Tumor Microenvironment Stimuli-Responsive Fluorescence Imaging and Synergistic Cancer Therapy by Carbon-Dot–Cu2+ Nanoassemblies

Article abstract of DOI:10.1002/anie.202007786

A method is developed to fabricate tumor microenvironment (TME) stimuli-responsive nanoplatform for fluorescence (FL) imaging and synergistic cancer therapy via assembling photosensitizer (chlorine e6, Ce6) modified carbon dots (CDs-Ce6) and Cu²⁺. The as-obtained nanoassemblies (named Cu/CC nanoparticles, NPs) exhibit quenched FL and photosensitization due to the aggregation of CDs-Ce6. Their FL imaging and photodynamic therapy (PDT) functions are recovered efficiently once they entering tumor sites by the stimulation of TME. Introducing of Cu²⁺ not only provides extra chemodynamic therapy (CDT) function through reaction with hydrogen peroxide (H₂O₂), but also depletes GSH in tumors by a redox reaction, thus amplifying the intracellular oxidative stress and enhancing the efficacy of reactive oxygen species (ROS) based therapy. Cu/CC NPs can act as a FL imaging guided trimodal synergistic cancer treatment agent by photothermal therapy (PTT), PDT, and thermally amplified CDT.

Full text of DOI:10.1002/anie.202007786

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How to cite:
International Edition: doi.org/10.1002/anie.202007786
German Edition: doi.org/10.1002/ange.202007786
Nanoassemblies
Tumor Microenvironment Stimuli-Responsive Fluorescence Imaging
and Synergistic Cancer Therapy by Carbon-Dot–Cu2+ Nanoassemblies
Shan Sun, Qiao Chen, Zhongdi Tang, Chuang Liu, Zhongjun Li, Aiguo Wu, and Hengwei Lin*
Abstract: A method is developed to fabricate tumor micro-
environment (TME) stimuli-responsive nanoplatform for
fluorescence (FL) imaging and synergistic cancer therapy via
assembling photosensitizer (chlorine e6, Ce6) modified carbon
dots (CDs-Ce6) and Cu2+. The as-obtained nanoassemblies
(named Cu/CC nanoparticles, NPs) exhibit quenched FL and
photosensitization due to the aggregation of CDs-Ce6. Their
FL imaging and photodynamic therapy (PDT) functions are
recovered efficiently once they entering tumor sites by the
stimulation of TME. Introducing of Cu2+ not only provides
extra chemodynamic therapy (CDT) function through reaction
with hydrogen peroxide (H2O2), but also depletes GSH in
tumors by a redox reaction, thus amplifying the intracellular
oxidative stress and enhancing the efficacy of reactive oxygen
species (ROS) based therapy. Cu/CC NPs can act as a FL
imaging guided trimodal synergistic cancer treatment agent by
photothermal therapy (PTT), PDT, and thermally amplified
CDT.
advantages to enhance therapeutic efficiency with minimized
side effects. Although extensive researches have been con-
ducted for the design and fabrication of TME-triggered
theranostic platforms,[1a,5] the development of facile methods
to fabricate such systems with satisfactory outcomes are still
highly desirable.
Benefiting from the fast development of nanotechnology,
nanomaterials have become the most explored objects for
fabricating stimuli-responsive theranostic platforms under the
conditions of endogenous TME activation or exogenous
triggering.[6] Among them, carbon dots (CDs) have emerged
as a kind of promising candidate for realizing cancer diagnosis
and treatment simultaneously thanks to their superior bio-
compatibility, stable fluorescence (FL) emission, facile sur-
face functionalization, and more importantly, the potential
photo-mediated therapeutic functions, such as photodynamic
therapy (PDT) and photothermal therapy (PTT), that can
destroy tumor cells via cytotoxic reactive oxygen species
(ROS) or local hyperthermia converted from irradiation
light.[7] However, the small size character of CDs (usually less
than 10 nm) limits their efficient accumulation in tumor sites
due to the poor enhanced permeability and retention (EPR)
effect, consequently reducing theranostic outcomes.[8] Fur-
thermore, utilizing metal-ion-containing nanoparticles for
stimuli-responsive anticancer therapy has become an attrac-
tive area owing to their ion-interference role.[9] Of note, the
copper ion (Cu2+) as one of the most affluent metal ions in
biosystem possesses enormous potentials in chemodynamic
therapy (CDT) due to the Cu-catalyzed Fenton-like reactions
induced by overexpressed hydrogen peroxide (H2O2) in
tumor sites.[10] More importantly, it has been reported that
Cu2+ can consume supernormal intracellular glutathione
(GSH) via a redox reaction.[11] Since GSH scavenges pro-
duced ROS and consequently weakens the efficacy of photo-
and chemodynamic treatments,[11b,12] the GSH depletion
capacity of Cu2+ will aggravate the oxidative damages to
pathological sites and amplify the therapeutic efficiency.
