ROS-responsive cyclodextrin nanoplatform for combined photodynamic therapy and chemotherapy of cancer

Article abstract of DOI:10.1016/j.cclet.2020.11.052

Cyclodextrin (CD) has special spatial structure and well biological safety, so it has been widely used for constructing CD-based nanoplatforms. Through functionalization, cyclodextrin can form various stimulus-response nanoplatforms, such as pH, temperatu

Full text of DOI:10.1016/j.cclet.2020.11.052

Chinese Chemical Letters 32 (2021) 162–167
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
ROS-responsive cyclodextrin nanoplatform for combined
photodynamic therapy and chemotherapy of cancer
Die Jiaa,b,1, Xianbin Maa,b,1, Yi Lua,b, Xinyi Lia,b, Shengxin Houa,b, Yuan Gaoa,b
,
Peng Xuea,b, Yuejun Kanga,b, Zhigang Xua,b,
*
a
State Key Laboratory of Silkworm Genome Biology, School of Materials and Energy, Southwest University, Chongqing 400715, China
b
Chongqing Engineering Research Center for Micro-Nano Biomedical Materials and Devices, Southwest University, Chongqing 400715, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Cyclodextrin (CD) has special spatial structure and well biological safety, so it has been widely used for
constructing CD-based nanoplatforms. Through functionalization, cyclodextrin can form various stimulus-
response nanoplatforms, such as pH, temperature, redox, light and magnetic fields. In this study, we
designed a highly sensitive reactive oxygen species (ROS)-responsive polymer PCP which encapsulated
doxorubicin (DOX) and purpurin 18 (P18) to achieve the synergy of photodynamic and chemotherapy. The
highcontentofreactiveoxygen species(ROS)inthetumor microenvironment(TME)triggers thecleavageof
theboratebondofMPEG-CD-PHB(PCP),therebypromotingthereleaseofdrugs. Whenirradiatedwithnear-
infrared laser, the photosensitizer P18 released by polymer micelles can produce reactive oxygen species to
promote cell apoptosis. Compared with monotherapy, a series of experiments confirmed that our micelles
had enhanced anti-cancer activity. This work was beneficial to the design of ROS-responsive materials and
provides an effective strategy for the application of collaborative anti-tumor therapy.
Received 14 September 2020
Received in revised form 25 November 2020
Accepted 25 November 2020
Available online 1 December 2020
Keywords:
Chemotherapy
Photodynamic therapy
Synergistic therapy
Cyclodextrins
ROS-responsive
© 2020 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
In recent years, more and more researchers have paid attention
to the use of nanoparticles as drug carriers to construct drug
delivery systems [1,2]. Targeted therapy of nanomedicine was
considered to be a promising strategy [3–5]. By constructing
intelligent targeted drugs, therapeutic and imaging drugs can be
specifically delivered to the tumor site. However, there were still
some shortcomings in the treatment of tumors, such as poor drug
targeting, low drug delivery efficiency and weak drug penetration
in tissues [6–8]. Studies had found that the tumor microenviron-
ment has the characteristics of low oxygen, low pH, high reactive
oxygen species (ROS) levels and high expression of enzymes [9–
12]. Using these characteristics of the tumor microenvironment as
stimulating factors, designing a sensitive nano-drug system can
improve the therapeutic effect of anti-tumor drugs [13–19].
Cyclodextrin (CD) had been widely used to construct drug
delivery systems due to harmlessness and excellent biocompati-
bility [20,21]. Cyclodextrin had a special spatial structure, so it can
form inclusion compounds with a variety of drugs, and further
change the physical, chemical and biological properties of the
drugs. For example, increased the solubility of hydrophobic drugs,
improved the stability and bioavailability of the drugs, and reduced
the toxicity and side effects of the drugs. At present, many
stimulus-responsive nanomedicines have been designed using
cyclodextrin, such as pH [22], temperature [23], redox [24–26] and
enzymes, etc. [27,28]. But based on the characteristics of high
reactive oxygen species (ROS) level, there were relatively few
reports on cyclodextrin nanomedicine systems that respond to ROS
[29]. Combined the cyclodextrin inclusion technology with the
nano-drug delivery technology can not only utilize the unique
inclusion principle of cyclodextrin, but also improve the solubility,
stability and absorption of the drug, and reduce the stimulating
effect of the drug. At the same time, nano prodrugs can further
make drugs working synergistically to achieve sustained release,
targeting and stability.
