Cu[x]O but not NE exhibited a significant
increase in LVEF and FS compared to untreated AMI mice
(Figure [106]3B), indicating the restoration of myocardial functions.
Particularly, the NE‐ABR‐Cu[x]O group showed significant therapeutic
improvement compared with the NE‐Cu[x]O and NE‐ABR groups
(Figure [107]3B).
Figure 3.
Figure 3
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The treatment of NE‐ABR‐Cu[x]O promotes cardiac function and reduces
infarction size in AMI mice. A) Representative M‐mode echocardiograms
at baseline and 2 days after AMI. B) Cardiac function was assessed by
the measurement of EF (%), FS (%), LVIDS (mm), LVIDD (mm), LVPWS (mm),
and LVPWD (mm) in AMI mice receiving different treatments. C,D)
Representative TTC staining images C) and bar charts D) showed the
infarcted sizes in heart harvested 2 days after AMI. The infarction
size was defined by the percentage of left ventricular volume
(infarcted area %). The statistical difference between the groups was
analyzed with the Kruskal‐Wallis test followed by a Dunn's multiple
comparisons. Data are represented as mean ± SD. * and # indicted P <
0.05, ** and ## indicted P < 0.01, *** and ### indicted P < 0.001.
To evaluate the heart structure, left ventricular internal diameter
systole (LVIDS), left ventricular posterior wall thickness in diastole
(LVPWD), and left ventricle posterior wall thickness in systole (LVPWS)
were measured by TTC analysis. Compared to the control, AMI ‐induced
LVIDS increased (Figure [109]3B). The treatment of NE‐Cu[x]O, NE‐ABR,
or NE‐ABR‐Cu[x]O but not NE displayed a significant decrease in LVIDS,
LVPWD, and LVIDS compared to untreated AMI mice. NE‐ABR‐Cu[x]O showed
superior performance in reducing LVIDS than the other two groups, which
might be attributed to the combined effect of ABR and antioxidant
Cu[x]O. Consistently, a significantly reduced infraction size was
detected in NE‐ABR‐Cu[x]O‐treated mice compared to the NE‐Cu[x]O and
NE‐ABR groups (Figure [110]3C,D). Overall, these results demonstrate
that NE‐ABR‐Cu[x]O nanocomplexes exhibit powerful cardioprotective
effects by enhancing heart function and reducing infarction size in the
context of AMI.
2.6. Suppression of AMI‐Induced Granulopoiesis Mediated by the Treatment of
NE‐ABR‐Cu[x]O
AMI‐induced granulopoiesis plays an essential role in mediating
systemic and local inflammatory responses post ‐AMI, and the
neutrophils and macrophages are the main pro‐informatory cells that
promote inflammation.^[ [111]^20 ^] Previous investigations have proven
that ABR regulates AMI‐induced inflammation mainly by inhibiting
granulopoiesis. Thus, we performed flow cytometry analysis to
investigate the effect of nanocomplexes on granulopoiesis
(Figures [112]S15 and [113]S16, Supporting Information). Our data
indicated that the treatment of NE‐ABR significantly reduced the
frequencies of neutrophils and monocytes infiltrated the heart
(Figure [114] 4A–G), indicating the dampening effect on AMI‐induced
granulopoiesis. AMI mice were treated with nanocomplexes immediately
and one day after the AMI operation, and mice were sacrificed on
post‐operative day two. Meanwhile, the treatment of NE‐Cu[x]O displayed
a comparable therapeutic effect in inhibiting the infiltration of
neutrophils and monocytes. Strikingly, NE‐ABR‐Cu[x]O showed the most
potent effect on reducing infiltrated neutrophils in the heart
(Figure [115]4C,D). Regarding the macrophages in the heart, all the
above treatments did not reduce the infiltrating CD11b^+ F4/80^+ Ly6G^−
macrophages, while the subtype analysis (Figure [116]4E–G) suggested
that NE‐ABR‐Cu[x]O significantly promoted the population of CD86^+
M2‐type macrophages (anti‐inflammatory) but not CD206^+ M1‐type
macrophages (pro‐inflammatory). By contrast, NE‐ABR and NE‐Cu[x]O did
not alter the frequencies of both phenotypic macrophages.
Figure 4.
Figure 4
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The treatment of NE‐ABR‐Cu[x]O suppresses AMI‐induced granulopoiesis.