Considering the coordination capability of Cu2+ and
abundant functional groups on the surface of CDs,[13] herein,
versatile nanoparticles (named Cu/CC NPs) with TME
stimuli- responsive and GSH depletion capacities are facilely
prepared via assembling Cu2+ and photosensitizer (chlorine
e6, Ce6) modified CDs (CDs-Ce6) (Scheme 1a). Through
such a simple assembly step, the corresponding product can
be endowed numerous remarkable features: 1) in contrast to
the CDs-Ce6, the particle size of Cu/CC NPs is obviously
increased, which should be more favorable for their accumu-
lation in tumor sites due to the EPR effect;[14] 2) FL and
photosensitization of Cu/CC NPs are in quenched state under
neutral condition, but the FL imaging, PDT and CDT
Introduction
Tumor microenvironment (TME), featuring angiogenesis,
maladjusted biosynthesis intermediates, acidosis, and hypo-
xia,[1] provides interior environment for cancer cells origina-
tion and residence, which takes the main responsibility for
tumor progression, aggressive metastasis and high drug
resistance,[2] and should be paid crucial attention for exploring
novel anticancer strategies. The distinguished characteristics
of TME could offer effective therapeutic targets specifically
towards carcinoma cells.[3] To realize precision cancer treat-
ments, employment of intelligent materials that holding TME
stimuli-responsive theranostic capacities has been proposed
as one of the most ideal ways,[4] which exhibit obvious
[*] Dr. S. Sun, Q. Chen, Z. Tang, C. Liu, Prof. A. Wu, Prof. H. Lin
Cixi Institute of Biomedical Engineering, Chinese Academy of
Science (CAS) Key Laboratory of Magnetic Materials and Devices &
Zhejiang Engineering Research Center for Biomedical Materials,
Ningbo Institute of Materials Technology and Engineering, CAS
1219 ZhongGuan West Road, Ningbo 315201 (China)
E-mail: linhengwei@nimte.ac.cn
Prof. H. Lin
International Joint Research Center for Photo-responsive Molecules
and Materials, School of Chemical and Material Engineering
Jiangnan University, Wuxi 214122 (China)
Prof. Z. Li
College of Chemistry and Molecular Engineering
Zhengzhou University, Zhengzhou 450001 (China)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202007786.
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Scheme 1. Illustration of a) the synthesis process of Cu/CC nanoassemblies, and b) their features for enhancing tumor accumulation, TME
stimuli-responses and synergistic therapy.
functions (from CDs, Ce6 and Cu2+, respectively) could be
selectively activated only in tumors due to the lower pH and
overexpressed GSH and H2O2 stimulation of TME, conse-
quently minimizing the adverse effects to normal tissues;
3) the contained Cu2+ in the nanoassemblies not only to react
with endogenous H2O2 to produce highly toxic COH, but also
enables to significantly deplete GSH, and meanwhile trigger
the disassembly, leading to amplified oxidative stress and
improved therapeutic efficacy; 4) disassembly of the Cu/CC
NPs would occur under the stimulation of TME, which is
beneficial for deep penetration and cell internalization and as
well as elimination from the body; and 5) the intrinsic
photothermal and recovered photosensitization functions
from CDs-Ce6 would further enhance therapeutic efficacy
under laser irradiation by synergistic PTT, PDT, and CDT
(Scheme 1b). All these results have been confirmed by in
vitro and in vivo experiments. This work develops a facile
method to fabricate an ingenious FL imaging guided syner-
gistic tumor therapy platform, which may provide a promising
strategy for achieving enhanced therapeutic efficacy with
minimized sides effects.