In this study, a ROS responsive CD-based micelles was proposed
to deliver anti-tumor drugs (Fig. 1A). Mono(6-amino-6-deoxy)-β-
cyclodextrin and hydrophilic polymPEG-NHS are first used for
polymerization to generate MPEG-CD. Then MPEG-CD reacted
with the compound PHB-CDI which contained borate bond to
generate ROS responsive MPEG-CD-PHB (PCP) for wrap doxorubi-
cin (DOX) and purpurin-18 (P18). Due to the high H2O2
concentration in the tumor microenvironment, when the drug
* Corresponding author at: State Key Laboratory of Silkworm Genome Biology,
School of Materials and Energy, Southwest University, Chongqing 400715, China.
E-mail address: zgxu@swu.edu.cn (Z. Xu).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.cclet.2020.11.052
1001-8417/© 2020 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.

D. Jia, X. Ma, Y. Lu et al.
Chinese Chemical Letters 32 (2021) 162–167
Fig. 1. (A) The synthesis route of β-CD-derived H2O2 responsive material (detailed chemical structure is shown in Supporting information), the self-assembly process of
nanoparticles and their photo-activated combined treatment process. (B) TEM image and DLS size distribution histogram of PDP micelles. (C) Mean diameter distribution of
PDP micelles under different incubation times. (D) H2O2 triggered DOX release of PDP micelles in PBS solution and 100 mmol/L of H2O2 solution. Data are presented as
mean Æ SD (n = 3).
entered the tumor microenvironment, DOX and P18 will be
released from the PDP micelles. After the PDP micelles entered the
tumor cell via enhanced permeability and retention (EPR) effect,
the loaded DOX and P18 are released under the excitation of high
concentration of ROS in tumor microenvironment (TME), which
can produce cytotoxic ROS under NIR laser, thus achieved chemo-
photodynamic therapy. Systematic experiments demonstrated
that this PDP micells exhibited excellent anti-tumor efficacy and
negligible systemic toxicity. Compared with other reported redox
drug delivery systems [30,31], our nanocarrier had the following
advantages: excellent stability, strong biocompatibility, stimulus-
responsive release, fast phagocytosis, and effective tumor perme-
ability, outstanding biological safety and excellent anti-cancer
effect.
The micelles size and morphology of PCP micelles were
measured by Transmission electron microscope (TEM) and
dynamic light scattering (DLS), respectively. As shown in Fig. 1B,
the average particle size of the PDP micelles was 148.4 nm. PDP
micelles showed a nearly spherical morphology (TEM image),
which might be due to the expansion of micelles in water. The
corresponding zeta potentials of free DOX, free P18 and PDP were
13.2 Æ 1.0, À11.5 Æ 0.3 and À7.5 Æ 7.5 mV, respectively (Fig. S5 in
Supporting information). Then the hydrated particle size of the
micelles was tested every two days, and there was no significant
change in micelles particle size within 13 days (Fig. 1C). Under the
effect of increasing permeability and retention rate (EPR), micelles
had obvious advantages in extending the blood circulation time
and increasing the permeability of drugs in tumor tissues [32,33].
The UV–vis spectrum was used to measure the encapsulation of
drugs, and the UV–vis spectrum of PDP micelles showed all typical
absorption peaks of DOX, P18 and PDP micelles (Fig. S6 in
Supporting information). The fluorescence spectrum of PDP
showed the characteristic emission peaks of DOX and P18, which
meant that DOX and P18 had been successfully encapsulated
(Fig. S7 in Supporting information). Similarly, the DLC of DOX and
P18 were calculated to be 10.8% and 11.9%, respectively.
PCP was synthesized and characterized by the following
steps. Firstly, as shown in Figs. S1-S4 (Supporting information),
which show the 1H NMR results of MPEG-CD, PHB-CDI and PCP,
respectively. As shown in Fig. S2, 1H NMR of PEG-CD in DMSO-d6
showed the hydroxyl signal c (5.71 ppm), d (5.66 ppm) from β-CD
and the methyl signal a (3.38 ppm) and methylene signal b
(3.65 ppm) of MPEG-NHS, indicating the successful synthesis of
MPEG-CD. Secondly, PHB reacts with N,N-carbonyldiimidazole
(CDI) to form PHB-CDI, and the 1H NMR result showed in Figs. S3
and S4 indicated that the final product PCP had been successfully
synthesized.
