Abstract
The engineered nanoformulation that can be activated by intracellular
tumor microenvironment, including acidic pH, overexpressed H[2]O[2],
and high concentration of glutathione (GSH), features high efficacy to
eradicate tumor cells with the intrinsic specificity and therapeutic
biosafety. However, the relatively slow reaction rate of traditional
Fe^2+-mediated Fenton reaction induces the low production amount of
reactive oxygen species (ROS) and subsequently the limited therapeutic
outcome against tumors. Here, we established Cu (II)-based
two-dimensional (2D) metal–organic framework (MOF) nanosheets as a
distinct chemoreactive nanocatalyst for GSH-triggered and
H[2]O[2]-augmented chemodynamic therapy (CDT), depending on the “AND”
logic gate, for significant tumor suppression. After internalization by
tumor cells, the MOF catalytic nanosheets reacted with local GSH for
inducing GSH consumption and reducing the Cu^2+ into Cu^+.
Subsequently, abundant hydroxyl radicals (·OH) generation was achieved
via Cu^+-mediated Fenton-like catalytic reaction. The dual effects of
·OH production and GSH depletion thus enhanced ROS production and
accumulation in tumor cells, leading to significant cellular apoptosis
and tumor inhibition, which was systematically demonstrated in both 4T1
and MDA-MB-231 tumor models. Therefore, GSH and H[2]O[2], serve as an
“AND” logic gate to trigger the Cu^+-mediated Fenton-like reaction and
reduce GSH level for augmented CDT with high therapeutic specificity
and efficacy, thus inducing cellular apoptosis primarily through
ferroptosis at the RNA sequence level.
Graphical Abstract
[35]graphic file with name 12951_2022_1250_Figa_HTML.jpg
Supplementary Information
The online version contains supplementary material available at
10.1186/s12951-022-01250-x.
Keywords: Metal–organic framework, Nanocatalytic therapy, Fenton
reaction, Tumor therapy, Nanomedicine
Introduction
The initiated production of toxic reactive oxygen species (ROS) with
the aid of nanoformulations has been exploited as an effective
therapeutic modality for tumor treatment [[36]1–[37]5]. In particular,
chemodynamic therapy (CDT) typically utilizes ferrous ions (Fe^2+) to
catalyze the production of highly oxidative hydroxyl radicals (·OH)
[[38]6–[39]9] through Fenton or Fenton-like chemical reactions with
excess hydrogen peroxide (H[2]O[2]) in tumor microenvironment (TME)
[[40]10–[41]12]. In comparison with other external energy
input-initiated therapeutic strategies, CDT activated by the intrinsic
chemical energy conversion in tumorous tissues rather than external
energy circumvents the rapid attenuation in energy input during the
therapeutic processes [[42]13]. Furthermore, the
transition-metal-containing nanocatalysts respond to the unique TME
characteristics for CDT [[43]14, [44]15], featuring low invasiveness
and high treatment specificity. However, the Fe^2+-mediated Fenton
reactions proceed effectively in highly acidic milieu (pH 2–4)
[[45]16]. In addition, even at the desirable reaction surroundings, the
relatively slow reaction rates of Fe^2+-mediated Fenton reactions
contribute to low capability in inducing ROS generation. Therefore, it
is highly desirable and necessary to engineer the iron-free
transition-metal-containing nanoformulations [[46]17, [47]18] that
feature a high therapeutic specificity and favorable catalytic
performance in weakly acidic TME for efficient CDT-based cancer
treatment.
By contrast, the Cu-based Fenton-like nanocatalysts are preliminarily
demonstrated to be the potential candidates for CDT because of their
high efficiency in weakly acidic TME [[48]19, [49]20]. Especially, the
reaction rate of Cu^+ was calculated to be 1 × 10^4 M^−1 s^−1 [[50]21,
[51]22], almost 160-fold than that of Fe^2+ (~ 63 M^−1 s^−1) [[52]23,
[53]24]. However, Cu^+ is extremely unstable and easily oxidized into
Cu^2+ attributing to the low redox potential of Cu^2+/Cu^+ [[54]25].