NPs with CDT ability, and as well as plays the role to deplete
GSH in tumors. First of all, the successful assembly of CDs-
Ce6 and Cu2+ into nanoassemblies is determined by trans-
mission electron microscopy (TEM). As shown in the
Supporting Information, Figures S1 and S2, both CDs and
CDs-Ce6 are observed to be well-dispersed quasi-sphere NPs
with average diameters of 2.40 and 2.49 nm, respectively. The
high-resolution TEM (HR-TEM) images exhibit both of them
containing the same lattice fringes with a spacing of 0.21 nm,
demonstrating the existence of graphite-like structures.[6d,15]
However, the average size of Cu/CC NPs is increased to
40.1 nm (Figure 1a) under the optimal preparation condi-
tions, indicating the successful obtaining of nanoassemblies
with an appropriate particle size for tumor tissue accumu-
lation.[14] The HR-TEM and element mapping results further
indicate that the Cu/CC NPs are composed of CDs-Ce6 and
Cu2+ due to the identical lattice spacing with free CDs-Ce6
(Figure 1b) and evenly distribution of C, O, N, and Cu
elements (Figure 1c). The size change induced by the
formation of nanoassemblies is also confirmed by dynamic
light scattering (DLS) measurements. As shown in Figure 1d,
the hydrodynamic diameter of Cu/CC NPs is found to
obviously increase to about 47.5 nm. The observed slightly
larger particle sizes from DLS than that of from TEM are
generally attributed to the formation hydration layers on the
surface of NPs.[16]
Results and Discussion
Preparation and Characterization of Cu/CC NPs
Subsequently, Fourier transform infrared (FTIR), ultra-
violet-visible (UV/Vis) absorption, zeta potential, and X-ray
photoelectron spectroscopy (XPS) are carried out to inves-
tigate the surface conditions and chemical compositions of
these materials. In the FTIR spectra (Figure 1e), CDs exhibit
The preparation of Cu/CC NPs is schematic illustrated in
Scheme 1a and the detailed procedures are referred to the
Experimental Section in the Supporting Information. In this
design, the photosensitizer Ce6 modified CDs (CDs-Ce6) are
taken for FL imaging and PTT/PDT bimodal phototherapy.[6c]
Cu2+ is introduced as glue for assembling and endows Cu/CC
obvious peaks at around 1350, 1650, 3200, and 3390 cmÀ1 and
À1
À
=
a shoulder peak at about 1720 cm , assigning to C N, C N,
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Figure 1. a) TEM image (inset: size distribution histogram), b) HRTEM image and, c) element mapping images of Cu/CC NPs. d) Hydrodynamic
diameter of Cu/CC NPs (PDI=0.267). e) FTIR spectra of CDs, CDs-Ce6, and Cu/CC NPs. f) XPS survey spectrum and, g) high-resolution Cu 2p
spectrum of Cu/CC NPs. h) UV/Vis absorption spectra of CDs, CDs-Ce6, Cu/CC NPs, and free Ce6. i) FL emission spectra of CDs-Ce6 and Cu/CC
NPs under the excitation wavelength of 550 nm with same concentration (that is, the emission channel of CDs).
À
=
N H, O < C- < H, and C O stretching vibrations, respective-
coupling due to the introduction of -COOH from the
photosensitizer. As expected, the surface charge of Cu/CC
NPs is increased in comparison with CDs-Ce6 (from À34.7 to
À25.6 mV), ascribing to the effects of positively charged Cu2+.
The UV/Vis absorption spectra of CDs, CDs-Ce6, Cu/CC
NPs, and Ce6 are presented in Figure 1h. The characteristic
absorption bands of CDs at around 380 and 540 nm as well as
typical absorbance peaks of Ce6 at 405 and 640 nm can all be
found in the spectrum of Cu/CC NPs, this not only confirming
the formation of assembles with reserved absorption proper-
ties of CDs and Ce6, but also revealing potentials of the
assemblies for NIR-light-triggered photothermal imaging and
therapy.[20] Interestingly, the FL of CDs-Ce6 at optimal
emission channel of CDs (lex = 550 nm) and Ce6 (lex =
405 nm) are dramatically quenched when assembled into
Cu/CC NPs (Figure 1i; Supporting Information, Figure S5),
which is mainly ascribed to the Cu2+ induced aggregation of
CDs-Ce6 and the electron/energy transfer processes.[13a,21]
This phenomenon also indicates that the FL imaging and
photosensitization functions of CDs-Ce6 in the nanoassem-
blies are in quenched state under neutral condition. Finally,
stability of Cu/CC NPs is evaluated. As shown in the
Supporting Information, Figure S6a, no evident aggregation
can be found from the Cu/CC NPs dispersion in water, PBS,
and cell culture medium even after standing for 3 days.
Finally, hydrodynamic diameters of Cu/CC NPs dispersed in
PB buffer (pH 7.4) and simulated body fluid (SBF) keep
constant during the whole observation period from 2 to 120 h
À
À
ly, suggesting the existence of carboxyl ( COOH), amino (
NH2), and hydroxy ( OH) groups.[6c] Compared with CDs, the
À
FTIR spectrum of CDs-Ce6 shows an additional shoulder
peak at about 1570 cmÀ1 attributed to N H bending vibration
À
of amide bond and a decreased peak at about 3200 cmÀ1
À
ascribed to N H stretching vibration of amino, demonstrating
the successful conjugation of Ce6 on the CDs. The loading
content of Ce6 on CDs can be calculated to be about 11.66%
(mass percentage) based on the calibration curve (Supporting
Information, Figure S3 and Table S1). Besides, the newly
emerged peak in the FTIR spectrum of Cu/CC NPs at about
1045 cmÀ1 arises from stretching vibration of metal–ligand
bond,[17] indicating the coordination between Cu2+ and CDs-
Ce6. XPS results further confirm the chemical compositions
of Cu/CC NPs. The survey spectrum exhibits four character-
istic peaks of C 1s (284.8 eV), N 1s (399.8 eV), O 1s
(531.7 eV), and Cu 2p (934.4 eV; Figure 1 f). High-resolution
XPS spectrum of Cu 2p (Figure 1g) reveals two dominant
peaks at 934.3 and 954.2 eV, assigning to Cu 2p3/2 and Cu 2p1/2
,
respectively.[18] Furthermore, satellite peaks at the range of
938–948 eV are also observed, which have been proved to be
the typical peaks of Cu2+,[19] verifying again the existence of
Cu2+ in the Cu/CC NPs. The amounts of Cu2+ in Cu/CC NPs is
determined to be 3.6% (mass percentage) using inductively
coupled plasma optical emission spectrometry (ICP-OES).