Compared with normal cells, the high level of H2O2 in cancer
cells can decompose the material and accelerate the release of
drugs [34,35]. The in vitro drug release behavior of PDP micelles
was tested by dialysis. The release rate of DOX from the PDP NP was
measured by dialysis in different concentration of H2O2, which was
used to simulate the reducing and oxidizing conditions in TME. As
shown in Fig. 1D, when the concentration of H2O2 was 0 mmol/L,
DOX is rarely released, which was related to the stability of PDP NP
in physiological environment. However, when the H2O2 concen-
tration increased to 100
mmol/L, more than 90% of the drug is
released. This result showed that in the presence of H2O2, the
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D. Jia, X. Ma, Y. Lu et al.
Chinese Chemical Letters 32 (2021) 162–167
rupture of the borate bond in PDP micelles could accelerate the
release of P18 and DOX.
efficiency of PDP micelles was time-dependent. Fig. 2B showed
that the DOX and P18 fluorescence intensity of PDP micelles
gradually increased over time. At the same time, the cell uptake
rate of PDP micelles was measured by flow cytometry (FCM).
Initially, 4T1 cells were seeded in a 24-well culture dish at a density
of 5 Â104 per well, and cultured overnight at 37 ꢁC in 5% CO2. The
complete DMEM with 0.4 mL PDP micelles (DOX concentration is
The reactive oxygen species (ROS) produced by photosensitizer
under laser irradiation can oxide the phospholipids on the cell
membrane and DNA in the nucleus [36]. Singlet oxygen (1O2) is the
main component of ROS, which can destroy targeted cancer cells in
PDT [37]. DPBF can be rapidly oxidized to phthaloylbenzene under
the presence of 1O2. Therefore, the generation of singlet oxygen can
be judged according to the change in absorbance of DPBF probe at
415 nm [38]. As shown in Fig. S8 (Supporting information), the
absorbance of DPBF probe gradually decreased within 0–6 min, it
indicated that the PDP micelles could generate 1O2 under laser
irradiation [39]. As the irradiation time increases, the absorbance
gradually decreases, which indicated that PDP micelles can
produce a large amount of 1O2 to cause DPBF decomposition.
To further investigate the compatibility of PDP micelles, we
conducted a blood hemolysis assay to determine the damage to red
blood cells. As shown in Fig. S9 (Supporting information), the
supernatants of the negative control group and the drug treatment
group were transparent, the status of PDP micelles was almost
consisted with the PBS group and the supernatants was transpar-
8
mg/mL) were added, and were incubated at a constant
temperature. 4T1 cells were cultured with PDP micelles for 1 h,
2 h and 4 h. After reaching the time point, the cells were washed
with PBS, and then digested with trypsin, and DMEM terminated
the digestion. PBS containing calcium and magnesium was added
and collected for FCM analysis, the cell uptake rate of PDP micelles
reached 96.87% after 4 h. (Fig. 2C). It could be seen that the
phagocytosis rate increased with the increase of culture time. It
showed that micelles could be swallowed quickly.
In order to study the anti-cancer properties of PDP micelles, we
used 4T1 cells to evaluate in vitro cytotoxicity. Free DOX, Free P18
and PDP micelles were added to 4T1 cells respectively. The groups
(Free P18 and PDP micelles) were treated with laser irradiation
after incubated with 4T1 cells for 4 h, and the cells without
treatment were set as controls. All experimental groups showed a
concentration-dependent ability to induce cancer cell death and
PDP with laser group showed more toxic than other groups. For
ent. When the concentration was 250 mg/mL, the hemolysis rate
was still less than ꢀ3%. These results indicate that the PDP micelle
had good blood compatibility in blood circulation.
