Abstract
Cuproptosis is an emerging mode of programmed cell death for tumor
suppression but sometimes gets resisted by tumor cells resist under
specific mechanisms. Inhibiting copper transporter ATPase (ATP7A) was
found to disrupt copper ion homeostasis, thereby enhancing the effect
of cuproptosis and eventually inhibiting tumor invasion and metastasis.
In this study, we develop a multifunctional nanoplatfrom based on
Cu[9]S[8] (CAPSH), designed to enhance cuproptosis in tumor cells by
specifically targeting ATP7A interference, while combining
thermodynamic therapy with immune effects. The release of copper ions
from CAPSH and the copper homeostasis interference by siRNA
cooperatively increases the concentration of copper ions in tumor
cells, which induces effectively cuproptosis and activates immune
responses for suppressing development and metastasis of tumor. This
nanoplatform simultaneously regulates cuproptosis from both principles
of onset and development, facilitating the application of cuproptosis
in tumor therapy.
Subject terms: Biomedical materials, Drug delivery, Genetic vectors
__________________________________________________________________
Cuproptosis is an emerging mode of programmed cell death for tumor
suppression but limited by the copper ion regulatory mechanisms in most
tumor cells. Here, this group develops a Cu9S8-based nanoplatform for
breast cancer-targeting and cuproptosis-induction via ATP7A
interference, thereby eliciting thermodynamic cancer therapy.
Introduction
Programmed cell death is a regulated process of cell death, including
apoptosis, necrosis, pyroptosis, and ferroptosis^[44]1,[45]2. As an
emerging programmed cell death, cuproptosis is caused by
copper-dependent mitochondrial dysfunction^[46]3, whose key triggering
factor is the accumulation of copper ions in cells^[47]4. It directly
binds to the lipoylated components in the tricarboxylic acid (TCA)
cycle, leading to the aggregation of lipoylated mitochondrial proteins
and the disappearance of Fe-S cluster proteins, thereby inducing
protein toxicity stress and ultimately leading to cell
death^[48]5,[49]6. Such direct ion-induced cell death has good
controllability and precision, which is helpful for application in
tumor therapy. However, the special mechanisms of copper transport and
homeostasis in most tumor cells maintain a low concentration of copper
homeostasis in the cytoplasm, thus resisting them from undergoing
copper-induced death^[50]7. Specifically, copper transporter proteins
represented by ATPase copper transporting alpha (ATP7A), are able to
pump excess copper ions to the extracellular compartment, while ATP7A
also regulates lysyl oxidase (LOX) activity thereby participating in
the process of tumor invasion and metastasis^[51]8. Hence, the
inhibition of ATP7A expression serves as an effective strategy to
perturb the copper transport mechanism and homeostasis, ultimately
leading to an increase in intracellular copper ions concentration and
the inhibition of tumor development^[52]9. As a promising gene
regulation technology for tumor therapy, small interfering RNA (siRNA)
offers precise and selective targeting of a specific gene’s mRNA while
preserving the normal cellular function^[53]10,[54]11. However, the
precise delivery of siRNA in tumor cells faces significant challenges
due to its inability to spontaneously traverse cell membranes, low
bioavailability, and susceptibility to degradation by nucleases,
thereby hindering its clinical applicability^[55]12. Nanocarriers offer
several advantages in addressing the siRNA delivery challenge,
encompassing highly controlled loading, protection of siRNA from
degradation, and precise targeted delivery, among others^[56]13. A
range of nano-delivery systems, including nanoparticles, liposomes, and
viral vectors, has been devised to tackle the siRNA delivery
issue^[57]14,[58]15. Consequently, delivering siRNA via nanocarriers
for the regulation of protein expression, such as ATP7A in tumor cells,
can precisely disrupt their copper ions' metabolic homeostasis, thereby
achieving the induction of cuproptosis in tumor cells^[59]16.
As a versatile, easily prepared, and readily modifiable nano-delivery
platform, Cu[9]S[8] holds significant promise and advantages in the
field of tumor gene therapy^[60]17,[61]18, including: (1) Cu[9]S[8] is
an excellent reservoir of copper ions in nano form, which is capable of
releasing a large number of copper ions and triggering Cu death in
tumor cells^[62]19–[63]21; (2) Cu[9]S[8] has a mesoporous structure,
and its high specific surface area is conducive to cargo, especially
siRNA loading, which can achieve the silencing of ATP7A gene, thus
preventing the exocytosis of copper ions and further increasing the
intracellular concentration of copper ions^[64]22; (3) Cu[9]S[8] has a
large number of Cu defects, which generates a localized surface plasmon
resonance effect^[65]23–[66]25, and is able to absorb a large number of
near-infrared region II (NIR-II) lasers (1064 nm) and undergo a strong
photothermal conversion to kill the tumor cells. In comparison to
visible light (390 nm-750 nm) and the near-infrared region I (NIR-I,
750–1000 nm), NIR-II excitation light offers superior tissue
penetration depth, reduced light scattering, and lower
absorption^[67]26. This makes it more suitable for the treatment of
deep-seated tumors^[68]27. In summary, Cu[9]S[8] represents a versatile
nano-delivery system with the ability to integrate multiple therapeutic
strategies, including copper-induced cell death, gene therapy, and
photothermal therapy. It presents a more promising avenue for the
development of effective tumor treatments^[69]28–[70]30.
In this work, we construct a multifunctional Cu[9]S[8]-based
nanoplatform (CAPSH) to precisely target tumor tissues, effectively
enhance cuproptosis by ATP7A interfering, and integrate thermodynamic
therapy with immune effects (Fig. [71]1). CAPSH is supported by hollow
mesoporous Cu[9]S[8] nanoparticles, loaded with alkyl radical precursor
2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH), and
coated with poly(arylene propylene amine) hydrochloride (PAH) to
electrostatically adsorb siRNA (siATP7A). The outer layer is coated
with hyaluronic acid (HA) to prevent siRNA degradation and provide
tumor targeting. The hyaluronidase in the tumor microenvironment
degrades HA to promote the endocytosis of CAPSH, and further releases
AIPH, siATP7A, and a large amount of copper ions. Through 1064 nm laser
irradiation, Cu[9]S[8] undergoes photothermal conversion, induces tumor
cell death upon heating, and decomposes AIPH to produce
oxygen-independent alkyl radicals. siATP7A enhances cuproptosis and
inhibits tumor metastasis by blocking copper ion efflux and suppressing
LOX activity. Both in vivo and in vitro experiments verify that the
copper death strategy mediated by CAPSH elicits a robust antitumor
immune response, effectively curbing tumor growth and demonstrates its
promising potential in cancer treatment^[72]31–[73]34.
Fig. 1. Synthesis and antitumor mechanism of CAPSH.
[74]Fig. 1
[75]Open in a new tab
a Schematic diagram of the synthesis of CAPSH. b Schematic diagram of
copper sulfide-based nanocarriers for the regulation of copper
homeostasis in tumors enabling synergistic treatment of breast cancer.
Figure 1b was created with BioRender.com released under a Creative
Commons Attribution 4.0 International license
([76]https://creativecommons.org/licenses/by/4.0/).
Results
Synthesis and characterization of CAPSH
The synthetic scheme of CAPSH was shown in Fig. [77]1a. The preparation
of Cu[9]S[8] nanoparticles (NPs) was composed of synthesis and in situ
sulfidation of Cu[2]O. The synthesis method was optimized by
characterizing the UV-vis absorption spectrum, hydrodynamic size, and
zeta potential of Cu[9]S[8] (Supplementary Figs. [78]1–[79]4). The
results obtained from transmission electron microscopy (TEM,
Fig. [80]2a) and scanning electron microscopy (SEM, Supplementary
Fig. [81]5) showed that the synthesized Cu[9]S[8] NPs had a spherical
and hollow mesoporous structure. The average diameter of the Cu[9]S[8]
NPs was ~100 nm, with a shell thickness of 20 nm. This unique structure
was believed to be a result of the “Kirkendall effect” induced by
sulfurization. The TEM image of CAPSH exhibited negligible alterations
when compared to that of Cu[9]S[8] NPs. The results of the TEM
elemental mapping of CAPSH showed that the Cu and S elements had high
signal intensities in the shell region and low signal intensities in
the central cavity, which was consistent with the hollow structure of
Cu[9]S[8] NPs. Within CAPSH, alongside the presence of Cu and S
elements, the emergence of elements such as N and P provided
unequivocal confirmation of the effective encapsulation of both AIPH
and siRNA (Fig. [82]2a).
Fig. 2. Characterization of CAPSH.