Flow cytometric analysis shows the representative dot plots (left
panel) and quantification (right panel) of neutrophils, monocytes, and
macrophages in the heart A–G) and blood H–K) at 2 days post AMI in
Sham, AMI and nanocomplexes‐treated AMI mice. Cells in the hearts were
normalized to 100 mg of heart tissue. The statistical difference
between the AMI groups was analyzed with the Kruskal‐Wallis test
followed by a Dunn's multiple comparisons. Data are represented as mean
± SD. * and # indicted P < 0.05, ** and ## indicted P < 0.01, *** and
### indicted P < 0.001.
Consistently, CD115^+ Ly6G^− monocytes and CD115^− LyG6^+ neutrophils
circulating in the blood showed a similar trend in number reduction
after each intervention (Figure [118]4H,J; Figure [119]S17, Supporting
Information). As shown in Figure [120]4J, the monocytes in the blood
were significantly reduced in the NE‐Cu[x]O group (P < 0.01) and
NE‐ABR‐Cu[x]O group (P < 0.001), as compared to the PBS‐ and NE‐treated
groups. NE‐ABR‐Cu[x]O‐treated mice displayed further decreased
monocytes when compared to the NE‐Cu[x]O group (P < 0.01). Further
phenotyping analysis indicated that the inhibition of the
proinflammatory monocytes (Ly6C‐high monocytes) contributed to the
decreased number of total monocytes (Figure [121]4I–K), while no
significant differences regarding the number of anti‐inflammatory
monocytes (Ly6C‐low monocytes) were found among all the groups
(Figure [122]4K, all P > 0.05). These results implied that
NE‐ABR‐Cu[x]O nanocomplexes played remarkable anti‐inflammatory effects
compared to a single treatment by effectively suppressing the
infiltration of pro‐inflammatory subsets of innate immune cells.
2.7. NE‐ABR‐Cu[x]O does not Target the BONE marrow to Inhibit the AMI‐Induced
Granulopoiesis
Despite the fact that previous investigations have proven that ABR
targets cardiac neutrophils to inhibit AMI‐induced granulopoiesis,^[
[123]^4a ^] we observed that the immune cell counts in the above
experiment diminished in both the heart and the blood. The question
raised whether the target was the infarcted heart or the bone marrow
(BM). Next, we sought to answer this question by tracking the
distribution of NE‐ABR‐Cu[x]O‐Cy5.5 in BM post‐administration.
Figure [124]S18 (Supporting Information) showed no noticeable
fluorescence signals of NE‐ABR‐Cu[x]O‐Cy5.5 in BM, indicating that the
nanocomplexes did not enter BM. This result essentially ruled out the
possibility that NE‐ABR‐Cu[x]O inhibited the egress of immune cells
from the hematopoietic niches during AMI. In addition, we further
conducted experiments to check whether NE‐ABR‐Cu[x]O could inhibit the
pharmacologically induced release of immune cells from the BM
(Figure [125]S19a, Supporting Information). The results demonstrated
that granulocyte colony‐stimulating factor (G‐CSF) significantly
promoted the release of neutrophils from the BM in the blood
(Figure [126]S19b, Supporting Information) but not BM
(Figure [127]S19c,d, Supporting Information). However, the
NE‐ABR‐Cu[x]O made no significant effects on the G‐CSF‐induced
promotion of neutrophils (P > 0.05). Overall, the therapeutic effect of
NE‐ABR‐Cu[x]O in alleviating granulopoiesis w achieved by targeting the
infarcted heart rather than the BM.
2.8. Potent Inhibition Effect of NE‐ABR‐Cu[x]O on Myocardial Apoptosis and
Inflammation in AMI Mice
After AMI, ROS are released immediately by ischemic cardiomyocytes,
promoting the expression of chemokines and pro‐inflammation cytokines,
leading to apoptosis and necrosis of cardiomyocytes and, eventually,
the decline in cardiac function. The proinflammatory cytokines,
interleukin 6 (IL‐6), IL‐1β, and MCP‐1(monocyte chemoattractant
protein‐1), play a central role in AMI pathophysiology,^[ [128]^21 ^]
thus we next explored the changes of these key chemokines and cytokines
via western blotting (WB) and immunohistochemistry at 2 days after AMI,
to understand the therapeutic mechanism of NE‐ABR‐Cu[x]O. AMI‐induced
apoptosis (detected by TUNEL staining) was significantly ameliorated
after the treatment of NE‐Cu[x]O (NE‐Cu[x]O group versus PBS and NE
groups, both P < 0.01) (Figure [129] 5A,B; Figure [130]S20, Supporting
Information). A significant difference was detected between the
NE‐ABR‐Cu[x]O and NE‐ABR group, and a similar tendency was found
between NE‐ABR‐Cu[x]O and NE‐Cu[x]O, suggesting that the significant
improvement of NE‐ABR‐Cu[x]O in protecting myocardial cells from
apoptosis was contributed by the combined therapeutic effect from ABR
and Cu[x]O.
Figure 5.
Figure 5
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NE‐ABR‐Cu[x]O nanocomplexes inhibit apoptosis and inflammation in AMI
heart. A) Representative TUNEL staining images and B) the statistical
difference of TUNEL positive cells in heart tissues at 2 days post AMI
from Sham, AMI, and nanocomplexes‐treated AMI mice. C,D) Western
blotting and E) representative immunofluorescence staining images of
pro‐inflammatory cytokines, IL‐1β, IL‐6 and MCP‐1 in AMI hearts from
mice receiving distinct treatments as indicated (the quantitative
results seen in Figure [132]S21, Supporting Information). The
statistical difference was analyzed with the Kruskal‐Wallis test
followed by a Dunn's multiple comparisons. Data are represented as mean
± SD. * and # indicted P < 0.05, ** and ## indicted P < 0.01, *** and
### indicted P < 0.001.
Regarding the expression of inflammatory cytokines, WB analysis
(Figure [133]5C,D) indicated that the treatments of NE‐ABR‐Cu[x]O,
NE‐ABR, and NE‐Cu[x]O significantly decreased the protein expression of
IL‐1β, IL‐6, and MCP‐1. Among these treatment groups, NE‐ABR‐Cu[x]O
showed a superior inhibition effect on the expression of these
inflammatory cytokines as compared to the NE‐ABR and NE‐Cu[x]O groups.