As illustrated in Figure S4 (SI), the Zeta potential of CDs
negatively shifts from À18.1 to À34.7 mV after the Ce6
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(Supporting Information, Figure S6b,c and S6c), indicating
CC NPs in comparison with the CDs-Ce6 and blank,
high stability of Cu/CC NPs in dispersed state.
indicating the remarkable GSH consumption capacity of
Cu/CC NPs. This property is significant for improving ROS-
based cancer therapy efficiency, because GSH is a well-known
ROS scavenger in biosystem. Furthermore, it has been
reported that copper-involved materials could effectively
catalyze H2O2 and generate hydroxyl radicals (COH) even in
mild acidic media via Harber–Weiss and Fenton-like reac-
tions (Eqs. (1)–(3) in Figure 2c).[10e] The COH production
property of Cu/CC NPs can be determined using disodium
terephthalate (TA) as a specific probe (that is, TA being
transformed into 2-hydroxy-terephalic acid (TAOH) with
a typical FL peak at around 435 nm after reacting with COH).
As depicted in Figure 2d, the FL intensity from TA, H2O2 and
Cu/CC NPs mixture is found to be much stronger than that of
other control groups, verifying the efficient H2O2-responsive
generation of COH and consequent CDT capacity of Cu/CC
NPs. Therefore, the lower pH, overexpressed GSH and H2O2
of the TME can trigger multiple functions of Cu/CC NPs, such
as FL recovery, particle disassembly, GSH consumption, and
COH generation, demonstrating great potentials of the Cu/CC
NPs as a smart cancer theranostic agent.
Stimuli-Responsive Properties
As a designed specific antitumor theranostic agent,
responses of Cu/CC NPs towards internal stimuli of TME
(such as lower pH, overexpressed GSH, and H2O2) are
evaluated. As shown in Figure 2a and the Supporting
Information, Figure S7, FL emission of CDs-Ce6 (CDs and
Ce6 channels, respectively) from the Cu/CC NPs is observed
to be gradually recovered with increasing concentrations of
GSH in an acidic medium (pH 6.3 of PB). FL recovery as the
function of time is also investigated to evaluate the responsive
process (Supporting Information, Figure S8). This phenom-
enon could be attributed to the disassembly of Cu/CC NPs,
being confirmed by TEM and DLS measurements (Support-
ing Information, Figures S9 and S10). Similar FL recovery
and morphology change performances of Cu/CC NPs dis-
persed in SBF induced by the addition of GSH are recorded in
the Supporting Information, Figures S11 and S12, verifying
the stimuli-responsive disassembly feature of Cu/CC NPs. It is
known that Cu2+ can be reduced to Cu+ by GSH (inset
equation in Figure 2a), and meanwhile the electrostatic
repulsion will be enhanced between H+ and metal ions in
acidic media, which would weaken the coordination capa-
bility between Cu2+ and CDs-Ce6, subsequently leading to the
disassembly of Cu/CC NPs. Moreover, the redox reaction
between Cu2+ and GSH also causes GSH depletion, which can
be proved by the variations of GSSG/GSH ratio in GSH
solutions using the commercial assay kit. As shown in
Figure 2b, 4–6 times enhancement of GSSG/GSH ratio are
observed from the GSH solutions after incubation with Cu/
Considering the broad absorption of Cu/CC NPs tailed up
to NIR region, their feasibility acting as a PTTagent is further
evaluated. The laser dose-dependent temperature change of
Cu/CC NPs at a fixed concentration (200 mgmLÀ1) is firstly
investigated. As revealed in the Supporting Information,
Figure S13, the temperature increases up to 15.98C under
continuous irradiation by 660 nm laser for 10 min at
0.6 WcmÀ2, which meets the requirement for cancer cell
damage.[22] Thus, this laser dose is employed for the following
experiments. Temperatures elevation of the nanoassemblies
dispersions quickly rise and finally reach to 7.7 to 15.98C with
the concentrations from 50 to 200 mgmLÀ1, whereas the
temperature of water as control increases only 0.38C (Fig-
ure 3a). Furthermore, the photothermal conversion efficiency
(h) of Cu/CC NPs is calculated to be 45.7% based on the
reported method and acquired data in the Supporting
Information, Figure S14.[23] These results demonstrate excel-
lent photothermal property of Cu/CC NPs and which can be
applied as the PTT agent. Noteworthily, in contrast with the
mixture of Cu/CC NPs and TA (COH indicator) without laser
irradiation, the probe FL intensity significantly increased
after laser irradiation (Figure 3b; Supporting Information,
Figure S15), indicating the thermally amplified effect of Cu/
CC NPs-induced production of COH. According to our