[83]Fig. 2
[84]Open in a new tab
a TEM images of Cu[9]S[8] and CAPSH, along with an element mapping
image of CAPSH. Images are representative of three independent
experimental replicates. Scale bar: 100 nm. b XRD pattern of Cu[9]S[8].
c, d XPS energy spectra of elemental Cu and elemental S of Cu[9]S[8]. e
UV-vis absorption spectra of Cu[9]S[8] and Cu[2]O. f Nitrogen
adsorption and desorption curves and (illustration) pore size
distribution of Cu[9]S[8]. g Hydration diameter of Cu[9]S[8], CAP,
CAPS, and CAPSH. Data were presented as the means ± SD (n = 3
independent experiments). h Zeta potential of Cu[9]S[8], CAP, CAPS, and
CAPSH. Data were presented as the means ± SD (n = 3 independent
experiments). i UV-vis absorption spectra of Cu[9]S[8], CA, CAP and
CAPH. j Delayed determination of siRNA by agarose gel electrophoresis
at different w/w ratios of Cu[9]S[8] to siRNA. Images are
representative of three independent experimental replicates. k Zeta
potentials of Cu[9]S[8] and siRNA with different w/w. Data were
presented as the means ± SD (n = 3 independent experiments). l XPS
energy spectra of elemental P of Cu[9]S[8]. m XPS energy spectra of
elemental P of CAPS. Source data are provided as a Source Data file.
The crystal structure of Cu[9]S[8] NPs was investigated using an X-ray
diffractometer (XRD, Fig. [85]2b). The results showed that Cu[9]S[8]
NPs had diffraction peaks at 29.3°, 31.9°, and 48.0°, which were
consistent with the (0022), (1013), and (111) crystal planes of
Yarrowite Cu[9]S[8] (PDF#36-0379). This suggested that a
non-stoichiometric copper sulfide with copper vacancies was produced.
To demonstrate this, Cu[9]S[8] NPs were tested for X-ray photoelectron
spectroscopy (XPS, Fig. [86]2c, d). The coexistence of Cu^+ and Cu^2+
suggests the formation of a non-stoichiometric copper sulfide. The
UV-vis-NIR spectrophotometer was employed to examine the absorption
spectra of Cu[2]O and Cu[9]S[8] NPs (Fig. [87]2e). The absorption band
of Cu[9]S[8] NPs exhibited a “V” shape and notable absorption in the
near-infrared area, in contrast to the continuous absorption band of
Cu[2]O. This phenomenon arises from the localized surface plasmon
resonance effect (LSPR) correlated with copper vacancies present within
the Cu[9]S[8] NPs. Notably, the Cu[9]S[8] dispersion exhibits
comparatively enhanced absorbance within the NIR-II window compared to
the NIR-I window. This heightened absorbance profile facilitates
improved penetration into biological tissues, enabling the utilization
of light within the NIR-II window for more effective excitation.
Through nitrogen adsorption-desorption experiments, the pore size
distribution of Cu[9]S[8] was analyzed (Fig. [88]2f). The results
revealed a distinct hysteresis loop in the adsorption isotherm of
copper sulfide, indicating the presence of mesopores with
interconnected channels in the material. The specific surface area of
Cu[9]S[8] nanoparticles was calculated to be 25.3 m^2/g, and the pore
diameter was 33.0 nm. Additionally, we performed small-angle XRD
experiments (Supplementary Fig. [89]6; The detailed testing conditions
are shown in Supplementary Table [90]1). The results showed diffraction
peaks at low scattering angles (typically less than 5 degrees), further
confirming the presence of mesoporous structures in the sample. To
demonstrate the importance of mesoporous structures in improving drug
loading capacity and therapeutic efficacy, we synthesized
non-mesoporous CuS (Supplementary Fig. [91]7) and compared their drug
loading capacity with that of hollow mesoporous Cu[9]S[8]. The results
indicated that the drug loading capacity of hollow mesoporous Cu[9]S[8]
was 3.67 times higher than that of non-mesoporous CuS (Supplementary
Fig. [92]8). A higher drug loading capacity can enhance therapeutic
efficacy and reduce the frequency of administration.
The hydrodynamic size of Cu[9]S[8] NPs and CAPSH was about
112.8 ± 2.0 nm and 218.2 ± 2.1 nm respectively, which further proved
loading the drug (Fig. [93]2g). The surface charge of Cu[9]S[8] NPs was
changed from −22.4 ± 0.43 mV to 22.1 ± 0.71 mV after loading AIPH and
PAH, demonstrating that the successful encapsulation of PAH facilitated
the electrostatic adsorption of negatively charged siRNA. The surface
charge further changed to −10.6 ± 0.98 mV and −18.3 ± 0.76 mV
separately after additional loading of siRNA and encapsulation of
HA-formed CAPSH (Fig. [94]2h). Comparison of the absorption spectra of
Cu[9]S[8] NPs, CA, CAP and CAPH (Fig. [95]2i), CA, CAP, and CAPH
exhibited a prominent absorption peak at 220 nm after AIPH loading. The
capacity of Cu[9]S[8] NPs to load siRNA was confirmed through
electrophoresis experiments using agarose gel (Fig. [96]2j). The
outcomes indicated that Cu[9]S[8] NPs was effective at loading siRNA at
a mass ratio of 40:1 and beyond (Fig. [97]2k). As the mass ratio of
Cu[9]S[8] NPs to siRNA rose, the surface charge of CAPS underwent a
gradual trend which first decreased and then increased. By comparing
the XPS spectra of Cu[9]S[8] NPs with CAPSH, it was further confirmed
that siRNA was successfully loaded into Cu[9]S[8] NPs. Since
nucleotides and phosphates in siRNA molecules make up a significant
portion of the P elements in siRNA, CAPSH possessed unique peaks in P
spectra, while no P signals were found in Cu[9]S[8] NPs (Fig. [98]2l,
m). The XPS spectra of Cu and S elements in CAPSH exhibited no
significant changes, indicating that the modifications with PAH and HA
did not affect the structure of Cu[9]S[8] NPs and the loading of siRNA
(Supplementary Fig. [99]9).
Additionally, we conducted in vitro stability simulation experiments on
CAPSH. CAPSH was co-incubated with H[2]O, PBS, NaCl, DMEM, 1640, 10%
fetal bovine serum (FBS), 50% FBS, and 100% FBS. No significant
precipitation was observed in any of the solutions (Supplementary
Fig. [100]10a). The hydrated particle size (Supplementary
Fig. [101]10b), zeta potential (Supplementary Fig. [102]10c), and
polydispersity index (PDI) (Supplementary Fig. [103]10d) in PBS, DMEM,
and DMEM + 50% FBS showed no significant changes over 12 days. After
soaking CAPSH in simulated body fluid, the pH values of the solution
measured at different time points did not show significant changes,
indicating that CAPSH has favorable chemical stability and
biocompatibility. (Supplementary Fig. [104]10e).
In vitro performance evaluation of CAPSH
The photothermal properties of Cu[9]S[8] were studied using a
near-infrared thermal imaging camera. A range of Cu[9]S[8] dispersion
at concentrations of (0–100 μg/mL) were exposed to a 1064 nm laser
(0.75 W/cm^2), while concurrently monitoring the temperature via an
infrared camera (Fig. [105]3a, b). Under 1064 nm laser irradiation, the
temperature of water increased by 12.5 °C in 300 s. The temperature of
Cu[9]S[8] dispersion (100 μg/mL) increased rapidly by 42.7 °C in 300 s
and showed a concentration-dependent temperature increase of 30.9,
37.9, and 40.9 °C, respectively (the concentration of Cu[9]S[8]
dispersions were 12.5, 25, and 50 μg/mL). In addition, at the same
concentration, the warming is more pronounced at higher power densities
(Fig. [106]3c, d). The temperature profiles resulting from repeated
laser exposure on Cu[9]S[8] dispersions vividly illustrated the
commendable stability of its photothermal characteristics
(Fig. [107]3e). The time constant (τ) of Cu[9]S[8] was 128.5 s
(Fig. [108]3f). The photothermal conversion efficiency of Cu[9]S[8] was
calculated as 42.3% using the given equation. Moreover, the synthesized
Cu[9]S[8] displayed excellent photothermal performance, making it
suitable for research on tumor photothermal therapy.
Fig. 3. Photothermal, radical generation, and drug/copper ions release of
CAPSH.
[109]Fig. 3
[110]Open in a new tab
a, b Temperature versus time curves and thermal imaging results of
Cu[9]S[8] (0–100 μg/mL) under 1064 nm laser irradiation (0.75 W/cm^2).
Data were presented as the means ± SD (n = 3 independent experiments).
c, d Temperature versus time curves and thermal imaging results of
Cu[9]S[8] at 100 μg/mL under 1064 nm laser irradiation
(0.25–0.75 W/cm^2). Data were presented as the means ± SD (n = 3
independent experiments). e Temperature rise and fall curves of
Cu[9]S[8] irradiated repeatedly with a 1064 nm laser irradiation
(0.75 W/cm^2). f Photothermal conversion efficiency curve of Cu[9]S[8].
g Free radical production was induced by irradiating CAPH at different
times using a 1064 nm laser. h ESR spectra in AIPH, Cu[9]S[8], and CAPH
solutions irradiated with a 1064 nm laser using POBN as a spin-trapping
agent. i AIPH release from CAPH in PBS at different pH (n=independent
experiments). j Copper ions release of CAPSH in PBS at different pH
(n = 3 independent experiments). Source data are provided as a Source
Data file.
AIPH rapidly decomposes when exposed to heat and produces alkyl
radicals. We utilized the photothermal effect of Cu[9]S[8] to control
the decomposition of AIPH. Next, the generated free radicals were
detected by UV-vis and ESR spectroscopy.