Moreover, excess free Cu^+ may cause severe systemic toxicity
[[55]26–[56]28]. In addition, the ROS produced through Cu^+-catalyzed
Fenton-like reaction would be reduced and quenched by excessive
reductive molecules in tumorous cells, which thus lowers the
therapeutic efficacy of CDT. Therefore, it is crucially significant to
spatiotemporally confine the nanoformulations at tumor regions to avoid
the production of free Cu^+ during the blood circulation [[57]29].
Furthermore, it is conceived that we can fabricate Cu^2+-containing
nanoformulations with high stability in physiological media, which can
be specifically reduced to Cu^+ at tumor sites by excess glutathione
(GSH) in TME [[58]30–[59]35]. Subsequently, efficient ROS production
can be achieved through Cu^+-catalyzed Fenton-like reaction with
overexpressed H[2]O[2] in tumorous tissues.
As one type of burgeoning porous nanoformulations, nanosized
metal–organic frameworks (MOFs) have been extensively utilized in
catalysis, biosensing, and theranostic applications [[60]36–[61]40],
attributing to their unique superiorities of large specific surface
area as well as structural tunability and diversity. Considering the
high specific surface area and active centers of nanosized MOF, we
assumed that a rationally tailored nanosized MOF with dual functions
should be qualified to decrease the GSH level and successively activate
Cu^+-mediated Fenton-like reaction, thus augmenting the therapeutic
efficacy of CDT depending on the “AND” logic gate. Here, we designed
and constructed a nanocatalyst, i.e., Cu (II)-based nanosized MOF
(PEG/Cu-BDC; BDC^2− = 1,4-benzenedicarboxylate), for achieving the
augmented and TME-activated CDT against tumors. Once endocytosis by
tumor cells, the released Cu^2+ in PEG/Cu-BDC MOF was reduced to Cu^+
along with the transformation from GSH to oxidized GSH (GSSG)
(Eq. [62]1), followed by H[2]O[2] consumption, ·OH generation, and
oxidation from Cu^+ to Cu^2+ through Fenton-like reaction (Eq. [63]2).
The double effects of ·OH generation and GSH consumption elevate the
ROS level in tumor cells, inducing the effective cell apoptosis and
tumor suppression. Therefore, GSH and H[2]O[2], overexpressed in
tumorous tissues, serve as an “AND” logic gate to activate the
Cu^+-catalyzed Fenton-like reaction and decrease the GSH level for
augmented CDT with high therapeutic efficiency and tumor specificity.
This work introduces a novel copper nanosized metal–organic framework
responsive to the TME, which may have immense potential in chemodynamic
cancer therapy (Fig. [64]1).
[MATH:
Cu2++GSH→Cu++GSSG. :MATH]
1
[MATH:
Cu++H2O2→Cu2++·OH+OH-. :MATH]
2
Fig. 1.
[65]Fig. 1
[66]Open in a new tab
The fabrication of PEG/Cu-BDC nanocatalyst and its therapeutic
application for augmented CDT through the activation of Cu^+-catalyzed
Fenton-like reaction and reduction of GSH levels
Results and discussion
Design, synthesis and characterization of 2D PEG/Cu-BDC nanocatalysts
There are two main procedures in the synthesis of PEG/Cu-BDC, with the
color change from transparent to yellow in the first step owing to the
generation of Cu[2]O and yellow to blue in the conversion process of
Cu^+ to Cu^2+ [[67]41]. The morphology, composition, structure, and
porosity of the as-obtained Cu-BDC nanosheets were confirmed by diverse
characterization techniques. As observed by transmission electron
microscopy (TEM), Cu[2]O showed cubic morphology with a mean size of
60 nm, whereas PEG/Cu-BDC nanosheets were square with a diameter of
approximately 90 nm (Fig. [68]2a, b). As for X-ray diffraction (XRD)
patterns, the disappearing of Cu[2]O peaks and emerging of the main (2
0 − 1) crystallographic planes in Fig. [69]2c illustrated the complete
conversion of Cu[2]O to Cu-BDC nanosheets. In addition, the X-ray
photoelectron spectroscopy (XPS) analysis affirmed that the Cu-BDC
nanosheets were composed of Cu, C, and O elements (Fig. [70]2d). The Cu
2p core peak of Cu-BDC nanosheets revealed two main components at
954.4 eV and 934.4 eV, with a satellite peak located at 962.6 eV and
943.7 eV, respectively, validating the presence of Cu^2+ in the
structure of Cu-BDC nanosheets (Fig. [71]2e) [[72]42, [73]43].