previous study, photosensitization function of Ce6 (that is,
1
light-triggered O2 generation) is preserved even after modi-
fication onto CDs.[6c] The photosensitization properties of the
Cu/CC NPs are subsequently evaluated using singlet oxygen
sensor green (SOSG) as the FL probe. As shown in Figure 3c,
stronger FL emissions from SOSG in the Cu/CC NPs
dispersion are observed in the presence of GSH than that of
without GSH under the laser irradiation (660 nm,
0.6 WcmÀ2), indicating more 1O2 being produced in the
former case. The higher photosensitization performance of
Cu/CC NPs in the presence of GSH might be attributed to
their disassembly and lower quenched ability of Cu+ that
caused by GSH. This result is interesting because which
Figure 2. a) FL recovery of Cu/CC NPs with increasing addition of
GSH at CDs channel (lex =550 nm) in PB buffer (pH 6.3). b) GSSG/
GSH ratios of GSH solution after incubation with PB buffer (blank),
CDs-Ce6, and Cu/CC NPs, respectively. c) Possible reaction equations
of Cu2+ and H2O2 with the production of COH. d) FL emission spectra
of TA mixes with H2O2, Cu/CC NPs and both of them, respectively, and
FL spectrum of the mixture of H2O2 and Cu/CC NPs (lex =315 nm).
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means that the PDT function of Cu/CC NPs will keep in
a quenched state in neutral condition, consequently reducing
their phototoxicity. It is also found that the FL intensity of
SOSG in Cu/CC NPs dispersion is obviously increased in the
presence of GSH under laser irradiation, but the same
concentration of GSH induces certain SOSG FL decrease in
CDs-Ce6 dispersion (Figure 3d), demonstrating that the Cu/
CC NPs can play better PDT role than CDs-Ce6 in tumor
environment. It is worth noting that the GSH depletion
capacity of Cu/CC NPs benefit not only for effective
generation of 1O2, but also for COH via Fenton-like reactions,
and this would enhance the ROS-relevant therapeutic
efficacy. Taken together, the multiple stimuli-responsive
properties of Cu/CC NPs enable to achieve FL imaging
guided cancer synergistic CDT, PTT and PDT.
In Vitro FL Imaging and Synergistic Therapy
Figure 3. a) Temperature elevation of Cu/CC NPs dispersions with
various concentrations (from 50 to 200 mgmLÀ1) under the irradiation
of 660 nm laser at 0.6 WcmÀ2. b) FL intensity variation of TA mixes
with Cu/CC NPs at different concentrations with and without laser
irradiation (660 nm, 0.6 WcmÀ2, 5 min), in which F and F0 represent
probe intensity with and without laser irradiation. c) FL intensity of
SOSG in Cu/CC NPs solution in the presence and absence of GSH
with laser irradiation of 660 nm (0.6 WcmÀ2) at different time points
(lex =488 nm, lem =530 nm). d) FL emission spectra of SOSG in Cu/
CC NPs and CDs-Ce6 dispersions after incubation with or without
GSH and then exposed to laser irradiation (660 nm, 0.6 WcmÀ2) for
5 min.
Based on the unique TME stimuli-responsive properties
of Cu/CC NPs, their feasibility for FL imaging and synergistic
therapy is firstly investigated at cellular level. FL imaging of
Cu/CC NPs towards three types of carcinoma cell lines is
examined via confocal laser scanning microscope (CLSM).
Considering the more superior photostability of CDs than
Ce6, CDs channel is chosen for subsequent FL imaging. As
revealed in Figure 4a, 4T1 cells with bright red emission can
be distinguished easily, validating the efficient cell penetra-
Figure 4. a) CLSM images of 4T1 cells treated with Cu/CC NPs (50 mgmLÀ1) and Hoechst (5 mgmLÀ1) (CDs: lex =552 nm, lem =600–720 nm;
Hoechst: lex =405 nm, lem =420–480 nm). b) CLSM images of Cu/CC NPs treated 4T1 cells with or without pre-incubation with ALA, respectively,
and corresponding quantitative result of normalized mean FL intensity (lex =552 nm, lem =600–720 nm). c) Cell viability of MRC-5, A549 and 4T1
cells after treatment with various concentrations of Cu/CC NPs (from 0 to 200 mgmLÀ1) for 24 h. d) Calcein-AM/P staining images of 4T1 cells
after incubation with PBS (blank), H2O2, Cu/CC NPs and the mixture of Cu/CC NPs and H2O2, respectively. e) Cell viability of 4T1 cells treated
with various concentrations of Cu/CC NPs (from 0 to 200 mgmLÀ1) without and with laser irradiation at two different conditions (0.01 WcmÀ2 for
3 min and 0.6 WcmÀ2 for 10 min). f) CLSM images of 4T1 cells pre-incubated with DCFH-DA followed by treatments of PBS (blank), Cu/CC NPs
and Cu/CC NPs+laser, respectively, and corresponding quantitative result of normalized mean FL intensity (lex =488 nm, lem =508–550 nm). All
scale bars: 25 mm.