2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt)
(ABTS) could react with free radicals to form ABTS^+•, which has
characteristic absorption peaks in the 400–1000 nm range of the UV-vis
absorption spectrum. The distinctive absorption peaks of ABTS^+•
gradually intensified as the laser exposure time extended, indicating
that the concentration of ABTS^+• was gradually increased by the
generation of free radicals in the solution (Fig. [111]3g). The same
conclusion was reached using methylene blue to detect reactive oxygen
species (Supplementary Fig. [112]11). Additionally, we employed POBN to
capture the free radicals generated by AIPH (Fig. [113]3h). The CAPH
group exhibited distinct characteristic signals under laser
irradiation. This further confirms that the photothermal effect of
Cu[9]S[8] could trigger the decomposition of AIPH and produce alkyl
radicals.
Next, we investigated the release of AIPH and copper ions from CAPSH
under acidic conditions. The drug release from CAPSH after 48 h was
found to be 67.5 ± 1.6% at pH 5.4, while it was only 36.1 ± 0.9% at pH
7.4 (Fig. [114]3i). After replacing AIPH with DOX, the drug release
capability in an acidic environment was further verified by detecting
the fluorescence of DOX. The results of the UV-vis absorption spectra
(Supplementary Fig. [115]12a) and fluorescence spectra (Supplementary
Fig. [116]12b) showed that DOX-loaded Cu[9]S[8] exhibited the same
characteristic absorption peaks and fluorescence emission peaks as DOX.
This phenomenon clearly confirms the successful loading of DOX onto
Cu[9]S[8]. The release experiment results indicated that DOX was
significantly released under acidic conditions. This finding further
demonstrates the ability of CAPSH to release drugs in an acidic
environment (Supplementary Fig. [117]13). Inductively Coupled Plasma
Mass Spectrometry (ICP-MS) analysis of CAPSH’s ability to release
copper ions under acidic conditions can be found in Fig. [118]3j,
revealing that the release of copper ions was 2964.4 ± 55.2 μg/L at pH
5.4, while the release was only 941.8 ± 26.9 μg/L at pH 7.4. The
characteristic of CAPSH to release AIPH and copper ions in response to
acidic conditions contributed to minimizing the potential cytotoxicity
to healthy tissues.
Cytotoxicity and combined therapy in vitro
The impact of CPH (Cu[9]S[8]-clad PAH and HA) on the survival of 4T1
cells was analyzed through MTT (Fig. [119]4a). After incubating 4T1
cells with CPH at concentrations ranging from 0 to 60 μg/mL for 24 h,
the viability of all cells remained above 95%. However, when the
concentration was increased to 100 μg/mL, the survival rate dropped
significantly to only 48.4%, which demonstrated the high
biocompatibility of CPH at low concentrations. Two concentrations, 40
and 80 μg/mL, of CAPSH were chosen to investigate the combined impact
of their treatment. At a concentration of 40 μg/mL (Fig. [120]4b), the
survival rate of 4T1 cells remained above 80% without NIR laser
exposure. However, under light exposure, the cell survival rate dropped
to 22.1%. On the other hand, at an 80 μg/mL concentration of CAPSH
(Fig. [121]4c), the survival rate of 4T1 cells was still above 48%
without NIR laser irradiation. Nevertheless, under light exposure, the
cell survival rate decreased to 15.3%. Additionally, we conducted
toxicity tests on 3T3 and RAW cells using CAPSH and found that the
cytotoxicity of CAPSH was lower at low concentrations (0–20 μg/mL), but
significantly increased at high concentrations (100 μg/mL)
(Supplementary Fig. [122]14).
Fig. 4. Therapeutic effects of the CAPSH at the cellular level.
[123]Fig. 4
[124]Open in a new tab
a MTT for CPH toxicity on 4T1 cells. Data were presented as the
means ± SD (n = 4 independent experiments). b, c Cell survival of 4T1
cells after different treatments. The concentration of AIPH in (b) was
10 μg/mL, and the concentration of Cu[9]S[8] in CPH, CAPH, and CAPSH
was 40 μg/mL. Data were presented as the means ± SD (n = 5 independent
experiments). The concentration of AIPH in (c) was 20 μg/mL, and the
concentration of Cu[9]S[8] in CPH, CAPH, and CAPSH was 80 μg/mL. Data
were presented as the means ± SD (n = 5 independent experiments). d
Flow cytometry was used to detect the intake of CAPSH by 3T3 and 4T1
cells at different time points. The gate strategy is shown in
Supplementary Fig. [125]37. e Fluorescence images of intracellular free
radicals. Images are representative of three independent experimental
replicates. Scale bar: 50 μm. f Images of 4T1 cells co-stained with
calcineurin-AM (green)/PI (red) after different treatments. Images are
representative of three independent experimental replicates. Scale bar:
100 μm. g Flow cytometry for cell survival at 4T1 after different
treatments. Images are representative of three independent experimental
replicates. The gate strategy is shown in Supplementary Fig. [126]37.
Significance between the two groups in (b, c) was assessed by unpaired
two-tailed Student’s t-test. Source data are provided as a Source Data
file.
We studied the uptake of CAPSH by 4T1 and 3T3 cells. CAPSH was added to
the medium of 3T3 and 4T1 cells, and cells were co-cultured and
collected at different time points (2, 4, and 8 h). ICP-MS analysis of
copper content in the cells can be found in Supplementary Fig. [127]15,
revealed that 4T1 cells uptake more CAPSH than 3T3 cells at the same
time points. Flow cytometry further confirmed this finding
(Fig. [128]4d). This indicated that due to the targeting effect of HA,
CAPSH uptake was more pronounced in cancer cells than in normal cells.
The release of the drug at the cellular level was demonstrated by
detecting the fluorescence of DOX, and a large amount of DOX entered
the cells within 6 h, showing that the hollow mesoporous structure of
Cu[9]S[8] was successful in achieving effective drug loading and
release (Supplementary Fig. [129]16).
Furthermore, we investigated the generation of free radicals using
DCFH-DA. As depicted in Fig. [130]4e, neither the control group nor the
AIPH group demonstrated the capability to initiate free radical
production. In contrast, the most intense green fluorescent signal was
observed in 4T1 cells subjected to CAPH treatment followed by laser
irradiation. This observation suggested that the photothermal impact of
Cu[9]S[8] facilitated the breakdown of AIPH, leading to the formation
of alkyl radicals. The viability of 4T1 cells following various
treatments was assessed through fluorescence imaging using
calcineurin-AM/PI staining (Fig. [131]4f). In both the control and
AIPH-treated groups, vibrant green fluorescence (indicating live cells)
persisted even after laser irradiation, underscoring robust cell
activity. In stark contrast, the CAPH and CAPSH treatment groups
exhibited a considerable population of cells emitting red fluorescence
(indicative of cell death) post laser irradiation. Notably, the CAPSH
group displayed the highest observed cell mortality rate among the
treatment groups. This observation underscored that a synergistic
combination of multiple treatments could significantly enhance the
efficiency of tumor cell eradication. The flow cytometry data showed
that the cell necrosis rate in the CAPSH+Laser group was 40.8% higher
than that in the control group (Fig. [132]4g). These results further
confirmed that the CAPSH+Laser group had a great antitumor effect.
Mechanism of cell death mediated by CAPSH
Through bioinformatics analysis, we have discovered a significant
upregulation in the expression levels of ATP7A and the copper-related
apoptosis gene dihydrolipoamide S-acetyltransferase (DLAT) in breast
invasive carcinoma (BRCA) compared to normal tissues (Fig. [133]5a, b).
These findings suggest that breast cancer may exhibit sensitivity to
copper apoptosis therapy, with high levels of ATP7A expression
potentially exerting a negative regulatory effect on copper apoptosis.
In addition, we observed higher survival in patients with lower levels
of ATP7A and DLAT over 8 years and lower survival in patients with
higher levels of ATP7A (Fig. [134]5c) and DLAT (Fig. [135]5d). PCR
analysis further confirmed the high expression of ATP7A (Fig. [136]5e)
and DLAT (Fig. [137]5f) in tumor tissues and their low expression in
normal tissues. These results strongly indicated that a potentially
promising approach to breast cancer treatment might involve the
combined application of copper apoptosis therapy and ATP7A
intervention.
Fig. 5. Mechanism of cell death mediated by CAPSH.
[138]Fig. 5
[139]Open in a new tab
a Bioinformatics analysis of ATP7A in BRCA. b Bioinformatics analysis
of DLAT in BRCA. c, d Patient survival analysis of ATP7A and DLAT in
BRCA. e, f mRNA levels of ATP7A and DLAT in 4T1 and 3T3 cells. Data
were presented as the means ± SD (n = 3 independent experiments). g A
functional model of ATP7A in tumor growth and metastasis. h Protein
content of ATP7A in 4T1 cells after different treatments. Data were
presented as the means ± SD (n = 3 independent experiments). i LOX
activity of 4T1 cells after different treatments. Data were presented
as the means ± SD (n = 3 independent experiments). j DLAT, FDX1, and
LIAS protein levels after treatment of 4T1 cells with different
samples. Images are representative of three independent experimental
replicates. The samples of the same group were derived from the same
experiment, and the gels/blots were processed in parallel. k, l
Immunofluorescence analysis of the expression of HMGB1 and CRT in 4T1
cells after different treatments, DAPI was used to stain nuclei (blue).