The cell uptake behavior was performed by confocal micro-
scope. Using 4T1 cells, after incubating with PDP micelles (DOX
example, different pharmaceutical formulations containing 5
mg/
mL DOX, 8.7 g/mL P18, free DOX, PDP, P18 + laser, PDP + laser
m
concentration of 15
m
g/mL) for 1 h and 4 h, the nucleus were
interacted with 4T1 cells for 4 h, and then were irradiated with
660 nm (0.5 W/cm2) laser for 5 min. The cell survival rates of free
DOX, PDP, P18 + laser, and PDP + laser were about 40.89%, 33.74%,
61.28% and 17.17%, respectively. It could be seen from the
stained with DAPI. It could be seen from Fig. 2A that the released
DOX had entered the nucleus. The released P18 molecules
remained in the cytoplasm. It showed that the cell uptake
Fig. 2. Cellular uptake, cytotoxicity and ROS generation of PDP micelles in vitro. (A) Confocal images and (B) average fluorescence intensity of 4T1 cells incubated with PDP
micelles for 1 h and 4 h. Scale bar: 50 m. (C) Flow cytometry analysis of the cellular uptake of DOX-loaded PDP micelles for 1 h, 2 h and 4 h, respectively. (D) Cell viabilities of
m
4T1 cells treated with different concentrations of Free P18 + laser (P18+L), PDP + laser (PDP+L), PDP, and Free DOX for 24 h under 660 nm laser irradiation (0.5 W/cm, 5 min). (E)
Quantification of ROS produced in cells. Data are presented as mean Æ SD (n = 3).
164

D. Jia, X. Ma, Y. Lu et al.
Chinese Chemical Letters 32 (2021) 162–167
experimental results that when the drug concentration was low,
the tumor cell survival rate was 17.17%, indicating that the
combination of PDT and chemotherapy had significant tumor
ablation effect (Fig. 2D).
the ability of micelles to penetrate solid tumor tissue. As shown in
Fig. 3A, after incubateing with 4T1 multicellular spheres for 6 h, the
red fluorescence of P18 and green fluorescence of DOX could be
detected at different depths by scanning confocal images. Fig. 3B
showed the average fluorescence values of DOX and P18 at
different depths of tumor spheres, which indicated that multi-drug
micelles had good permeability to solid tumors. Fig. 3C showed the
fluorescence signal intensity distribution of DOX and P18 channels
Because of its highly selective 1O2, the singlet oxygen (SOSG)
fluorescent probe was applied to assess ROS level in cells. In the
presence of singlet oxygen, the probe could emit green fluores-
cence similar to fluorescein. As shown in Fig. 2E and Fig. S10
(Supporting information), after 4 h of incubation with DOX, P18
and PDP drugs, P18 and PDP were given a certain amount of light
(660 nm, 0.5 W/cm2, 5 min). It could be seen that the PDP plus laser
treatment group green fluorescence was the strongest, while green
fluorescence in other controls was much weaker.
at a depth of 40 mm.
The distribution of PDP micelles were measured by fluores-
cence imaging on the 4T1 tumor-bearing mice [40]. PDP and free
P18 were injected through the tail vein of mice at the same dose of
P18, and whole-body fluorescence imaging was performed 24 h
later, as shown in Fig. 3D. It could be seen from the imaging
diagram that 24 h after the injection of the drug, the mice in the
PDP group presented obvious fluorescence, indicating that PDP
micelles could be enriched in the tumor lesion. Fig. 3E and Fig. S13
(Supporting information) showed the quantification of the
separated tumor and major organs (heart, liver, spleen, lung and
kidney). It could be seen from this figure that PDP micelles could
effectively accumulate than free P18 through prolong blood
circulation at the tumor site.
In addition, the in vivo biocompatibility test of free P18, free
DOX and PDP micelles was carried out on healthy Kunming mice.
Typical blood indicators including WBC, LYM, RBC, RDW, HCT,
MCHC, HGB, MPV and PLT were tested. The control groups were
injected with normal saline and the biochemical parameters of the
mice were tested after 7 days of treatment with PDP micelles.
Compared with the PBS group, the parameters of the PDP
The cellular internalization pathways of PDP micelles were also
conducted by staining lysosome and mitochondria. As shown in
Figs. S11 and S12 (Supporting information), lysosomes were
stained with Lyso-Tracker Blue, and mitochondria were stained
with Mito-Tracker Orange. After 1 h and 4 h of drug incubation, the
fluorescence intensity of DOX and P18 increased slightly with the
increase of time, indicateing that the time-dependent process of
PDP micelles entering the cells, which was consisted with our
above FCM results. Fig. S11B showed the average fluorescence
intensity of DOX and P18 at different times in the mitochondrial
colocalization experiment. Fig. S12B showed the average fluores-
cence intensity of DOX and P18 at different times in the lysosomal
colocalization experiment.