Moreover, Fourier-transform infrared spectroscopy (FTIR) spectrum of
Cu-BDC nanosheets was shown in Fig. [74]2f. The characteristic peaks at
1578, 1501, 1156, and 1017 cm^−1 belonging to the benzene rings of the
ligands were observed. The characteristic bands at 1624 and 1439 cm^−1
were indexed to the symmetric and antisymmetric stretching vibrations
of –COOH group. Furthermore, thermogravimetric analysis (TGA) was
performed to investigate the thermal stability of Cu-BDC nanosheets. As
depicted in Fig. [75]2g, the obtained TGA profile of Cu-BDC nanosheets
under air (or N[2]) atmosphere illustrated that the structure of Cu-BDC
remained stable up to 300 °C. The weight loss was determined to be 16
wt% by heating to 300 °C, which was attributed to the liberation of the
coordinated N, N-dimethylformamide (DMF) molecule. The occurrence of
weight loss was observed ranging from 300 to 330 °C, owing to the
decomposition of the BDC ligand of Cu-BDC nanosheets. Furthermore, the
Brunauer–Emmett–Teller (BET) surface areas were determined to be 53.2,
254.7, and 307.6 m^2 g^−1 for the Cu-BDC nanosheets pretreated at 120,
200, and 250 °C for 12 h before examination, respectively, indicating
the porosity evolution after heat treatments by getting rid of
the guest DMF molecules.
Fig. 2.
[76]Fig. 2
[77]Open in a new tab
Structure, morphology, and composition characterization of PEG/Cu-BDC.
a TEM images of Cu[2]O. b TEM images of PEG/Cu-BDC. c XRD patterns of
Cu[2]O and PEG/Cu-BDC. d XPS spectrum of the PEG/Cu-BDC. e XPS spectrum
of Cu 2p in PEG/Cu-BDC. f FTIR spectrum of PEG/Cu-BDC. g TGA curves of
PEG/Cu-BDC in air and N[2]. h N[2] absorption–desorption isotherms of
PEG/Cu-BDC activated at 120, 200, and 250 ℃ for 12 h prior to the
tests, respectively
In vitro Fenton-like reaction enabled by 2D PEG/Cu-BDC nanocatalysts
There were two necessary procedures in the Fenton-like reaction
mediated by PEG/Cu-BDC, GSH consumption and ·OH generation. The GSH
depletion was confirmed with the assistance of an indicator, DTNB
[5,5′-dithio-bis-2-(nitrobenzoic acid)], which generates yellow
compound (5-thio-2-nitrobenzoic acid) showing a specific peak at 405 nm
after reaction with GSH. As displayed in Fig. [78]3a, the GSH level
kept declining with the elevating concentration of PEG/Cu-BDC. In
addition, the GSH depletion was evidently accelerated after incubation
of PEG/Cu-BDC at pH 6.5 under the same condition (Fig. [79]3b–d). The
results illustrated that the obtained PEG/Cu-BDC significantly consumed
GSH in acidic TME. Furthermore, the ·OH generation property of
PEG/Cu-BDC at different pHs was also assessed using a ·OH indicator,
3,3′,5,5′-tetramethylbenzidine (TMB), which exhibits the characteristic
peak at 652 nm after oxidation by ·OH. As depicted in Fig. [80]3e,
negligible absorbance change at 652 nm can be detected after incubation
of TMB with H[2]O[2]. In contrast, PEG/Cu-BDC accelerated the ·OH
generation in the presence of GSH and H[2]O[2] by measuring the
absorbance of TMB at 652 nm, while a slight increment in the absorbance
at 652 nm can be observed after incubation of TMB with PEG/Cu-BDC in
the presence of GSH. In addition, the ·OH generation enhanced with the
extension of reaction durations as well as the increasing proportion of
H[2]O[2] (Fig. [81]3f–h). Furthermore, the electron spin resonance
(ESR) spectroscopy was also recorded to confirm the ·OH production
using a ·OH capture agent, 5, 5-dimethyl-1-pyrroline-Noxide (DMPO). The
presence of the characteristic signals in the ESR spectra validated the
high capability of PEG/Cu-BDC in generating ·OH (Fig. [82]3i). All
these results confirmed that GSH and H[2]O[2] can serve as an “AND”
logic gate to activate the Cu^+-catalyzed Fenton-like reactions for
efficient ·OH generation in the presence of PEG/Cu-BDC nanocatalysts.