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tion and TME-stimuli responsive FL recovery of Cu/CC NPs.
therapeutic efficacy by the combination of PDT and PTT.
Hoechst is utilized for nucleus targeted staining, further
confirming the counterstain compatibility of the nanoassem-
blies. Similar imaging properties are also observed from the
CLSM images towards A549 and MCF-7 cells (Supporting
Information, Figure S16). Furtherly, GSH effects to FL
imaging from Cu/CC NPs is also studied. As presented in
Figure 4b, 4T1 cells pre-incubated with alpha lipoic acid
(ALA, a GSH promoter) display 1.8-fold enhanced FL
intensity than that of without treatment, which is in good
consistence with the FL recovery result caused by the addition
of GSH in Figure 2a, demonstrating GSH would improve the
FL imaging performance of the nanoassemblies in living cells.
For in vitro CDT, cell viability of carcinoma and normal
cells after being incubated with Cu/CC NPs is investigated via
cell counting kit-8 (CCK8) assay. The cytotoxicity of Cu/CC
NPs at various concentrations towards the normal cell line
(that is, MRC-5) and two kinds of cancer cell lines (A549 and
4T1) is displayed in Figure 4c. From which one can see that
the survival rate of MRC-5 cells still keeps more than 80%
even with the concentration of Cu/CC NPs reaching to
200 mgmLÀ1. By contrast, cell viabilities of A549 and 4T1 cells
drop quickly with the concentration of Cu/CC NPs increasing,
namely, 51% of A549 cells and 38% of 4T1 cells being
remained alive after treatment with Cu/CC NPs at
200 mgmLÀ1, indicating higher cytotoxicity of the nanoassem-
blies towards carcinoma cells than normal cells. This finding
can be mainly attributed to the promoted COH production via
Fenton-like reactions and GSH depletion under the specific
TME conditions. Moreover, negligible cytotoxicity of CDs-
Ce6 is observed to all the three kinds of cells (Supporting
Information, Figure S17), confirming the CDTefficacy of Cu/
CC NPs basically resulted from the contained Cu2+. Calcein-
AM/PI double stain experiments are performed to further
prove the specific CDTefficacy of Cu/CC NPs using 4T1 cells
as an example. As shown in Figure 4d, the distribution of live
(green) and dead (red) 4T1 cells in blank and Cu/CC NPs
treatment groups agrees well with the cytotoxicity results.
Compared with H2O2-only treated group exhibiting no
obvious cell death, great majority of cells that cultured with
the mixture of Cu/CC NPs and H2O2 are killed, verifying
H2O2 triggered CDT. As expected, the Calcein-AM/PI co-
stained MRC-5 cells exhibit that only a small number of dead
cells appear after the incubation with Cu/CC NPs (Supporting
Information, Figure S18).
Furthermore, cellular ROS level is further estimated using
2,7-dichlorofluorescin diacetate (DCFH-DA) as the FL
indicator. As illustrated in Figure 4 f, no significant FL is
observed in blank group, but cells cultured with 25 mgmLÀ1 of
Cu/CC NPs for 4 h display distinct green FL, indicating the
generation of COH. Notably, the FL emission of Cu/CC NPs
treated cells becomes much stronger under the laser exposure,
suggesting an effective elevation of ROS stress from the
combination of COH and 1O2. Quantitatively, average FL
intensity of the Cu/CC NPs treated group and the laser
irradiated Cu/CC NPs incubated group are about two and
four-fold higher than the blank group, respectively. Overall,
the Cu/CC NPs exhibit TME stimuli-responsive FL imaging
and synergistic CDT, PTT and PDT antitumor features
in vitro with favorable selectivity.
In Vivo Imaging and Antitumor Effect
Encouraged by the excellent TME-triggered FL imaging
and synergistic therapy performance of Cu/CC NPs at cellular
level, their imaging and antitumor effects in vivo are further
investigated. Firstly, blood compatibility of the Cu/CC NPs is
evaluated by hemolysis assay to determine their safety in
antitumor applications. As shown in the Supporting Informa-
tion, Figure S19, in contrast to the positive control group, no
obvious color of red blood cells is observed in different
concentrations of Cu/CC NPs, implying high hemocompati-
bility of the nanoassemblies. Then, in vivo bio-distribution of
Cu/CC NPs is measured using IVIS spectrum imaging system.