Images are representative of three independent experimental replicates.
Scale bar: 50 μm. m Quantitative analysis of HSP70 expression in 4T1
cells after different treatments using flow cytometry. Images are
representative of three independent experimental replicates. The gate
strategy is shown in Supplementary Fig. [140]38. Significance between
two groups in (e, f) was assessed by unpaired two-tailed Student’s
t-test, and between each of the multiple groups in (h, i) was
calculated using one-way ANOVA. Source data are provided as a Source
Data file. Figure 5g was created with BioRender.com released under a
Creative Commons Attribution 4.0 International license
([141]https://creativecommons.org/licenses/by/4.0/).
Copper levels within the body were intricately regulated through a
complex network. When intracellular copper concentrations became
elevated, the copper transport protein ATP7A, situated in the
trans-Golgi network, relocated to the plasma membrane and facilitates
the efflux of copper (Fig. [142]5g)^[143]8. This mechanism served to
avert the excessive accumulation of copper ions, a pivotal step in
maintaining intracellular copper balance. Disturbing ATP7A’s function
enhanced the accumulation of intracellular copper ions, disrupting
copper homeostasis. Simultaneously, ATP7A contributed copper ions to
diverse copper-dependent enzymes. When its function was impaired, it
could result in the dysfunction of multiple enzymes, subsequently
affecting the organism’s normal physiological processes. Research
indicated that silencing ATP7A hampers LOX activity, thereby impeding
the phosphorylation of focal adhesion kinase (FAK)^[144]35. LOX, an
amine oxidase responsible for the oxidative deamination of lysine
residues in collagen, interacted with epithelial-mesenchymal
translational transcription factors, playing a role in tumor invasion
and metastasis^[145]36.
To explore CAPSH’s potential for delivering siRNA into cells and
silencing target genes, we initially examined the mRNA levels of ATP7A
in 4T1 cells subjected to various treatments via fluorescence
quantitative PCR experiments. Notably, the mRNA levels of ATP7A in the
CAPSH-treated group exhibited a substantial reduction (Supplementary
Fig. [146]17). Furthermore, we conducted a precise quantification of
ATP7A using ELISA (Fig. [147]5h). The protein levels of ATP7A were
significantly reduced in the CAPSH-treated group, indicating effective
gene silencing. Additionally, we measured the activity of LOX in cells
after different treatments. The results showed that CAPSH effectively
inhibited LOX activity in 4T1 cells (Fig. [148]5i). Next, we
investigated the effect of ATP7A deletion on cell migration by in vitro
scratch assay (Supplementary Fig. [149]18). The CAPSH-treated group
showed significantly weaker cell migration compared with the CPH- and
CAPH-treated groups. The cell migration rate in the blank group was
91.7%, while in CAPSH, it was only 29.3% (Supplementary Fig. [150]19).
It indicated that inhibition of ATP7A expression could suppress the
migration and metastatic ability of tumors. Subsequently, extracting
proteins from 4T1 cells enabled us to study the expression of copper
toxicity-related proteins at the protein level through Western blotting
experiments (Fig. [151]5j). We investigated the CAPSH-mediated copper
death phenomenon by comparing the expression of relevant proteins in
4T1 cells after different sample treatments. The protein expressions of
DLAT, ferredoxin 1 (FDX1), and lipoic acid synthetase (LIAS) were
significantly decreased after CAPSH and CAPH treatments. And the
expression of DLAT and LIAS in the CAPSH-treated group was lower than
that in the more CAPH-treated group. This suggested that the knockdown
of ATP7A by siRNA contributed to the enhancement of the copper death
strategy.
We further investigated the accumulation of copper ions in 4T1 cells
(Supplementary Fig. [152]20). The results showed that, compared to the
control group, the copper ions content in the Lipo-siRNA group
increased (cultured in a medium with low copper ions content).
Additionally, the copper ions content in the CAPSH group was higher
than in the CAPH group. The study demonstrated that when the ATP7A gene
in tumor cells was knocked out, copper ions significantly accumulated
within the cells. This accumulation significantly enhanced the
copper-induced cell death effect, thereby achieving effective killing
of tumor cells. More importantly, this copper-induced cell death
(cuproptosis) was not limited to the direct killing of tumor cells but
could also trigger immunogenic cell death (ICD). When ICD occurred,
characteristic danger-associated molecular patterns (DAMPs) were
exposed or released, including changes in calreticulin (CRT), high
mobility group box 1 (HMGB1), heat shock protein 70 (HSP70), and
adenosine triphosphate (ATP). Immunofluorescence results indicated
(Fig. [153]5k, l) that 4T1 cells in the CPSH group significantly
induced the release of HMGB1 from the nucleus and the surface exposure
of CRT, exceeding the levels observed in the CPH group. Flow cytometry
was used to quantitatively analyze the expression of HMGB1, CRT
(Supplementary Fig. [154]21), and HSP70 (Fig. [155]5m). The results
showed that the positive rate of HMGB1 in the CPSH group was 16.1%
lower, the positive rate of CRT was 9.4% higher, and the positive rate
of HSP70 was 20.2% higher than in the CPH group. The ATP content in the
supernatant of the CPSH group was 1.48 times higher than that in the
CPH group (Supplementary Fig. [156]22). Overall, ATP7A knockdown
resulted in increased accumulation of intracellular copper ions and
promoted ICD. CAPSH combination therapy had a significant effect on the
promotion of ICD.
In vivo antitumor efficacy
In order to assess the safety profile of CAPSH, we conducted in vitro
tests to examine the stability of CAPSH in the blood (Supplementary
Fig. [157]23). The outcomes of the hemolysis assay revealed that even
after 3 h of co-incubation with 100 μg/mL of CAPSH, the rate of
erythrocyte hemolysis remained below 3%. This finding supported the
favorable applicability of CAPSH for potential in vivo use. Based on
the above experimental results, we established a subcutaneous injection
of the 4T1 tumor-bearing Balb/c mouse model to evaluate the antitumor
efficacy of CAPSH. The 4T1 tumor-bearing mice were randomly divided
into six groups: PBS, AIPH + L, CAPH, Cu[9]S[8] + Laser (Cu[9]S[8]+L),
CAPH + L, and CAPSH + L. After 24 h of injecting different samples, the
mice in the laser group were irradiated at the tumor sites using a
near-infrared laser (1064 nm, 0.75 W/cm^2, 5 min) (Fig. [158]6a). Among
them, the tumor volume of mice in the PBS and AIPH + L groups increased
rapidly. The slow increase in the CAPH group was attributed to
apoptosis induced by copper ions. Mice in the Cu[9]S[8] + L, CAPH + L,
and CAPSH + L groups exhibited significant tumor suppression under NIR
laser irradiation (Fig. [159]6b–d). The treatment effect was most
significant in the CAPSH + L group. The tumors of the mice were weighed
after the treatment, which lasted for 25 days, and the weight of the
tumors further supported this result (Fig. [160]6e). There were no
significant changes in the body weight of all mice during the
treatment, indicating that the samples had good biosafety
(Fig. [161]6f). The temperature changes at the tumor sites of mice were
monitored through thermal imaging, and there was no difference in the
temperature changes at the tumor sites of the Cu[9]S[8] + L and
CAPSH + L groups 12 h after the injection of the samples into the tail
vein. In contrast, 24 h after sample injection, the temperature at the
tumor site in the CAPSH + L group was significantly higher than that in
the Cu[9]S[8] + L group, which was attributed to the targeting effect
of HA resulting in a higher accumulation of CAPSH at the tumor site
than that of Cu[9]S[8] (Fig. [162]6g and Supplementary Fig. [163]24).
Fig. 6. Subcutaneous tumor model of breast cancer.
[164]Fig. 6
[165]Open in a new tab
a Transplanted tumor model in mice. b Photographs of representative
tumors after different treatments, n = 5 mice per group. c Tumor volume
after different treatments. Data were presented as the means ± SD
(n = 5 mice per group). d Tumor volume change curves for each mouse
after different treatments. e Tumor weights obtained after different
treatments. Data were presented as the means ± SD (n = 5 mice per
group). f Changes in body weight of mice after different treatments.
Data were presented as the means ± SD (n = 5 mice per group). g Thermal
imaging of Cu[9]S[8] + L and CAPH + L groups at different times. h H&E
sections and immunofluorescence staining (Ki-67 and Tunel) of tumor
tissues from mice of different treatment groups. Images are
representative of three biologically independent mice. Scale bar:
100 μm. i mRNA levels of TNF-α in different treated tumors. Data were
presented as the means ± S.D. (n = 3 mice per group). j mRNA levels of
IFN-γ in different treated tumors. Data were presented as the
means ± SD (n = 3 mice per group). k Flow cytometry analysis of DC
cells in the tumor. Images are representative of three independent
experimental replicates. The gate strategy is shown in Supplementary
Fig. [166]39. l Flow cytometry analysis of CD8^+ T cells in the tumor.
Images are representative of three independent experimental replicates.
The gate strategy is shown in Supplementary Fig. [167]39. m Survival
rate of breast cancer model mice after different treatments, n = 10
mice per group. n Changes of Cu in blood over time. Data were presented
as mean ± 95% confidence interval (n = 3 mice per group). The
significance between each of the multiple groups in (c, e, i, j) was
calculated using one-way ANOVA. Source data are provided as a Source
Data file.