Fig. 3.
[83]Fig. 3
[84]Open in a new tab
In vitro GSH depletion and ·OH generation enabled by PEG/Cu-BDC. a
UV–Vis absorption spectra of DTNB solution containing different
proportions of GSH and PEG/Cu-BDC. b, c UV–Vis absorption spectra of
DTNB solution containing GSH and PEG/Cu-BDC at (b) pH 6.5 and (c) pH
7.4. d The comparison of GSH consumption rate under pH 6.5 and 7.4,
respectively. e UV–Vis absorption spectra of oxidized TMB solution
after various treatments. f, g UV–Vis absorption spectra of oxidized
TMB solution containing the proportions of PEG/Cu-BDC to H[2]O[2] of
(f) 1: 1 and (g) 1:2. h The ·OH generation rate in different
proportions of PEG/Cu-BDC to H[2]O[2]. i EPR spectra of DMPO containing
different proportions of PEG/Cu-BDC to H[2]O[2]
In vitro tumor cell-killing activity of 2D PEG/Cu-BDC nanocatalysts
The intracellular uptake of 2D PEG/Cu-BDC nanocatalysts was assessed
using confocal laser scanning microscopy (CLSM) observation after
incubation of 4T1 and MDA-MB-231 breast tumor cells with fluorescein
isothiocyanate (FITC)-labeled PEG/Cu-BDC for different durations. On
the basis of the obtained CLSM images, the green fluorescence of
FITC-labeled PEG/Cu-BDC enhanced with the extension of the incubation
duration, which demonstrated that PEG/Cu-BDC could be efficiently
endocytosed by 4T1 and MDA-MB-231 breast tumor cells (Figs. [85]4a,
[86]5a). After confirmation of the efficient internalization of
PEG/Cu-BDC, the ROS generation in 4T1 and MDA-MB-231 breast tumor cells
was evaluated after incubation with various concentrations of
PEG/Cu-BDC using 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) as
an indicator. The green fluorescence signal brightened with the
increasing concentration of PEG/Cu-BDC, and the signal reached the
maximum at an incubation concentration of 100 μg mL^−1, demonstrating
the efficient ROS generation enabled by PEG/Cu-BDC nanocatalysts (Figs.
[87]4b, [88]5b).
Fig. 4.