The immunofluorescence analysis (Figure [134]5E; Figure [135]S21,
Supporting Information) suggested a similar trend in cytokine
expression with WB results, further supporting the potent
anti‐inflammation effect of NE‐ABR‐Cu[x]O nanocomplexes.
2.9. Long‐Term Ameliorating Effect of NE‐ABR‐Cu[x]O on Progressive Heart
Remodeling and Loss of Function Post‐MI
To validate the long‐term therapeutic efficacy of NE‐ABR‐Cu[x]O, we
next examined the ventricular remodeling (Masson staining for
myocardial fibrosis) and changes in cardiac function (transthoracic
echocardiography) at 28 days after MI. The nanocomplexes were injected
immediately after surgery and once per day within the three
post‐operative days. The results demonstrated that the treatment of
NE‐ABR‐Cu[x]O nanocomplexes significantly relieved the long‐term
decline in EF and FE percentages (Figure [136] 6A,B), implying durable
restoration of cardiac functions. Similarly, NE‐ABR‐Cu[x]O
nanocomplexes maintained a marked reduction in LVIDS, LVIDD, LVPWS, and
LVPWD, suggesting a longstanding effect on heart remodeling.
Pathological examination revealed that the chronic myocardial fibrosis
size in the heart was significantly reduced after the intervention of
NE‐ABR‐Cu[x]O (Figure [137]6C,D). Collectively, these results verified
the long‐term therapeutic effect of NE‐ABR‐Cu[x]O with dramatic
promotion of heart remodeling and function restoration, representing a
potent therapeutic modality with clinical potential for treating AMI
diseases.
Figure 6.
Figure 6
[138]Open in a new tab
NE‐ABR‐Cu[x]O nanocomplexes exhibit a long‐term inhibition effect on
the loss of cardiac function and myocardial fibrosis post MI. A)
Representative echocardiograms at 28 days after MI. B) Cardiac function
was assessed by the measurements of EF%, FS%, LVIDS (mm), LVIDD (mm),
LVPWS (mm) and LVPWD (mm). C) Representative images and D) statistical
difference of myocardial fibrosis sizes stained by Masson staining at
28 days after AMI. The statistical difference was analyzed with the
Kruskal‐Wallis test followed by a Dunn's multiple comparisons. Data are
represented as mean ± SD. * indicted P < 0.05, ** indicted P < 0.01,
*** indicted P < 0.001.
2.10. Molecular Mechanism Studies of NE‐ABR‐Cu[x]O using the Transcriptomic
Analysis
To reveal the detailed mechanisms underlying the protective action of
NE‐ABR‐Cu[x]O on the infarcted hearts, RNA sequencing analysis was
performed. Compared to the control group (PBS‐treated group), 7286
differently expressed genes (DEGs) were detected. In the Sham group,
1655 genes were up‐regulated, while the remaining 5631 genes were
down‐regulated (Figure [139] 7A). By contrast, the DEG numbers were
obviously decreased with only 1801 DEGs (929 up‐regulated and 872
down‐regulated genes), 1141 DEGs (426 up‐regulated and 715
down‐regulated genes), and 3591 DEGs (2273 up‐regulated and 1318
down‐regulated genes) between NE‐Cu[x]O versus PBS, NE‐ABR versus PBS,
and NE‐ABR‐Cu[x]O versus PBS, respectively.
Figure 7.
Figure 7
[140]Open in a new tab
Transcriptome profiles of untreated and treated infarcted hearts reveal
potential therapeutic mechanism. A) Up‐ and down‐regulated DEG in
different groups. B) GSEA depicts the significant Th17 cell
differentiation mediated inflammatory pathway in infarcted hearts
treated with PBS. C) KEGG map shows the most down‐regulated
inflammatory pathways, particularly IL‐17 signaling pathway and Th17
cell differentiation, in NE‐ABR‐Cu[x]O treated group by comparting with
untreated group in infarcted hearts. D,E) A heatmap D) shows expression
of MI related genes in infarcted hearts with distinct treatments. Red
marks the genes correlated with neutrophilic activation, inflammation,
endothelial activation, cardiac Tert activation E).