In addition, we observed the tumor tissues through Hematoxylin and
Eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated
dUTP nick end labeling (Tunel) assay, and immunofluorescence (IF)
staining of Ki-67, respectively. As shown in Fig. [168]6h, H&E staining
revealed the most severe apoptosis in the CAPSH + L group. The red
fluorescence of Ki-67 was significantly reduced, while the green
fluorescence of Tunel staining was significantly elevated, which
further confirmed the highly efficient tumor-suppressing property of
the combined treatment. We further evaluated the therapeutic effect of
the combination therapy at the cytokine level. First, we examined the
concentrations of TNF-α and IFN-γ in tumor tissues after different
treatments. At the end of treatment, TNF-α (Fig. [169]6i) and IFN-γ
(Fig. [170]6j) were significantly increased in the CAPSH + L group
compared to the PBS group.
Next, we measured the expression levels of HMGB1 and CRT in tumor
tissues as well as the changes in T cells and DC cells. The
experimental results showed that in the CAPSH+Laser group, tumor cells
significantly released a large amount of HMGB1 and CRT during the
process of cell death (Supplementary Fig. [171]25 a, b). These
immunogenic molecules not only acted as “danger signals” to activate
the body’s immune system but also served as potent adjuvants, guiding
and promoting the recruitment and activation of T cells and DC cells in
the spleen, lymph nodes, and tumor (Fig. [172]6k, l and Supplementary
Fig. [173]25 c–e). In the tumor tissues, the percentage of DC cells and
CD8^+ T cells significantly increased in the CAPSH + L treatment group,
reaching 15.5 and 20.8%, respectively. It showed that the combination
treatment had an immune-activating effect and contributed to tumor
treatment. In addition, the CAPSH group exhibited the highest long-term
survival rate (Fig. [174]6m).
Cu was used as a tracer to investigate the distribution and metabolism
of CAPSH in the blood, major organs, and tumor sites in mice. The
results indicated that CAPSH had a relatively long circulation time in
the blood (t[1/2] = 4.88 h), which was advantageous for the targeted
transport of CAPSH to tumor sites (Fig. [175]6n). The distribution and
metabolism of CAPSH in vivo showed time-dependent characteristics with
different effects on different organs (Supplementary Fig. [176]26). In
addition, the prolonged retention time of CAPSH in tumor tissues
provided an effective therapeutic window.
In situ models closely resemble natural lesions histologically and
cytologically as they develop in situ, mimicking the growth of tumor
cells in their original location. To further investigate, we
established an in situ breast cancer tumor model and treated tumor-mice
using the same treatment as the transplanted tumor model
(Fig. [177]7a). The therapeutic effect was assessed through the
monitoring of tumor volume in the mice, as well as the weight of the
tumors and the body weight of the mice. Among them, mice in the
CAPH + L group and CAPSH + L group showed significant tumor inhibition
(Fig. [178]7b–d). Tumor growth before and after treatment was monitored
by bioluminescence of tumor cells expressing luciferase. As expected,
the CAPH + L group and the CAPSH + L group showed the highest tumor
inhibition efficiency during treatment, whereas none of the other
treatments were effective in inhibiting tumor growth (Fig. [179]7e).
After the treatment lasted for 14 days, the tumors of the mice were
weighed, and the tumor weights further confirmed this result
(Fig. [180]7f). There was no significant change in the body weight of
all mice during the treatment period (Fig. [181]7g). We further
evaluated the therapeutic effect of the combination therapy at the
cytokine level. we examined the concentrations of TNF-α and IFN-γ in
tumor tissues after different treatments. At the end of treatment,
TNF-α (Fig. [182]7h) and IFN-γ (Fig. [183]7i) were significantly
increased in the CAPSH + L group compared to the PBS group.
Furthermore, the robust tumor-suppressive effects of the combination
treatment were further validated through an array of investigative
techniques. This included the examination of tumor tissues via H&E
staining, Tunel assay, and (IF) staining for Ki-67, CD8, and CD4
markers (Fig. [184]7j). Finally, we evaluated the therapeutic effect of
CAPSH by detecting the mRNA levels of ATP7A and the copper
death-related genes DLAT and FDX1 in tumor tissues after treatment. As
shown in Fig. [185]7k, for CAPH, Cu[9]S[8] + L, and CAPH + L, the mRNA
level of ATP7A was substantially increased, probably due to the
increase in intracellular copper ions concentration, which led to the
increase in the expression of ATP7A to resist the copper death of tumor
cells. In the CAPSH + L group, the mRNA level of ATP7A was
substantially reduced, indicating that siATP7A had a certain silencing
effect on ATP7A. mRNA levels of LIAS (Fig. [186]7l) and FDX1
(Fig. [187]7m) in the CAPSH + L group were reduced to a certain extent
compared with those of CAPH + L, indicating that inhibition of ATP7A
could promote cuproptosis of tumor cells. In addition, we examined the
changes of monovalent copper ions content in tumor tissues of mice
after tail vein injection of PBS and CAPSH for 12 h. The results showed
that the monovalent copper ions content in the tumor tissues was
significantly increased after injection of CAPSH, and the fluorescence
intensity was 1.9 times higher than that in the PBS group
(Supplementary Fig. [188]27).
Fig. 7. In situ tumor model of breast cancer.
[189]Fig. 7
[190]Open in a new tab
a In situ tumor model in mice. b Photographs of representative tumors
after different treatments, n = 4 mice per group. c Tumor volume after
different treatments. Data are presented as the means ± SD (n = 4 mice
per group). d Tumor volume change curves for each mouse after different
treatments. e In vivo bioluminescence imaging of mice before and after
treatment. f Tumor weights obtained after different treatments. Data
were presented as the means ± SD (n = 4 mice per group). g Changes in
body weight of mice after different treatments. Data were presented as
the means ± SD (n = 4 mice per group). h mRNA levels of IFN-γ in
different treated tumors. Data were presented as the means ± SD (n = 4
mice per group). i mRNA levels of TNF-α in different treated tumors.
Data were presented as the means ± SD (n = 4 mice per group). j H&E
sections and immunofluorescence staining (Tunel, Ki-67, CD8, and CD4)
of tumor tissues from mice of different treatment groups. Images are
representative of three biologically independent mice. Scale bar:
100 μm. k mRNA levels of ATP7A in different treated tumors. Data were
presented as the means ± SD (n = 4 mice per group). l mRNA levels of
LIAS in different treated tumors. Data were presented as the means ± SD
(n = 4 mice per group). m mRNA levels of FDX 1 in different treated
tumors. Data were presented as the means ± SD (n = 4 mice per group).
The significance between each of the multiple groups in (c, f, h, i, k,
l, m) was calculated using one-way ANOVA. Source data are provided as a
Source Data file. Figure 7a was created with BioRender.com released
under a Creative Commons Attribution 4.0 International license
([191]https://creativecommons.org/licenses/by/4.0/).
In order to further evaluate the biosafety of CAPSH application in
vivo, we conducted blood routine, blood biochemistry, organ
coefficient, and histological examinations of major organs after
administering CAPSH injections to mice. The results of the experiments
revealed that there were no significant changes in seven indices: white
blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), platelets
(PLT), alanine aminotransferase (ALT), aspartate aminotransferase
(AST), and urea nitrogen (UREA) (Supplementary Fig. [192]28). Organ
coefficients were used to assess the relative health or damage of
various organs. We weighed the organs of the mice and calculated the
organ coefficients (Organ weight/body weight × 100%) (Supplementary
Fig. [193]29). The results revealed that the spleen coefficients and
liver coefficients of the mice in the PBS group were significantly
higher compared to those in the CAPSH group. However, there was no
significant difference in the coefficients of other organs. These
findings suggest that the spleen and liver in the PBS group exhibited
abnormal cells, leading to significant organ changes. H&E staining of
the heart, liver, spleen, lungs, and kidneys’ tissues of the mice were
observed, and none of them exhibited significant changes in tissue
structure, which further demonstrated the favorable biosafety of CAPSH
(Supplementary Fig. [194]30).
Inhibition of metastasis and long-term immune memory
Encouraged by the excellent performance of CAPSH + Laser in knocking
out ATP7A and stimulating antitumor immune response, we further
evaluated its impact on long-term metastasis inhibition in mice
(Fig. [195]8a). On the 60th day after treatment, whole lung tissue
photos and H&E staining showed that the CAPSH + Laser group had the
least number of metastatic nodules (Fig. [196]8b). Compared with the
CPH group, the CPSH group significantly reduced the metastasis of 4T1
cells in the lungs, confirming the importance of ATP7A in tumor
invasion and metastasis. In addition, CAPSH not only inhibited
metastasis by inhibiting ATP7A, but also activated the immune system by
inducing ICD, further inhibiting metastasis. The experiment showed that
the positive rate of CD4^+ T cells in the spleen and lymph nodes was
higher in the CAPSH group, indicating that CAPSH can induce and
maintain the immune response of CD4^+ T cells, producing long-term
immune memory. The changes in CD8^+ T cells in the CAPSH group were not
as significant as in the short term, reflecting the dynamic adjustment
of immune cells (Fig. [197]8c). The results indicate that CAPSH+Laser
effectively inhibits long-term metastasis by inhibiting ATP7A and
activating the immune system.