[89]Fig. 4
[90]Open in a new tab
Cellular uptake and therapeutic efficacy of PEG/Cu-BDC against 4T1
breast tumor cells. a CLSM images of 4T1 breast tumor cells after
incubated with FITC-labeled PEG/Cu-BDC for various durations (1, 2, 4,
and 6 h). Scale bars: 30 μm. b Intracellular ROS generation in 4T1
breast tumor cells after cultured with various concentrations of
PEG/Cu-BDC (0, 20, 50, and 100 μg mL^−1). Scale bars: 50 μm. c Cellular
viability of the 4T1 breast tumor cells after incubated with PEG/Cu-BDC
for 24 and 48 h, respectively (n = 5 biologically independent samples).
d Live/dead staining of 4T1 breast tumor cells with calcein AM and PI,
respectively. Scale bars: 100 μm. e Flow cytometry analysis showing
apoptosis of 4T1 breast tumor cells after incubation with different
concentrations (0, 20, 50, and 100 μg mL^−1) of PEG/Cu-BDC for 24 h
Fig. 5.
[91]Fig. 5
[92]Open in a new tab
Cellular uptake and therapeutic efficacy of PEG/Cu-BDC against
MDA-MB-231 breast tumor cells. a CLSM images of MDA-MB-231 breast tumor
cells after incubated with FITC-labeled PEG/Cu-BDC for various
durations (1, 2, 3, and 4 h). Scale bars: 30 μm. b Intracellular ROS
generation in MDA-MB-231 breast tumor cells after cultured with various
concentrations of PEG/Cu-BDC (0, 10, 20, 50, and 100 μg mL^−1). Scale
bars: 50 μm. c Cellular viability of MDA-MB-231 breast tumor cells
after incubated with PEG/Cu-BDC for 24 and 48 h, respectively (n = 5
biologically independent samples). d Live/dead staining of MDA-MB-231
breast tumor cells with calcein AM and PI, respectively. Scale bars:
100 μm
The tumoricidal activity of PEG/Cu-BDC nanocatalysts against 4T1 and
MDA-MB-231 breast tumor cells was then assessed by the standard
counting kit-8 (CCK-8) assay, CLSM observation, and flow cytometry
(FCM) analysis. To further quantitively investigate the anti-tumor
efficiency, 4T1 and MDA-MB-231 breast tumor cells were cultured with
different concentrations of PEG/Cu-BDC for 24 and 48 h. As depicted in
Figs. [93]4c and [94]5c, the cell viabilities of 4T1 breast tumor cells
were 38.6% and 29.86%, respectively, corresponding to 44.23% and 33.07%
for MDA-MB-231 breast tumor cells after treatment with 100 μg mL^−1 of
PEG/Cu-BDC for 24 and 48 h. To confirm the high biocompatibility of
PEG/Cu-BDC nanocatalyst towards normal cells, the cell viability of
PEG/Cu-BDC nanocatalyst against normal endothelial cells was also
evaluated. As shown in Additional file [95]1: Fig. S1, the cell
viability was determined to be 90.3% after treatment with 100 μg mL^−1
for normal endothelial cells, which was significantly higher than that
of 38.6% for 4T1 cells. Additionally, a calcein acetoxymethyl ester
(calcein-AM) and propidium iodide (PI) assay was further applied to
distinguish the live and dead cells by means of color directly. As
presented in Figs. [96]4d and [97]5d, the red PI fluorescence signal
enhanced with the elevating concentration of PEG/Cu-BDC. Furthermore,
the flow cytometry analysis was performed to illustrate that the
therapeutic efficiency enhanced with the elevated concentration, which
was in accordance with the CLSM observation results (Fig. [98]4e).
RNA sequencing was further conducted to investigate the antitumor
mechanism of PEG/Cu-BDC by analyzing the mRNA profiling in 4T1 cells
after incubation with saline and PEG/Cu-BDC nanosheets, respectively.