For further understanding of the immunological regulation by
NE‐ABR‐Cu[x]O on AMI, Gene Scores Enrichment Analysis (GSEA) and Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
were conducted (Figure [141]7B,C). The DEGs between the NE‐ABR‐Cu[x]O
and PBS group were significantly enriched in the Th17 differentiation
pathway (Figure [142]7B), which represented a trigger of the
proliferation and recruitment of neutrophils by secreting IL‐17
cytokines.^[ [143]^22 ^] After the treatment with NE‐ABR‐Cu[x]O,
NE‐Cu[x]O, or NE‐ABR, Th17 cell differentiation and IL‐17 signaling
pathways, as well as other inflammation‐ and neutrophils‐ related
pro‐inflammatory pathways were downregulated, including the nod‐like
receptor signaling, chemokine signaling and Th1/Th2 cell
differentiation signaling pathways.
Previous investigations have identified a significant increase in
fibroblast growth factor receptor (FGFR) 1 and FGFR 4,^[ [144]^23 ^]
and the inflammatory factors such as c‐reactive protein (CRP),^[
[145]^24 ^] tissue inhibitors of metalloproteinases (TIMPS)^[ [146]^25
^] and matrix metalloproteinase‐9 (MMP9) after MI,^[ [147]^26 ^] all of
which exacerbated the inflammation to worsen the cardiac function.
According to the heatmap analysis (Figure [148]7D), the FGFR1/4 and the
inflammatory factors were detected as being down‐regulated after the
treatment of NE‐ABR, NE‐Cu[x]O, or NE‐ABR‐Cu[x]O. Notably,
NE‐ABR‐Cu[x]O treatment exhibited the strongest regulatory effect on
these genes among the three nanocomplex groups, representing one of the
mechanisms by which NE‐ABR‐Cu[x]O exerted cardioprotection after MI.
Beyond that, the telomerase (TERT) that was recently validated as a
protective factor after MI,^[ [149]^27 ^] was highly expressed in
NE‐ABR‐Cu[x]O and NE‐ABR groups but not NE‐Cu[x]O, implying a new
therapeutic targeting pathway of ABR on cardioprotection.
Collectively, the RNA sequencing analysis revealed the detailed
mechanism by which the NE‐ABR‐Cu[x]O down‐regulates the genes related
to neutrophilic activation, endothelial activation, and inflammation
while upregulates the genes associated with cardiac Tert activation,
thereby protecting cardiac functions after AMI with improved
ventricular function and reduced infarct scars (Figure [150]7E).
3. Discussion
Post MI, cardiomyocyte damage triggers the production of massive ROS
via the mitochondrial oxidative phosphorylation, which promotes the
influx of high frequencies of proinflammatory phenotypic immune cells
at the infracted area, mainly including neutrophils, monocytes and
macrophages circulated from the bloodstream.^[ [151]^6 ^] Excessive
inflammation aggravates cardiac ischemic injury and heart failure.
Among the cardiac immune cells, macrophages resident in the infracted
areas are mainly pro‐inflammatory M1 type, modulating the inflammatory
responses. The application of antioxidant nanomedicines is one of the
most attractive strategies for alleviating oxidative stress‐mediated
heart injury. For example, allomelanin nanoparticles were engineered to
effectively scavenge multiple free radicals, which thereby promoted the
shift of M1 to M2 type macrophages, and reduced the infiltration of
neutrophils.^[ [152]^28 ^] As the first responders, neutrophils secret
alarmins of S100A8/A9, which primes the TLR4‐inflammasome‐IL‐1β
signaling pathway in naïve neutrophils, fueling the infiltration of
other subsets of innate immune cells.^[ [153]^4a ^] Thus, recently
emergent nanoplatforms targeting neutrophil‐driven pathologies are an
alternative appealing approach for treating AMI.^[ [154]^29 ^]
Despite the advancements in anti‐inflammation nanotherapeutics,
single‐target nanomedicines often find it hard to fully revere the
progression of AMI, which is caused by the multiplicity of targets
orchestrating the elevated pathological inflammation. To overcome the
heterogenicity of the inflammatory network, our work modulates a broad
spectrum of targets (ROS and neutrophils) using one
immuno‐nanoplatform. Referring to previously reported studies in the
field of conjoint therapeutic nanoplatforms,^[ [155]^30 ^] we set up
saline, empty NP, NP‐Cu[x]O, and NP‐ABR as the control groups. Our data
indicated that the NE‐ABR‐Cu[x]O displayed the strongest therapeutic
effects in AMI compared to all the other groups, supporting the
enhanced effects of Cu[x]O and ABR loaded by NE‐ABR‐Cu[x]O. In addition
to the expected therapeutic targets, the innovative combination of
antioxidant Cu[x]O nanoparticles and S100A8/A9 inhibitor (ABR)
molecules suggests new mechanisms for restoring the functions of
cardiomyocytes. Our results imply that the immunocomplexes downregulate
the Th17‐IL‐17 signaling pathway, thereby reducing neutrophil
activities. Direct targeting the upstream of Th17 cells might offer
advanced treatment modalities. Beyond conferring effects on reversing
pathological inflammation and immunity, the nanocomplexes containing
ABR display a potential function in direct TERT activation ‐mediated
cardioprotection. The discovered heterogenous and complex network of
endothelia‐cardiomyocyte‐immune cells‐cytokines (Figure [156]7E)
provides new insights into AMI treatment.