Xenograft tumor-bearing mice are sacrificed before and after
intravenously (i.v.) injection with Cu/CC NPs at various time
points for collection of major organs and tumors. As
presented in Figure 5a, intense FL signal is detected from
tumors at 2 h after injection and remains at a relatively high
level until the end of the observation (24 h), validating the
efficient accumulation of the nanoassemblies at tumor sites. It
is noteworthy that liver and kidneys also exhibit obvious FL
signal especially at 24 h post-injection, suggesting Cu/CC NPs
could be cleaned out of the mice body through both renal and
hepatic pathways. According to the bio-distribution results,
photothermal performance of Cu/CC NPs is then evaluated
via IR thermal camera at the optimal time point of i.v.
injection (that is, 8 h; Supporting Information, Figure S20),
and the corresponding temperature variation curves are also
recorded. As shown in Figure 5b, Cu/CC NPs injected mouse
display the maximal temperature elevation at tumor site from
33.1 to 47.88C upon the laser irradiation of 660 nm
(0.6 WcmÀ2) for 10 min, while the limit temperatures at
tumor sites of PBS and CDs-Ce6 injected groups are 40.8 and
46.18C, respectively. Moreover, tumor temperature of Cu/CC
NPs treated mouse is observed to elevate much faster than
that of the PBS and CD-Ce6 treated groups (Figure 5c),
indicating enhanced accumulation capability and PTT effi-
ciency of the nanoassemblies in vivo due to their relatively
larger particle size.
Thereafter, Cu/CC NPs-mediated phototherapy to 4T1
cells is studied (Figure 4e). To minimize CDTeffect, material
incubation time is shortened to 4 h. Since lower laser dose
causes weaker photothermal response of Cu/CC NPs, laser
power of 0.01 WcmÀ2 is taken for the evaluation of PDT
effect. As the concentration of Cu/CC NPs increasing, the cell
survival rates gradually decrease upon the laser irradiation
(660 nm, 0.01 WcmÀ2 for 3 min) and finally reach to 15%
with the concentration of 200 mgmLÀ1, demonstrating con-
siderable PDTefficiency. Higher laser dose (0.6 WcmÀ2) with
prolonged irradiation time (10 min) is employed to evaluate
the PDT combined PTT effects. Under such conditions, the
cell viability sharply decreases to about 20% even at a low
concentration of 25 mgmLÀ1, indicating remarkably enhanced
Motivated by the excellent tumor homing capacity, the
synergistic therapeutic efficiency of nanoassemblies is per-
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Research Articles
Chemie
on day 1 and 14 further validate the tumor volume variation
in a more intuitive way (Figure 5e). Notably, black scar
caused by tumor necrosis almost falls off from the mice in
trimodal synergistic therapy group. In addition, to clarify the
corresponding tumor damaging effect induced by various
treatments, tumors from different groups are excised for
hematoxylin and eosin (H&E) staining at the end of the
monitoring period. Although apparent cell apoptosis can be
found in PTT+ PDT group, the highest tumor cell necrosis
ratio appears in CDT+ PTT+ PDT group. In vivo toxicity of
Cu/CC NPs is preliminarily evaluated via the mice body
weight change during the whole observation period. As
illustrated in the Supporting Information, Figure S21, al-
though mice body weight in CDT+ PTT+ PDT group slightly
decreased for the first few days, it gradually back to the
normal level in late observation period. Finally, major organs
of mice are gathered for H&E staining at 14 days post
treatment (Supporting Information, Figure S22). Neither
obvious inflammation nor injury is found in CDT+ PTT+
PDT group in comparison with that of blank group, further
indicating low toxicity of Cu/CC NPs in vivo. All these results
demonstrate the great potentials of Cu/CC NPs to serve as
a promising synergistic antitumor agent with negligible
adverse effects.
Figure 5. a) FL images of major organs (H: heart, Li: liver, S: spleen,
Lu: lung, and K: kidneys) and tumors (T) excised from mice before
and after i.v. injection with Cu/CC NPs at various time points.
b) Thermal graphs of tumor-bearing mice expose to laser irradiation
for 10 min after i.v. injection of PBS, CDs-Ce6, and Cu/CC NPs,
respectively, and, c) corresponding temperature variation curves.
d) Relative tumor volume variation of five treatment groups during the
monitoring period (*p<0.05 and **p<0.01, Student’s t test). e) Dig-
ital photographs of representative mice from different treatment
groups on days 1 and 14, and H&E staining of tumor tissues gathered
from representative mice at 14 days (scale bar: 100 mm).