Fig. 8. Inhibition of metastasis and long-term immune memory.
[198]Fig. 8
[199]Open in a new tab
a Schematic diagram of experimental design for transferring animals. b
Representative photos of lung tissue and H&E staining of middle lung
tissue in each group of mice. Images are representative of three
biologically independent mice. Scale bar: 2 mm (top) and 400 μm
(bottom). c Flow cytometry analysis of CD4^+ T cells in the lymph nodes
and spleen. Images are representative of three biologically independent
mice. Source data are provided as a Source Data file.
Safety evaluation of CAPSH
The main side effects of CAPSH might originate from the non-targeted
release of copper ions, causing damage to normal tissues. Copper ions
have an impact on the oxygenation ability and structural stability of
hemoglobin, which may disrupt its function and affect overall
physiology. To demonstrate the low toxicity of CAPSH, we tested its
interaction with hemoglobin. The results showed that there was no
significant change in the absorption peak of hemoglobin at 415 nm,
indicating that CAPSH had no significant effect on hemoglobin function
(Supplementary Fig. [200]31). ICP-MS analysis of copper ions content in
mouse serum can be found in the Supplementary Fig. [201]32, revealed
that the copper ions concentration in the CAPSH group was
8.45 ± 0.89 μmol/L, which was higher than that in the PBS group, but
did not exceed the safe concentration of 10–20 μmol/L. The Masson
staining results showed no significant changes in collagen fibers,
muscle tissue, cell nuclei, and other structures in the organs between
the experimental group and the blank group, indicating that CAPSH did
not cause damage to the organs (Supplementary Fig. [202]33).
We investigated the acute and chronic toxicity of CAPSH in mice. Acute
toxicity experiments have shown that doses of 80 mg/kg and above cause
rapid death in mice, with the 70 mg/kg group dying within 4 days. The
60 mg/kg group had a survival rate of 50%, while the 30 mg/kg group
showed no significant abnormalities, with a survival rate of 100%.
Chronic toxicity experiments showed that mice at doses of 30 and
15 mg/kg exhibited PLT reduction and abnormal elevation of liver and
kidney function indicators on the first day (Supplementary
Figs. [203]34, [204]35). However, by the seventh day, these indicators
returned to normal, indicating that the mice had the ability to
self-repair after experiencing early acute liver and kidney injury. The
experimental results indicate that a dose of 15 mg/kg is safe.
Utilizing proteomics, we conducted a comparative analysis to discern
alterations in the proteome composition and the expression profiles of
proteins in tumor tissues between the control and CAPSH treatment
groups. Our objective was to ascertain whether CAPSH intervention
modulates immune responses linked to metal ions beyond copper ions.
Through meticulous examination of proteomics data and the graphical
representation of differential protein expression via histograms and
volcano plots, we identified a total of 627 proteins exhibiting
differential expression, comprising 253 upregulated and 374
downregulated proteins (Supplementary Fig. [205]36a, b). Subsequently,
we screened metal ion-related proteins (e.g., ion channel proteins,
matrix metalloproteinases, and metal chaperonins) from these 627
differential proteins and displayed them in a Wayne diagram
(Supplementary Fig. [206]36c). We then proceeded to perform a KEGG
pathway enrichment analysis on the 149 differentially expressed
proteins that were associated with metal ions, revealing that 29 of
these proteins were intricately connected to the immune system
(Supplementary Fig. [207]36d). Further elucidation of the proteins that
intersect both metal ion biology and immune system involvement was
achieved through a heat map representation of their fold changes
(Supplementary Fig. [208]36e). Note that, upon functional annotation,
it was observed that these differentially expressed proteins, which are
associated with both metal ions and immune mechanisms, did not exert a
significant influence on the immune modulation pertinent to
oncotherapy. Consequently, our findings suggest that the CAPSH
intervention did not elicit additional metal-related immune responses
at the tumor site.
Discussion
In summary, we had successfully constructed a copper-based nanoplatform
(CAPSH) for the treatment of breast cancer. Mesoporous Cu[9]S[8]
nanoparticles with photothermal effect was used as a delivery platform
and copper reservoir, effectively delivering a large amount of copper
ions, AIPH, and SiRNA to the breast cancer region. In the near-infrared
II wavelength light (1064 nm) could not only release a large amount of
copper ions through photothermal conversion to cause disintegration,
but also the alkyl radicals generated by AIPH effectively killed local
tumor cells. By introducing siRNA, the expression of ATP7A protein was
successfully suppressed, thereby enhancing the sensitivity of tumor
cells to copper ions, and further improving the occurrence of
cuproptosis. Additionally, siRNA also helped inhibit tumor growth and
metastasis by suppressing the activity of LOX. The mouse model of in
situ breast tumors verified that the enhancement of cuproptosis
effectively activated immunogenic death in this tissue region based on
the release of tumor cell antigens, elevated the immune system response
at the end of the treatment, and effectively inhibited tumor
recurrence. This nanoplatform integrally enabled precise targeting of
the tumor region, elevated the onset of cuproptosis, inhibited tumor
cell self-regulatory mechanisms, and effectively activated the immune
response. In summary, we proposed a therapeutic strategy that both
promoted the occurrence of copper death and inhibited the
self-regulation of tumor cells, combining the deep photothermal effect
based on near-infrared II illumination as well as the generation of
alkyl radicals to achieve a multidimensional and efficient treatment
for breast tumors, and optimizing the application of cuproptosis in
tumor therapy.
Methods
Ethical statement
All animal experiments were approved by the Animal Experimentation
Ethics Committee of Huazhong University of Science and Technology
(IACUC Number: S904).
Animals
Female Balb/c mice (6-8 weeks old, 18–20 g) were purchased from Beijing
Vital River Laboratory Animal Technology Co., Ltd. Mice were housed in
an animal facility under constant environmental conditions (room
temperature, 22 ± 1 °C; relative humidity, 40–70%; and 12 h light-dark
cycle), and all mice had free access to food and water. Tumor volume
was calculated as 0.5 × (length × width × width). To minimize animal
discomfort, according to the Guideline of Assessment for Humane
Endpoints in Animal Experiments (Certification and Accreditation
Administration of the P. R. China, RB/T 173-2018), in general
experiments, the tumor burden should not exceed 5% of the animal’s
normal body weight; in therapeutic experiments, it should not exceed
10% of the animal’s body weight (10% indicated that the diameter of the
subcutaneous tumor on the back of a 25 g mouse reached 17 mm). At the
end of the mouse experiments, mice were euthanized according to animal
welfare standards (euthanasia of all animals was performed using
isoflurane in small animal anesthetics).
Materials
Polyvinylpyrrolidone (PVP), copper chloride dihydrate (CuCl[2]), sodium
sulfide nonahydrate (Na[2]S·9H[2]O),
2,2’-azinobis(3-ethylbenzothiazoline -6-sulfonic acid ammonium salt)
(ABTS), α-(4-pyridine-N-oxide)-N-tert butylnitrone (POBN),
2,2’-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH), and
3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT)
was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd
(Shanghai, China). Sodium hydroxide (NaOH), hydrazine hydrate solution
(NH[2]NH[2] · xH[2]O), ethanol absolute, dimethyl sulfoxide (DMSO)
isopropanol, sodium chloride, sodium dodecyl sulfate, anhydrous
methanol, concentrated nitric acid, and hydrochloric acid was purchased
from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Calcein
acetoxymethyl ester (Calcein AM), propidium iodide (PI), reactive
oxygen species assay Kit, BCA protein assay Kit, SDS-PAGE sample
loading buffer 5X, hydrophobic polyvinylidene fluoride, (PVDF),
penicillin–streptomycin, RNase-free dd H[2]O, 4% paraformaldehyde fix
solution and tween-20 was purchased from Shanghai Biyuntian
Biotechnology Co., Ltd (Shanghai, China). Polyacrylamide hydrochloride
(PAH) was purchased from Anhui Zesheng Technology Co., Ltd (Anhui,
China). Hyaluronic acid (HA) was purchased from Sigma-Aldrich
(Shanghai, China). RNA isolater, HiScript III All-in-one RT SuperMix
Perfect for qPCR Kit and Taq Pro Universal SYBR qPCR Master Mix Kit was
purchased from Vazyme Biotech Co., Ltd (Nanjing, China). ECL
Chemiluminescence Substrate Kit was purchased from Beijing Labgic
Technology Co., Ltd (Beijing, China). Pancreatin was purchased from
Gino Saber Biotechnology Co., Ltd (Zhejiang, China). Omni-Easy™
One-Step PAGE Gel Fast Preparation Kit was purchased from Shanghai
Yaenzyme Biopharmaceutical Technology Co., Ltd (Shanghai, China). Fetal
bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology
Co., Ltd (Zhejiang, China). PBS and DMEM was purchased from Thermo
Fisher Scientific Co., Ltd (Shanghai, China). Phenylmethanesulfonyl
fluoride, Tris(hydroxymethyl)methyl aminomethane, and Glycine was
purchased from Beijing Kehbio Technology Co., Ltd (Beijing, China). All
solutions were prepared using ultrapure water from the Milli-Q system
(≥18.20 M Ω).