In summary, we developed multifaceted immunomodulatory NE‐ABR‐Cu[x]O
nanocomplexes for the treatment of AMI. NE‐ABR‐Cu[x]O was found to
effectively alleviate oxidative stress by scavenging multiple free
radicals of DPPH·, PTIO·, ·OH, ·ABTS^+, ×O‐ 2, and H[2]O[2]. In
addition, the developed nanocomplexes enabled specific regulation of
neutrophilic inflammation by blocking S100A8/A9‐NLPR3‐IL‐1β signals,
thereby potently suppressing the infiltration of massive
proinflammatory innate immune cells post AMI. Particularly,
NE‐ABR‐Cu[x]O therapy contributed to a dramatic decrease in the
frequencies of Ly6C‐high monocytes and neutrophils while a significant
increase in the proportion of anti‐inflammatory phenotypic M2
macrophages. The combination of immunomodulatory ABR and antioxidant
Cu[x]O nanoparticles substantially improved the therapeutic effect in
restoring the function of damaged myocardial cells and reducing
fraction size in AMI compared to mono‐ABR or Cu[x]O ‐mediated therapy.
RNA sequencing analysis uncovered the detailed potential gene network
and new therapeutic cardiac Tert activation pathway. Our work offers a
promising strategy to integrate enzyme ‐mimicking nanomedicine therapy
with immunotherapy for durable, effective, and specific treatment of
AMI, holding substantial potential for clinical translation. In
follow‐up studies, we attempt to optimize the method and timing of
NE‐ABR‐Cu[x]O administration, which may represent one way to further
improve the therapeutic efficacy.
4. Experimental Section
Chemical Reagents
S100A8/A9 inhibitor (ABR215757, paquinimod) was purchased from the MCE
company (MedChemExpress). DSPE‐PEG‐SH was purchased from the BroadPharm
company. Copper (II) chloride dihydrate (CuCl[2]×2H[2]O) was bought
from General Reagent of Titan (Shanghai, China). L‐ascorbic acid (AA),
sodium hydroxide (NaOH), and N,N‐dimethylformamide (DMF) were ordered
from Sinopharm Chemical Reagent (Shanghai, China).
Preparation and Characterization of NE‐ABR‐Cu[x]O
Cu[x]O nanoparticles. The Cu[x]O was synthesized according to the
previous report.^[ [157]^13b ^] First, 10 mmol CuCl[2]×2H[2]O powder
was dissolved in 50 mL deionized water. Subsequently, a slow dropwise
addition of 0.1 M L‐AA solution (50 mL) was initiated. The pH of the
solution was then adjusted to 8–9 by 1 M NaOH. The resulting mixture
was continuously stirred at 80 °C in an oil bath for a duration of
12 h. Afterward, the supernatant was collected by centrifugation at
8000 rpm for 15 min. The obtained solution was subjected to dialysis
(Mw cutoff: 3000) for one day, purifying Cu[x]O nanoparticles.
NE‐ABR‐Cu[x]O
To prepare therapeutics‐loaded nanoemulsions, 30 mg DSPE‐PEG‐SH and
6 mg ABR were dissolved in 1 mL DMF. The solution was subjected to
ultrasonication until it became colorless and transparent.
Subsequently, the DMF solution was dropwise added to 4 mL deionized
water or phosphate‐buffered saline (PBS) containing 100 ng of Cu[x]O
nanoparticles. The mixture was stirred for a duration of 12 h.
Following the incubation period, the reaction mixture was dialyzed (Mw
cutoff: 3000) for 24 h.
NE‐ABR‐Cu[x]O‐Cy5.5
To achieve fluorescence labeling of NE‐ABR‐Cu[x]O, Cy5.5‐maleimide
(Life‐iLab, AP35L084) and DSPE‐PEG‐SH were dissolved in DMF with a
molar ratio of 1:10 to form DSPE‐PEG‐SH‐Cy5.5 via maleimide‐thiol
reactions. NE‐ABR‐Cu[x]O‐Cy5.5 was prepared as described in the
fabrication of NE‐ABR‐Cu[x]O by replacing DSPE‐PEG‐SH with
DSPE‐PEG‐SH‐Cy5.5.
Free Radical Scavenging Assessment of NE‐ABR‐Cu[x]O
The free radical scavenging ability of NE‐ABR‐Cu[x]O was detected with
various antioxidant activity assays and monitored by UV–vis
spectroscopy, including H[2]O[2] assay, ·O‐ 2 assay, ·OH assay, DPPH·
assay, PTIO· assay, and ·ABTS^+ assay. The inhibition percentage of
radicals was calculated as follows except for ·OH:
[MATH: Inhibition%=A0−<
mi>AA0×100% :MATH]
(1)
where A is the absorbance of NE‐ABR‐Cu[x]O after incubation with
various indicators, and A0 is the absorbance of a blank control that
replaced NE‐ABR‐Cu[x]O with deionized water.