As shown in Fig. [99]6a, the box plots in relation with 6 different
groups (3 groups for control and 3 groups for PEG/Cu-BDC) were at the
same level, which revealed the homogeneity of cell samples, laying
foundation for the following mechanism investigation. There were 5041
differentially expressed genes in both saline and PEG/Cu-BDC treated
groups, including 2608 up-regulated and 2433 down-regulated ones,
respectively (Fig. [100]6b, c). On the basis of the differentially
expressed genes, several key genes that are relevant to cell apoptosis
and proliferation were listed in the heat map after treatment with
PEG/Cu-BDC incubation. Among these differentially expressed genes
associated with ferroptosis [[101]44] and Hedgehog signaling pathway
[[102]45], 9 and 12 genes were up-regulated and down-regulated,
respectively (Fig. [103]6d). Furthermore, gene ontology (GO)
(Additional file [104]1: Figs. S2, S3) and Kyoto Encyclopedia of Genes
and Genomes (KEGG) (Fig. [105]6e, f) pathway enrichment analysis were
performed to understand how Cu-BDC nanosheets acted on tumor cells by
GSH “AND” H[2]O[2]-activated CDT. In the midst of these KEGG pathways,
ferroptosis is the main cause in inducing cell death of PEG/Cu-BDC, in
which the genetic expression in the glutathione metabolism pathway was
up-regulated, thus leading to GSH depletion and ROS elevation in 4T1
cells (Additional file [106]1: Fig. S4).
Fig. 6.
[107]Fig. 6
[108]Open in a new tab
Mechanistic study of 2D PEG/Cu-BDC nanocatalysts in inducing augmented
CDT effect. a The box plot of these 6 samples, in which all samples
were almost at the same level, indicating the quality homogenization of
the cell samples. b The chart showing the differentially expressed
genes in saline- and PEG/Cu-BDC-treated groups (n = 3 biologically
independent samples). c The gene expression heat-map of 4T1 cells in
saline- and PEG/Cu-BDC-treated groups (n = 3 biologically independent
samples). d The gene expression heat-map of the related genes in the
ferroptosis and Hedgehog signaling pathways. e, f The top 20 (e)
up-regulated, and (f) down-regulated KEGG pathways after treatment with
saline and PEG/Cu-BDC nanosheets (P-value < 0.05)
In vivo therapeutic efficacy of 2D PEG/Cu-BDC nanocatalysts
Biological behaviors comprehending blood half-life, biodistribution,
and biocompatibility were the preconditions of drug action for in vivo
application. As depicted in Fig. [109]7a, the blood half-life of 2D
PEG/Cu-BDC nanocatalysts was determined to be 1.16 h after intravenous
injection, which revealed the desirable pharmacokinetic performance of
PEG/Cu-BDC. In addition, the biodistribution investigation was also
performed to reveal the accumulation of PEG/Cu-BDC in tumor tissues and
major organs at 4, 8, and 24 h after intravenous administration. As
shown in Fig. [110]7b, PEG/Cu-BDC nanosheets mainly accumulated in the
liver tissues, and their accumulation in tumor tissues was determined
to be 3.98% at 24 h post-injection.
Fig. 7.
[111]Fig. 7
[112]Open in a new tab
In vivo biological behaviors and therapeutic efficacy of 2D PEG/Cu-BDC
nanocatalysts on 4T1 tumor-bearing mice. a In vivo pharmacokinetic
profile of PEG/Cu-BDC (n = 3 biologically independent samples). b The
biodistribution of PEG/Cu-BDC in tumor tissues and major organs (heart,
liver, lung, spleen, and kidney) at different durations post-injection
(4, 8, and 24 h) (n = 3 biologically independent samples). c Body
weight variations, d relative tumor volumes, e tumor growth rates, and
f tumor inhibition rates of the mice in different treatment groups (P
values: *P < 0.05, **P < 0.01, and ***P < 0.001). g H&E, TUNEL, and
Ki-67 staining of tumor sections (Scale bar: 100 μm), and h H&E
staining of the major organs in different treatment groups (Scale bar:
100 μm)
The systemic toxicity of 2D PEG/Cu-BDC nanocatalysts in vivo was
assessed by blood analysis and hematoxylin–eosin (H&E) staining of the
major visceral organs of the healthy female Kunming mice. As shown in
Additional file [113]1: Fig. S5, the routine blood parameters and serum
biochemical indexes exhibited no significant difference in all
treatment groups. In addition, the main organs in both control and
PEG/Cu-BDC-treated groups were collected for H&E staining. Negligible
inflammation lesions and damage signals were observed for the main
organs in all treatment groups, indicating high biocompatibility and
biosafety of the engineered PEG/Cu-BDC nanocatalysts for potential
therapeutic use (Additional file [114]1: Figs. S6, S7).