Cell lines
Mouse breast cancer cells (4T1: CRL-2539), mouse embryonic fibroblasts
(NIH-3T3: CRL-1658), and mouse monocyte macrophage leukemia cells
(RAW264.7: TIB-71) cell line was obtained from Wuhan Saikangte
Biotechnology Co., Ltd. Mouse breast cancer cells-luciferase labeled
(4T1-LUC: CRL-2539-LUC2) cells was cell line obtained from Wuhan
service Biotechnology Co., Ltd. All three cells were cultured in DMEM
medium containing 10% fetal bovine serum, 100 U/mL penicillin, and
0.1 mg/mL streptomycin. All cell lines in this study get tested without
mycoplasma contamination. Each cell line was morphologically confirmed
according to the information provided by the cell-source center, and
the main 4T1, NIH-3T3, and RAW264.7 cell lines were authenticated with
short tandem repeat (STR) analysis (Supplementary
Tables [209]2–[210]4).
Synthesis of Cu[9]S[8]
Weighing 480 mg of polyvinylpyrrolidone was dissolved in 50 mL of
ultrapure water, 200 μL of CuCl[2] solution (0.5 M) was added and
stirred for 5 min, then 50 mL of NaOH solution at pH = 9, and 16 μL of
hydrazine hydrate (85%) were added to form a bright yellow Cu[2]O
suspension. After stirring for 5 min, 400 μL of Na[2]S solution
(1.33 M) was added, and the reaction was carried out at 60 °C for 2 h.
At the end of the reaction, the solution was washed by centrifugation
with a water/ethanol solution with a volume ratio of 2:1 at 10,000×g
three times. The copper ions content was determined by ICP-MS and the
mass of Cu[9]S[8] was calculated to be 3.5 mg.
Synthesis of CPH, CAP, CAPH, CAPSH
To 1 mL of Cu[9]S[8] (1 mg/mL) solution, 0.1 mg of PAH was added and
stirred for 12 h. The solution was washed by centrifugation three
times. Adding 1 mg of HA and stirring for 8 h. Centrifuging and washing
three times to obtain CPH. To 1 mL of Cu[9]S[8] (1 mg/mL), adding 4 mg
of AIPH and stirring for 12 h. Adding 0.1 mg of PAH and stir for 12 h.
Centrifuging and washing three times to obtain CAP. About 1 mg of CAP
is stirred with 1 mg of HA for 8 h. Centrifuging and washing three
times to obtain CAPH. CAPS was obtained by incubating 0.12 mg of CAP
with 3 μg of siRNA at 4 °C for 2 h. About 0.12 mg of HA was added and
stirred for 8 h. The sample was washed by centrifugation three times to
obtain CAPSH. The sequence of siATP7A is as follows:
5′-CCCGAGUGAUAGCAGAGUUUAdTdT-3′, The siATP7A was synthesized by
Jinkairui Biotechnology Co., Ltd.
Photothermal properties of Cu[9]S[8]
About 200 µL of Cu[9]S[8] at different concentrations (12.5, 25, 50,
and 100 μg/mL) were taken in the wells of removable enzyme labeling
plates, and the samples were irradiated with a 1064 nm laser at
0.75 W/cm^2 for 5 min, and the temperature changes of the samples with
the laser irradiation were recorded with an infrared thermography
camera. The same method was used to determine the temperature change of
Cu[9]S[8] at a concentration of 100 μg/mL at different power densities
(0.25, 0.50, and 0.75 W/cm^2), and three parallels were set up for each
sample. To test the photothermal stability of the probes, 200 µL of
Cu[9]S[8] at a concentration of 100 μg/mL was placed in the wells of a
removable enzyme plate. The samples were then irradiated using a
1064 nm laser at 0.75 W/cm^2 for 5 min. After irradiation, the samples
were cooled for 5 min, and the temperature change was recorded using an
infrared thermography camera. The photothermal conversion efficiency of
Cu[9]S[8] was calculated using Eq. ([211]1):
[MATH:
η=hs(Tmax−Ts
urr)−QDisI(1−
10−A1064) :MATH]
1
hs = mc/τ; Q[Dis] = hs × (T[max of water] - T[surr]), T[max] represents
the peak temperature at which Cu[9]S[8] undergoes heating, T[surr]
denotes the surrounding ambient temperature, T[max] of the water
signifies the maximum temperature reached during the water heating
process. The mass of the solution is denoted by “m”, the specific heat
capacity of water is represented as “c”, the laser power is expressed
as “I”, and the absorption value at 1064 nm is indicated as A[1064].
Detection of free radicals
AIPH underwent thermal decomposition, resulting in the production of
alkyl radical groups that reacted with ABTS to form ABTS^+•. The
solution containing a mixture of CAPH and ABTS was exposed to a 1064 nm
laser with a power density of 0.75 W/cm^2 for 5, 10, and 15 min. After
centrifugation, the UV absorption spectrum of the solution was measured
using UV-vis absorption spectroscopy in the range of 400–1000 nm. To
determine the type of free radicals generated, we mixed 150 μL of AIPH,
Cu[9]S[8], and CAPH with 100 mM POBN. The illuminated group was then
irradiated with a NIR laser with a power density of 0.75 W/cm^2 for
5 min. On the other hand, the non-illuminated group was mixed for 5 min
and then directly detected. After the different treatments, we measured
the ESR signals of the samples at 25 °C using an ESR spectrometer.
Encapsulation rate and drug release studies of AIPH
In a 1 mL solution of Cu[9]S[8] (1 mg/mL), 4 mg of AIPH was introduced
and stirred for 12 h. Subsequently, 0.1 mg of PAH was added, and the
mixture was stirred for an additional 12 h. The absorption value of the
supernatant at 365 nm was determined through centrifugation, and the
mass of AIPH was calculated based on the standard curve. The
encapsulation rate was calculated using Eq. ([212]2):
[MATH:
Encapsu<
mi>lationrate=(massofinitiallyaddedAIPH
)−(massofAIPHinthesupernatant<
mo>)massofinitiallyaddedAIPH
*100% :MATH]
2
The CAPH was dialyzed using a 500 Da dialysis bag, and the pH of the
dialysate was adjusted to 5.4, 6.5, and 7.4. At regular intervals, the
external fluid of the dialysis bag was sampled, and the UV absorption
value was measured. The sample was then returned to the dialysis bag,
and this process was repeated until the absorption value remained
constant. The content of AIPH in the external fluid of the dialysis bag
was calculated using the standard curve of AIPH.
Release of copper ions
A solution of Cu[9]S[8] (1 mg/mL) was placed in a dialysis bag. The pH
of the dialysate was then adjusted to 5.4, 6.5, and 7.4. Samples were
collected at specific time intervals of 5, 10, 30 min and 1, 2, 6, 12,
24, 36 h. The amount of copper ions presented in the solution was
measured using ICP-MS.
ICP-MS testing conditions
Sample pretreatment: After weighing the tumor tissues, homogenize them
using a tissue homogenizer and digest them in concentrated nitric acid
until the solution is clear and transparent. Standard curve: Prepare a
standard curve using a standard solution of known concentration (the
concentration of the standard curve is 10, 20, 40, 60, 80 μg/L).
Instrument setting: set the element to be detected as copper, select
the standard mode, and optimize the parameters. The plasma power was
between 1000–1600 W. Set the argon flow rate at 0.8–1.2 L/min. Testing:
The sample was fed into the plasma through the feeding system, and the
signal intensity of the characteristic ions was recorded. Data
analysis: The standard curve was plotted according to the counts per
second (cps) of the standard solution versus concentration. The
concentration of the elements in the sample is calculated by comparing
the signal intensity of the sample with the standard curve.
Cytotoxicity assay
4T1 cells were inoculated in 96-well plates, with ~5 × 10^3 cells per
well and 5 parallels per group. Once the cells were fully attached to
the wall, different concentrations of CPH (0, 10, 20, 40, 60, 80, and
100 µg/mL) were added. After 12 h, the medium was removed, and the
cells were washed with PBS three times. Then, 100 µL of 1 mg/mL MTT
solution was added to each well. After 4 h, the medium and MTT solution
were aspirated out, and 100 µL of DMSO was added to each well. The
plate was shaken for 20 min to ensure even distribution. Subsequently,
the absorption at 490 nm of each well was measured using an enzyme
marker to determine cell viability.
In vitro combination therapy experiments
The 4T1 cells were uniformly inoculated in 96-well plates with
~5 × 10^3 cells per well. There were five parallel groups in each
group, and they were cultured for 24 h. The cells were then treated
with AIPH, CPH, and CAPH, respectively, for 12 h. After that, the
medium was removed, and the cells were washed three times with PBS. In
the illumination group, the cells were exposed to a 1064 nm laser at a
power density of 0.75 W/cm^2 for 5 min. Then, the medium was added
back, and the incubation continued for 6 h. Subsequently, 100 µL of
1 mg/mL MTT solution was added to each well. After 4 h, the MTT
solution was aspirated out, and 100 µL of DMSO was added to each well.
The 96-well plate was then placed on a shaking table and shaken
uniformly for 20 min. Finally, the absorption of each well at 490 nm
was measured with an enzyme marker to calculate the cell viability.