DPPH
1 mg of DPPH was dissolved in 24 mL of anhydrous ethanol and
ultrasonicated for 5 min to ensure complete dissolution. Subsequently,
DPPH solution (1 mL) was mixed with anhydrous ethanol (0.5 mL), and the
absorbance measured at 519 nm by a microplate reader was recorded as
A0. Then, DPPH solution (1 mL) was mixed with varying concentrations of
NE‐ABR‐Cu[x]O (6‐30 ng mL^−1), and after incubation at 37 °C for
30 min, the absorbance was measured and recorded as A.
PTIO
3 mg of PTIO was dissolved in deionized water (30 mL) under
ultrasonication for 5 min to ensure complete dissolution. Subsequently,
the prepared PTIO solution (0.8 mL) was mixed with deionized water
(0.2 mL) for dilution, and then the absorbance measured at 557 nm was
recorded as A0. Then, DPPH solution (0.8 mL) was mixed with varying
concentrations of NE‐ABR‐Cu[x]O and then incubated at 37 °C for 2 h.
Afterward, the absorbance was measured and recorded as A.
OH
27.8 mg of FeSO[4]·7H[2]O and 18 mg of 1,10‐Phenanthroline (PHEN) were
separately dissolved in deionized water (50 mL). Subsequently, 1 mL of
the FeSO[4]·7H[2]O solution, 0.2 mL of various concentrations of
NE‐ABR‐Cu[x]O, and 1 mL of 400 µM H[2]O[2] were incubated in the dark
for 3 min. Afterward, 1 mL of phenanthroline was added, and the
reaction proceeded in the dark for 30 min. Finally, the absorbance was
measured at 510 nm and recorded as A. In the same procedure, when
NE‐ABR‐Cu[x]O was replaced with water, the obtained value was recorded
as Ac, and when H[2]O[2] was replaced with water, the obtained value
was recorded as A0. Therefore, the formula for calculating the
inhibition percentage was as follows:
[MATH: Inhibition%=A−AcA0−Ac×100% :MATH]
(2)
ABTS^+
3 mg of ABTS diammonium salt was dissolved in 0.735 mL of deionized
water, and 1 mg of K[2]S[2]O[8] was separately dissolved in 1.43 mL of
water. Subsequently, 0.2 mL of each of the above solutions was mixed
and allowed to stand in the dark for 12 h. Afterward, it was diluted
20–30 times with anhydrous ethanol, and the diluted solution (0.8 mL)
was mixed with anhydrous ethanol (0.2 mL). The absorbance was then
measured at 734 nm and recorded as A0. After mixing with different
concentrations of NE‐ABR‐Cu[x]O, the absorbance was measured and
recorded as A.
O‐ 2
125 mM of L‐methionine, 750 µM of nitrotetrazolium blue chloride, and
20 µM of riboflavin were prepared. Subsequently, 0.3 mL of each
solution was mixed with 1.8 mL of PBS (pH 7.4). Afterward, the
resulting mixture was combined with varying concentrations of
NE‐ABR‐Cu[x]O and placed in a light incubator for 15 min. The
absorbance was then measured at 550 nm and recorded as A.
H[2]O[2]
According to a previous report,^[ [158]^31 ^] 2 g KI and 1 g potassium
hydrogen phthalate were separately dissolved in 30 and 48 mL of
deionized water, respectively. 20 mM H[2]O[2] solution was then
prepared. Subsequently, 0.9 mL of water, 35 µL of H[2]O[2], and 0.1 mL
of NE‐ABR‐Cu[x]O were mixed and incubated at 37 °C for 2 h. Afterward,
0.1 mL of KI and potassium hydrogen phthalate were added, and the
mixture was left to develop color for 30 min. The absorbance was then
measured at 350 nm and recorded as A.
In Vitro Experiments
Cell models: The primary cardiomyocytes were obtained and cultured as
previously described.^[ [159]^19 ^] One‐day‐old SD rat was quickly
sacrificed, and the hearts were digested with 0.03% trypsin and 0.04%
collagenase type II. Subsequently, the cardiomyocytes were isolated and
seeded in culture plates, the purity of which was verified via the
immunofluorescence staining of α‐actinin. To mimic the AMI‐induced
oxidative injury, 500 µM H[2]O[2] was added into the medium for 2 h.
Detection of Mitochondrial Function
The mitochondrial morphology was detected via MitoTracker Red CMXRos
staining according to the manufacturer's instructions (Invitrogen,
M7512). The function of the mitochondrial respiratory chain and the
antioxidant efficiency of nanocomplexes were measured by JC‐1 staining
according to the manufacturer's instructions (Thermo Fisher,
[160]M34152). Briefly, the cardiomyocytes were incubated in DMEM
containing PBS, NE‐ABR, NE‐Cu[x]O (50 ng Cu[x]O/mL) or NE‐ABR‐Cu[x]O
(50 ng Cu[x]O/mL) for 1 h and then H[2]O[2] was added. After further
incubating for 2 h, the medium was replaced with DMEM containing
MitoTracker Red CMXRos and Hoechst 38 450, or JC‐1 and Hoechst 38 450.
Following 30 min incubation, the cells were further photographed with a
confocal microscope (ZEISS, LSM710) or analyzed by flow cytometry.