Conclusion
An intelligent anticancer theranostic platform is fabricat-
ed via facile assembling of CDs-Ce6 and Cu2+ in this study,
which exhibit remarkable features of TME stimuli-responsive
FL imaging and synergistic treatment by PTT, PDT, and heat-
reinforced CDT. Significantly, the introducing of Cu2+ not
only provides CDT function (producing COH through reaction
with H2O2), but also effectively depletes GSH in tumors,
resulting in amplified intracellular oxidative stress and
enhanced ROS-relevant therapeutic efficiency. The activation
of FL imaging and ROS-involved therapy are observed
in vitro with enhanced therapeutic efficacy only towards
carcinoma cells. The in vivo results confirm improved accu-
mulation capability of Cu/CC NPs at tumor sites and superior
antitumor effect with insignificant systemic toxicity. This
study on one hand proposes a strategy to solve the problem of
small size limitation of CDs for their applications in cancer
biomedicine, and on the other hand develops a FL imaging
guided cancer synergistic treatment agent that could effec-
tively enhance therapeutic efficacy with minimized side
effects by fully taking advantage of the TME.
formed. Tumor-bearing mice are randomly divided into five
groups and implemented the following different treatments:
1) blank group (PBS + laser), 2) control group (CDs-Ce6),
3) CDT group (Cu/CC NPs without laser irradiation),
4) PTT+ PDT group (CDs-Ce6 + laser), and 5) CDT+
PTT+ PDT group (Cu/CC NPs + laser). All of the materials
are i.v. injected via the mouse tail vein with the concentration
of 2 mgmLÀ1 (100 mL). After the implement of five group
treatments, tumor volume and mouse body weight are
recorded regularly. Figure 5d presents the relative tumor
volume variation trend of different groups. In contrast with
blank group, negligible influence on tumor growth is observed
in control group that only injection with CDs-Ce6, demon-
strating almost no antitumor efficacy. Meanwhile, the mice of
CDT group exhibit observable tumor inhibition owing to the
CDTeffect of Cu/CC NPs. After exposure to laser irradiation,
mice in PTT+ PDT group displays significant inhibition to
tumor growth with the average relative tumor volume even
slightly shrinking (about 89% of the original volume),
implying considerable antitumor effect of PTT combined
PDT. As expected, maximum tumor inhibition is found in
CDT+ PTT+ PDT group with tumor volume remarkably
reduced to about 37% of the initial value, verifying the
highest efficacy by the trimodal therapy. Representative
photographs of mice administered with different treatments
Acknowledgements
The authors acknowledge the financial support from the
National Natural Science Foundation of China (51902323,
51872300 and U1832110), S&T Innovation 2025 Major
Special Programme of Ningbo (2018B10054), and the Zhe-
jiang Provincial Natural Science Foundation of China (Grant
No. LY20B050003).
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L. Jin, S. Zhang, Y. Zhao, S. Song, Y. Yang, H. Zhang, Adv.
Conflict of interest
Funct. Mater. 2019, 29, 1904678; e) L. Liu, D. Jiang, A.
McDonald, Y. Hao, G. L. Millhauser, F. Zhou, J. Am. Chem.
Soc. 2011, 133, 12229; f) X. Peng, Q. Pan, B. Zhang, S. Wan, S. Li,
K. Luo, Y. Pu, B. He, Biomacromolecules 2019, 20, 2372 – 2383;
g) Z. Wang, B. Liu, Q. Sun, S. Dong, Y. Kuang, Y. Dong, F. He, S.
Gai, P. Yang, ACS Appl. Mater. Interfaces 2020, 12, 17254 –
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Xing, T. Xu, H. Dong, X. Zhang, ACS Nano 2019, 13, 4267 –
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The authors declare no conflict of interest.
Keywords: carbon dots · nanoassemblies · stimulus–response ·
synergistic cancer treatment · tumor microenvironment
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Manuscript received: June 4, 2020
Revised manuscript received: July 22, 2020
Version of record online: && &&
,
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&&&&
These are not the final page numbers!

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Research Articles
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Research Articles
Nanoassemblies
A tumor microenvironment (TME) stim-
uli-responsive fluorescence imaging and
trimodal synergistic cancer treatment
nanoplatform is facilely constructed via
assembling carbon dots and Cu2+. This
could act as a potential strategy to
S. Sun, Q. Chen, Z. Tang, C. Liu, Z. Li,
A. Wu, H. Lin*
&&&& — &&&&
Tumor Microenvironment Stimuli-
Responsive Fluorescence Imaging and
Synergistic Cancer Therapy by Carbon-
Dot–Cu2+ Nanoassemblies
achieve enhanced cancer therapeutic
efficiency with minimized side effects.
&&&&
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Angew. Chem. Int. Ed. 2020, 59, 2 – 10
These are not the final page numbers!