The 4T1 cells were inoculated homogeneously in 24-well plates with
~1 × 10^5 cells per well and cultured for 24 h. The old medium was
discarded, and the cells were treated with AIPH, CPH, and CAPH,
respectively, for 12 h. After being washed three times with PBS, the
light group was exposed to a laser with a power density of 0.75 W/cm^2
at 1064 nm for 5 min and then cultured for 4 h. The cells were
co-stained using the Calcein AM/PI assay and observed under inverted
fluorescence microscopy.
Cell uptake
ICP-MS detection
Adding CAPSH to the culture medium of 3T3 and 4T1 cells and co-culture.
Collect the cells at 2, 4, and 8 h. After counting, nitric acid was
added for digestion, and ICP-MS was used to measure the copper content
within the cells.
Flow cytometry detection
Loading Cy5.5 onto CAPSH and adding it to the culture medium of 3T3 and
4T1 cells. Co-culture and collecting the cells at 2, 4, and 8 h. Using
flow cytometry to detect the fluorescence intensity of Cy5.5.
Detection of free radicals in cells
The 4T1 cells were uniformly inoculated in 24-well plates with
~1 × 10^5 cells per well and cultured for 24 h. The old medium was then
discarded, and the cells were treated with AIPH, CPH, and CAPH,
respectively, for a duration of 12 h. The intracellular content of
reactive oxygen species was assessed using the DCFH-DA reactive oxygen
species detection kit.
In vitro detection of ICD activation
Sterile coverslips were placed in a 12-well culture plate, and 4T1
cells were seeded and cultured for 24 h. After different treatments,
the cells were incubated for an additional 6 h. Subsequently, the
medium was removed, and the cells were washed twice with PBS. The 4T1
cells were then fixed with 4% paraformaldehyde and permeabilized with
0.1% Triton X-100. Next, the cells were blocked with 5% BSA at room
temperature for 20 min. Following blocking, the cells were incubated
overnight at 4 °C with anti-HMGB1, anti-HSP70, and anti-CRT antibodies.
After overnight incubation, the cells were washed three times with PBS
and incubated with secondary antibodies at room temperature for 1 h.
Finally, the cells were washed three times with PBS, and the coverslips
were carefully mounted on slides with DAPI antifade mounting medium for
confocal fluorescence imaging and flow cytometry detection. 4T1 cells
were seeded in a 12-well plate and cultured for 24 h. After different
treatments, the cells were incubated for an additional 6 h.
Subsequently, the ATP content in the culture medium was detected using
an ATP assay kit.
Copper distribution in tumors and organs
To study the distribution of copper in tumors and organs, 30
tumor-bearing mice were selected. Each mouse was administered an
intravenous injection of 150 μL of CAPSH (2 mg/mL) through the tail
vein. At the following time points: 0.5, 1, 2, 4, 6, 8, 12, and 24 h,
as well as 5 and 15 days post-injection, three mice were euthanized at
each time point. The heart, liver, spleen, lungs, kidneys, and tumor
tissues were then harvested. After weighing the tissues, each sample
was homogenized in a tissue grinder with 400 μL of PBS. Subsequently,
5 mL of concentrated nitric acid was added to the samples, which were
then stored in the dark for several hours. Once the samples were
completely digested at 120 °C in a digestion apparatus until the
solution was clear and transparent, the copper content was measured
using ICP-MS.
Blood half-life study
For the study of blood half-life, 150 μL of CAPSH (2 mg/mL) was
administered intravenously to three BALB/c mice via the tail vein.
Blood samples were collected at the following time points: 10 min,
30 min, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48, 72, and 96 h post-injection.
Each blood sample was then digested with concentrated nitric acid until
the solution was completely clear. The concentration of copper in the
blood was determined using ICP-MS.
RNA extraction
The adherent cells were washed three times with PBS after different
treatments, 1 mL of RNA isolator was added, and the cells were blown
down and collected in a centrifuge tube. Next, 200 µL of chloroform was
added to the solution. The mixture was vigorously shaken for 15 s to
form an emulsion and then left to stand on ice for 5 min. After that,
the solution was centrifuged at 12,000×g for 15 min at 4 °C. The upper
aqueous phase was aspirated into a new centrifuge tube. To this tube,
an equal volume of pre-cooled isopropanol was added. The mixture was
mixed upside down and left to stand on ice for 10 min. Subsequently,
the tube was centrifuged at 12,000×g for 10 min at 4 °C. Usually, a
white precipitate is visible at this stage. The supernatant was
removed, and 1 mL of 75% ethanol was added. The tube was inverted
several times and left at room temperature for 3–5 min. Then, it was
centrifuged at 12,000×g for 5 min at 4 °C, and the supernatant was
discarded. The precipitate was dried for 2–5 min, and an appropriate
amount of RNase-free dd H[2]O was added to dissolve it. Finally, the
ratios of A[206]/A[230] and A[260]/A[280] were measured using a
microspectrophotometer. The primer sequences used in quantitative
polymerase chain reaction experiments are provided in Supplementary
Table [213]5.
Detection of intracellular protein expression
The cell precipitate was collected, washed three times with PBS, and
the liquid was aspirated. Protein lysate (RIPA: PMSF = 1000:1) was
added to the cell precipitate and thoroughly mixed. It was then placed
on an ice bath and lysed for 1 h. Finally, it was centrifuged at
12,000×g, 4 °C for 5 min. The protein concentration in the supernatant
was determined by the BCA Protein Concentration Assay Kit and mixed
with loading buffer solution thoroughly, placed in heating denaturation
at 98 °C for 10 min, and placed in the refrigerator at −20 °C for
storage. The protein samples were separated by SDS-PAGE gel
electrophoresis and transferred to the PVDF membrane. The membrane was
blocked with 5% BSA in TBST for 1 h at room temperature and
immunoblotted with primary antibody overnight at 4 °C. The membrane was
then washed three times with TBST and incubated with HRP-labeled
secondary antibody at room temperature for 1 h. Finally, the protein
bands were visualized using the Enhanced Chemiluminescence (ECL)
Substrate Kit.
In vivo safety evaluation
To assess the biosafety of CAPSH, blood samples (400 µL per mouse) were
obtained from the heart after the completion of treatment. A portion of
300 µL was placed in a procoagulant tube containing separating gel and
centrifuged for 10 min. The resulting supernatant was used for liver
and renal function analysis, including AST, ALT and UREA. An additional
100 µL was collected in an anticoagulant tube for routine blood tests,
such as RBC, WBC, PLT, and HGB measurements. Furthermore, the heart,
liver, spleen, lung, and kidney of the mice were collected, weighed,
fixed in 4% paraformaldehyde, dehydrated, embedded, and sectioned for
H&E staining and histopathological examination using a light
microscope.
In vivo antitumor efficiency
Two tumor models were utilized to evaluate the effectiveness of
antitumor treatment in vivo. The subcutaneous tumor model was
established by injecting ~1 × 10^6 4T1 cells per mouse into the lower
right back of Balb/c mice. The in situ tumor model was created by
injecting ~1 × 10^6 4T1-LUC cells per mouse into the first pair of
mammary glands in the chest of Balb/c mice. Once the tumor volume
reached 80–100 mm^3, the mice in the subcutaneous tumor model were
randomly divided into six groups (seven mice per group): PBS, AIPH + L,
Cu[9]S[8] + L, CAPH, CAPH + L, CAPSH + L. Similarly, the mice in the in
situ tumor model were randomly divided into six groups (four mice per
group). Each group was administered 150 μL of different samples through
the tail vein, with a dosage of 15 mg/kg for Cu[9]S[8] in the
Cu[9]S[8] + L, CAPH, CAPH + L, CAPSH + L group, and 12 mg/kg for AIPH
in the AIPH + L, CAPH, CAPH + L, CAPSH + L group. After 24 h of sample
injection, the light group irradiated the tumor with a laser having a
wavelength of 1064 nm and a power density of 0.75 W/cm^2 for 5 min. The
changes in body weight and tumor volume were recorded every other day.
Tumor growth monitoring in mice with in situ tumor models using an
animal fluorescence imager.
Statistics and reproducibility
Data were expressed as mean ± SD. Significance between the two groups
was assessed by unpaired two-tailed Student’s t-test, and between each
of the multiple groups was calculated using one-way ANOVA. Values with
P < 0.05 were considered significant. Exact p values were provided
accordingly in the figures. All the statistical analyses were performed
using GraphPad Prism (9.5.0). In this study, the sample size was
determined based on prior experimental experience and standard
practices. We did not use statistical methods to predetermine the
sample size. During data analysis, no data were excluded. We have
provided a detailed description of the experimental methods to ensure
that other researchers can replicate the experiment and verify the
reproducibility of the results. Flow-cytometry data were analyzed with
FlowJo (ver. 10.8.1). ImageJ (ver. 1.4.3.67) were used to analyze
fluorescent grayscale. Alpha EaseFC 4.0 was used to analyze the WB.
Confocal images were analyzed with FluoView31S (ver.2.3.1.163).
Reporting summary
Further information on research design is available in the [214]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[215]Supplementary Information^ (3.2MB, pdf)
[216]Peer Review File^ (6.4MB, pdf)
[217]Reporting Summary^ (58.8KB, pdf)
Source data
[218]Source Data^ (3.6MB, xlsx)
Acknowledgements