Assessment of Intracellular ROS
In another parallel experiment, the DCFH‐DA was used to evaluate the
intracellular ROS according to the instructions (Sigma–Aldrich, D6883).
Briefly, the intracellular ROS oxidizes non‐fluorescent DCFH to
fluorescent form. Therefore, the fluorescent intensity indicates the
level of intracellular ROS. After the aforementioned incubation, the
cells were washed, and then 10 µM DCFH‐DA was added to the medium for
further incubation in the dark for 30 min. Afterward, the cells were
washed and imaged or subjected to a flow cytometry (Cytek Biosciences,
Fremont, CA) to quantify the intracellular ROS levels respectively.
Measurement of DNA Injury
In another parallel experiment, the injury of DNA was measured by
γ‐H2AX staining according to the instructions (Beyotime, C2035S). After
the treatment described above, the cardiomyocytes were fixed and
blocked and then incubated with anti‐γ‐H2AX primary antibody overnight.
Following washing, the cells were incubated with the secondary antibody
at room temperature for one hour.
Evaluation of Cell Viability
In another parallel experiment, the viability of cardiomyocytes was
measured by MTT Assay (Beyotime, C0009S). After the above‐mentioned
treatments, the cells were incubated with an MTT solution for 4 h.
After the dark blue formazan crystals were dissolved, the absorbance
was determined at 570 nm. Finally, the cell viability was shown as the
treated group/control group ratio (%).
Mice and Treatments
The adult male C57BL/6J mice aged 8–10 weeks were purchased from the
Shanghai SLAC Laboratory. The experiments were approved by the Animal
Care and Use Committee of Shanghai Chest Hospital (KS(Y)22 241) and
were performed in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals. Mice were
intraperitoneally injected (i.p.) with PBS, NE‐ABR (8 mg ABR/kg),
NE‐Cu[x]O (4 µg Cu[x]O/kg), or NE‐ABR‐Cu[x]O (8 mg ABR/kg and 4 µg
Cu[x]O/kg) nanocomplexes immediately after surgery and once per day
within postoperative three days. PBS served as the vehicle control.
Assessment of In Vivo Distribution
C57BL/6J mice were intraperitoneally injected with NE‐ABR‐Cu[x]O‐Cy5.5
(8 mg ABR/kg and 4 µg Cu[x]O/kg) and then sacrificed at 6 h, 48 h and 7
days. The organs and lower limb bones were dissected from mice treated
with ABR‐Cu[x]O‐Cy5.5 or PBS for ex vivo imaging using IVIS. Finally,
the fluorescence images and intensity were acquired by Living Image
(Perkin Elmer).
Biosafety Evaluation
The biosafety evaluation was conducted in C57BL/6J mice administrated
with PBS or NE‐ABR‐Cu[x]O nanocomplexes (8 mg ABR/kg and 4 µg
Cu[x]O/kg). The mouse blood and major organs were collected at 7 days
or 14 days after administration. The blood routine test of red blood
cell (RBC), white blood cell (WBC), platelet (PLT), and neutrophil
(Neu) was performed, and the major organs were collected for histology
studies. Meanwhile, the blood was centrifuged at 1500 rpm for 10 min
for further analysis of the levels of CK, AST, ALT, BUN, and CREA in
serum.
Mouse Model of Myocardial Infarction
At surgery, the mice were anesthetized with sodium pentobarbital
(60 mg kg^−1, i.p.) and maintained using 1% isoflurane. Briefly, after
the heart was exposed, the myocardial ischemia was induced by the
suture and ligation of LAD at a site of 3 mm from its origin. The sham
mice underwent the same procedure, with the exception that the LAD was
not tied.
Transthoracic Echocardiography
The transthoracic echocardiography (Biosound Esaote Inc.) was performed
at the indicated time points to evaluate cardiac function, and five
mice were subjected to the measurements in each group. The left
ventricle parameters were measured at both the long‐ and short‐axis
views. End‐systole or end‐diastole was achieved when the LV size was
the smallest or largest, respectively. LVESD and LVEDD were measured
using M‐mode tracing. The percentage of FS was calculated as
(LVEDD‐LVESD)/LVEDD × 100%.
Measurement of Myocardial Infarction Sizes and Scar Sizes
TTC staining in the acute phase. The heart tissue was harvested two
days after MI. After the section, the slices were immediately immersed
in 2% TTC at 37 °C for 30 min. The infarcted heart appeared
TTC‐negative (pale) after the staining and was outlined for further
calculation via ImageJ software. The infarcted area% was calculated as
the ratio of TTC‐negative area/total LV wall area × 100%. Masson's
trichrome staining in chronic phase. The heart was harvested at four
weeks after MI. After the routine process, the heart sections (5 mm
thick) were stained with Masson's trichrome to measure the cardiac
fibrosis and collagen deposition as previously described. The images
were acquired digitally and measured using ImageJ.