The antineoplastic activity of the engineered 2D PEG/Cu-BDC
nanocatalysts was assessed on female nude mice embedding with 4T1 and
MDA-MB-231 breast tumor cells subcutaneously. The mice in three groups
were injected with saline, doxorubicin (10 mg kg^−1), and PEG/Cu-BDC
(10 mg kg^−1), respectively. The body weights and tumor sizes of the
mice were recorded. During the therapeutic processes, whether in the
4T1 or MDA-MB-231 breast tumor-bearing mice, the quantitative values
and the variation tendency of the body weights in all groups were
almost identical, indicating that negligible toxicity in vivo was
caused by PEG/Cu-BDC administration (Figs. [115]7c, [116]8a). In 4T1
breast tumor-bearing mice, after 14-day treatment, the relative tumor
volume (V/V[0]) in the control and doxorubicin-treated groups reached
25.2 and 10.43 with the tumor growth rate of 100% and 41.37%,
respectively, while the value of V/V[0] in the PEG/Cu-BDC-treated group
was merely 5.59 with the growth rate of 22.17% (Fig. [117]7d). In terms
of MDA-MB-231 breast tumor-bearing mice, the value of V/V[0] and tumor
growth rate in the PEG/Cu-BDC treatment group approached half of that
in the saline-treated group (Fig. [118]8b, c). Additionally, the
average tumor weights, tumor sizes or growth inhibition rates in all
treatment groups intuitively validated that severe damage against tumor
tissues was caused by PEG/Cu-BDC administration (Figs. [119]7e, f,
[120]8d–f).
Fig. 8.
[121]Fig. 8
[122]Open in a new tab
The therapeutic efficacy of 2D PEG/Cu-BDC nanocatalysts on MDA-MB-231
tumor-bearing mice. a Body weight variations, b relative tumor volumes,
c tumor growth rates, d tumor inhibition rates, and f tumor weights of
the tumor-bearing mice in different treatment groups (P values:
*P < 0.05, **P < 0.01, and ***P < 0.001). e Photographs of the tumors
dissected from the tumor-bearing mice after various treatments. g H&E,
TUNEL, and Ki-67 staining of the tumor sections in different treatment
groups (Scale bars: 50 μm). h H&E staining of the major organs after
different treatments (Scale bars: 50 μm)
Furthermore, the histological evaluation of representative tumor
tissues of the mice in all treatment groups was conducted to
investigate the therapeutic mechanism of PEG/Cu-BDC through H&E and
terminal deoxynucleotidyl transferase uridine triphosphate nick end
labeling (TUNEL) staining. In comparison with doxorubicin-treated
group, more prominent histological damage signal was observed in the
PEG/Cu-BDC-treated group from H&E and TUNEL staining images (Figs.
[123]7g, [124]8g). Additionally, Ki-67 antibody staining assay was
performed to assess the tumor-cell proliferative property of the mice
in various treatment groups. By comparing the Ki-67 staining images in
other treatment groups, PEG/Cu-BDC presented conspicuous suppressive
effect on the proliferative activity of tumor tissues. To better
validate the therapeutic efficacy of PEG/Cu-BDC nanocatalyst, the
quantitative analysis of Ki-67 and terminal deoxynucleotidyl
transferase dUTP nick-end labeling (TUNEL) staining was performed. In
comparison with the control group, the percentages of Ki-67 positive
tumor cells were distinctly decreased to 23.3% and 12.5% for PEG/Cu-BDC
nanocatalyst-treated 4T1 and MDA-MB-231 tumor-bearing mice,
respectively, while the numbers of apoptotic cells were apparently
elevated (80.7% and 66.7% for 4T1 and MDA-MB-231 tumor-bearing mice,
respectively) after treatment with PEG/Cu-BDC nanocatalyst (Additional
file [125]1: Figs. S8, S9). Moreover, no obvious damage signal of the
main organs was detected from H&E staining images in different
treatment groups, demonstrating that PEG/Cu-BDC features negligible
adverse effect on the health of the mice (Figs. [126]7h, [127]8h).