The efficiency of anti-cancer micelles were affected by complex
tumor microenvironment in solid tumor. To further study this
feature, we established a multicellular sphere of 4T1 cells to study
Fig. 3. (A) Confocal microscopy imaging of PDP micelles penetrating the 4T1 tumor spheres for 6 h. Scale bar: 100
different depths in the tumor sphere. (C) Corresponding to the fluorescence signal intensity distribution of the DOX and P18 channels of the tumor sphere when the depth is
40 m. (D) Ex vivo fluorescence imaging of PDP micelles distribution in tumor, 4T1 tumor-bearing BALB/c mice after injection with free P18, PDP micelles (P18 concentration:
mm. (B) The average fluorescence value of DOX and P18 at
m
5 mg/kg) via tail veins. (E) Quantitative comparison of P18 signal intensity in different organs. Data are presented as mean Æ SD (n = 3).
165

D. Jia, X. Ma, Y. Lu et al.
Chinese Chemical Letters 32 (2021) 162–167
treatment group were kept within a reasonable range (Fig. S14 in
Supporting information). It showed that the designed micelles had
good blood compatibility and had no obvious negative effects on
the blood chemistry of mice. Simultaneously, the body weights of
the mice were recorded every day (Fig. S15 in Supporting
information). The weight of the mice did not fluctuate significantly,
indicating that the drug had good biological safety.
tumor tissue photos also verified the experimental results. Fig. S17
(Supporting information). Figs. 4E and F showed the quantification
of TUNEL and Ki67 images using Image J. It could be observed that
the PDP-treated tumors showed higher apoptosis. After treatment,
no obvious pathological abnormality was found in the H&E sites of
the heart, liver, spleen, lung and kidney (Fig. S18 in Supporting
information). All about these results proved that the prepared PDP
micelles had excellent biosafety in vivo.
Benefiting from excellent cytotoxicity in vitro and effective
accumulation of PDP micelles in tumor site, we further investigat-
ed the anti-tumor activity on 4T1 tumor-bearing mice. The
schematic diagram of anti-tumor experiments was shown in
Fig. 4A. Typically, when the tumor volume reached approximately
100 mm3, all of the mice were randomly divided into five groups:
(1) saline, (2) free DOX, (3) free P18 + laser, (4) PDP, (5) PDP + laser.
The tumor volumes were recoded every day. The drug was injected
every three days for a total of three injections, and the tumors were
removed from the mice sacrificed on the 14th day for further
analysis. As shown in Fig. 4B, it could be observed that the mouse
tumor volume in the saline group grew rapidly compared with
other groups. On the contrary, the PDP micelle group showed
significant tumor suppressor effect. A similar trend of results was
observed in the tumor weight in Fig. 4C. Fig. S16 (Supporting
information) showed the change in body weight during the
treatment, and the body weight of mice showed no significantly
fluctuate. In addition, as shown in Fig. 4D, hematoxylin and eosin
(H&E) and TdT-mediated dUTP nick-end labeling (TUNEL) stained
In short, we proposed a ROS-responsive biodegradable nano-
material PCP, which had the advantages of stimulating response,
synergistic treatment, low toxicity and good biocompatibility. By
encapsulating the photosensitizer P18 and the chemotherapeutic
drug DOX in the H2O2-responsive material PCP, a successful anti-
tumor therapy was obtained. Cell uptake and MTT experiments
were used to study the anti-cancer efficacy in vitro. Based on these
results, this work helped to provide effective strategies for the
application of synergistic anti-tumor therapy.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
This work was financially supported by the National Natural
Science Foundation of China (Nos. 51703187, 31671037) and the
Basic and Frontier Research Project of Chongqing (No. cstc2018jcy-
jAX0104).
Appendix A. Supplementary data
Supplementarymaterialrelatedtothisarticlecanbefound, inthe
online version, at doi:https://doi.org/10.1016/j.cclet.2020.11.052.
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