Flow Cytometry
Heart tissue: The single ‐cell suspension was prepared as previously
described to analyze the leukocytes in the heart. Briefly, the heart
was harvested, cleared of adhering tissues and atria, minced into three
pieces, transferred to GentleMACS C tubes containing collagenase type
II (1 mg mL^−1), and processed into a fine suspension using a tissue
dissociator (GentleMACS Octo Dissociator, Miltenyi Biotec). The
suspension was filtered through a 100 µm strainer, rinsed with FACS
buffer, and centrifuged at 500 g for 7 min at 4 °C. The resultant
pellet was resuspended in 1 mL buffer to determine cell count, followed
by staining for Live/Dead marker and surface proteins as described
above.
Data were acquired on an NL‐CLC flow cytometer (Cytek Biosciences,
Fremont, CA), and the analysis was performed with FlowJo software
(Ashland, OR). After excluding the doublets (by FSC‐H versus FSC‐A) and
dead cells (Live versus Dead), the neutrophils were identified as
CD45^+, CD11b^+, Ly6G‐hi cells, and the macrophages as CD45^+, CD11b^+,
Ly6G^−. To identify the polarization state, the macrophages were
further classified as CD86^+ cells (M1 activated macrophages) and
CD206^+ cells (M2 activated macrophages). The gating strategy was shown
in Figure [161]S12 (Supporting Information).
Peripheral Blood Leukocytes
The red blood cell lysed cells were washed and resuspended in 100 µL of
FACS buffer containing a cocktail of various antibodies directed
against different types of leukocytes. The stained cells were tested on
an NL‐CLC flow cytometer, and the data was analyzed via FlowJo
software. The neutrophils were identified as CD45^+, CD11b^+, and
Ly6G^+ cells, and the monocytes were identified as CD45^+, CD11b^+,
Ly6G^−, and CD115^+ cells, which were further classified as
pro‐inflammatory Ly6C‐hi monocytes and anti‐inflammatory Ly6C‐low
monocytes. The gating strategy was shown in Figure [162]S13 (Supporting
Information).
Immunofluorescence Assay
Immunofluorescence staining was performed to detect the expression of
IL‐1β, IL‐6, and MCP‐1 in formalin‐fixed sections of the infarcted
heart. The preparation of the sections was the same as in our previous
study.^[ [163]^19 ^] The prepared sections were incubated with the
primary antibody at 4 °C overnight (IL‐1β:Abcam, ab254360, 1:100;
IL‐6:Abcam, ab290735, 1:100; MCP‐1:Thermo Fisher, MA5‐17040, 1:300).
After incubating with secondary antibody (IL‐1β:Abmart, [164]M21014,
1:1000; IL‐6:Abmart, [165]M21012, 1:1000; MCP‐1:Abmart, M213408,
1:1000) for one hour at RT, the sections were washed and stained with
DAPI. The images were obtained by confocal photography.
Western Blotting
The procedure for protein extraction and the Western blotting
experiment was the same as the previous experiment. The antibodies used
were listed as following: IL‐1β‐Abmart, PK56327, 1:500; IL‐6‐Abmart,
TD6087, 1:1000; MCP‐1‐Abmart, TD7577, 1:1000; secondary
antibody‐Abmart, [166]M21002, 1:5000.
RNA Sequencing
The infarcted hearts were harvested two days after an infarction, and
Trizol extracted the total RNA. In the present study, the DESeq
algorithm to conduct the differential expression analysis was applied,
and the FDR‐corrected P < 0.05, log2FC > 1, or log2FC < −1 was
identified for the threshold for differentially expressed genes. To
further explore the functional and biological implications of the DEGs,
the GSEA and KEGG enrichment analyses were conducted.
Evaluation of the Effect on the Release of Neutrophils from the Bone Marrow
To perform the pharmacologically induced release of neutrophils from
the bone marrow, the mice were subcutaneously injected with a daily
dose of 250 µg kg we applied of recombinant human G‐CSF protein
(Proteintech, HZ‐1207) for 7 days.^[ [167]^32 ^] NE‐ABR‐Cu[x]O was i.p.
injected simultaneously once per day to check whether it inhibits the
G‐CSF‐induced release. Four hours after the last injection, the mice
were sacrificed to obtain peripheral blood and bone marrow for further
analysis. The count of neutrophils in blood was assessed by the routine
blood tests. The bone marrow was prepared as described previously and
analyzed by flow cytometry.^[ [168]^4a ^] After excluding the doublets
(by FSC‐H versus FSC‐A) and dead cells (Indo 1^+), the neutrophils were
identified as CD45^+ CD115^−, Gr‐1^+ cells.
Statistical Analysis
All data were presented as mean ± SD. For comparing the differences
between the two groups, the Mann‐Whitney test (U test) was applied. For
the comparisons of three or more groups, the Kruskal‐Wallis test,
followed by Dunn's multiple comparisons, was used. The P value ≤ 0.05
was considered statistically significant. All analyses were performed
via SPSS version 22.0, and the detailed analysis method was described
in each figure legend.
Conflict of Interest
The authors declare no conflict of interest.
Supporting information
Supporting Information
[169]ADVS-11-2402267-s001.docx^ (2.7MB, docx)
Acknowledgements