Conclusions
In summary, we have engineered a distinct 2D Cu (II)-based MOF
nanosheets as an iron-free nanocatalyst for GSH-activated and
H[2]O[2]-reinfored chemodynamic tumor therapy via sequential reaction
with intracellular GSH and H[2]O[2] to induce ·OH generation. The in
vitro and in vivo assessments verified that the achieved Cu-BDC
nanosheets efficiently induced cellular apoptosis and promoted tumor
inhibition in situ without apparent systematic toxicity, compared with
the inferior tumor suppression and high systemic toxicity of the
equivalent concentration of commercial chemotherapeutic drug, Dox.
Therefore, PEG/Cu-BDC exhibits high potential in the application of
non-ferrous high-performance Fenton catalyst for tumor CDT treatment as
well as broadens the application domain in MOF-based nanomaterials in
disease diagnosis and treatment.
Supplementary Information
[128]12951_2022_1250_MOESM1_ESM.docx^ (7.4MB, docx)
Additional file 1: Figure S1. Cell viability of 4T1 and normal
endothelial cells after incubated with various concentrations of
PEG/Cu-BDC for 24 h, respectively. Figures S2. The GO enriched pathways
ranked top ten in terms of credibility in biological process. (P-value
< 0.05). Figures S3. The GO descending pathways ranked top ten in terms
of credibility in biological process. (P-value < 0.05). Figures S4.
Illustration of ferroptosis signaling pathway. The red blocks represent
up-regulated genes. Figure S5. Routine blood parameters and serum
biochemical indexes of female Kunming mice after intravenous injection
with 10 or 20 mg kg^−1 of PEG/Cu-BDC at the 0, 3rd, 7th, 15th, and 30th
day, respectively (red and yellow for 10 mg kg^−1, blue and green for
20 mg kg^−1). Figure S6. H&E staining images of major organs (heart,
liver, spleen, lung, and kidney) from female Kunming mice after
injection with 10 mg kg^−1 of PEG/Cu-BDC at the 0, 3rd, 7th, 15th, and
30th day (Scale bar: 100 μm). Figure S7. H&E staining of major organs
(heart, liver, spleen, lung, and kidney) from female Kunming mice after
intravenous injection with 20 mg kg^−1 of PEG/Cu-BDC at the 0, 3rd,
7th, 15th, and 30th day (Scale bar: 100 μm). Figure S8. Quantitative
analysis of Ki-67 and apoptosis-positive tumor cells of 4T1
tumor-bearing mice in different treatment groups. Figure S9.
Quantitative analysis of Ki-67 and apoptosis-positive tumor cells of
MDA-MB-231 tumor-bearing mice in different treatment groups.
Authors’ contributions
SZ, YC, and LW did the experiments. SZ, HX, YC, LW, JZ, and YC wrote
the manuscript. All authors read and approved the final manuscript.
Funding
This study was supported by National Natural Science Foundation of
China (Grant Nos. 81971598, 32171391, and 51902336), Program of
Shanghai Subject Chief Scientist (Grant Nos. 18XD1404300, 21XD1420900),
and the Shanghai Shuguang Program (No. 19SG06).
Availability of data and materials
All data generated or analyzed during this study are included in the
manuscript and supporting information.
Declarations
Ethics approval and consent to participate
All the animal experiments were approved by the Department of
Laboratory Animal Science of Fudan University (20171531A483).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Contributor Information
Huijing Xiang, Email: xianghuijing@shu.edu.cn.
Yu Chen, Email: chenyuedu@shu.edu.cn.
Jun Zhang, Email: zhangjun_zj@fudan.edu.cn.
References