ROS-Responsive Camptothecin Prodrug Nanoparticles for On-Demand Drug Release and Combination of Chemotherapy and Photodynamic Therapy

Article abstract of DOI:10.1002/adfm.202005918

Minimizing drug leakage in systemic circulation, synchronizing the in vivo fate of multiple drugs, and precisely controlling tumor locoregional drug release, remain challenging for nanomedicine-based cancer therapy. Here, a reactive oxygen species (ROS)-responsive camptothecin (CPT) prodrug delivery system (MPEG-(TK-CPT)-PPa) is developed, in which CPT and photosensitizer pyropheophorbide-a (PPa) are concurrently conjugated to the same poly(ethylene glycol) methyl ether (MPEG) via ROS-responsive thioketal (TK) and lipid linkage. The synthesized MPEG-(TK-CPT)-PPa conjugate self-assembles to form nanoparticles (NPs) (43.6 ± 0.8 nm) in solution. The covalently conjugated prodrug prevents drug leakage during systemic circulation and synchronizes the in vivo distribution of the two drugs. The generated fluorescence signal of PPa helps precisely track and locate the NPs at tumor sites. Under the guidance of imaging, a near-infrared laser locally irradiates tumor tissue upon reaching the strongest fluorescence. The ROS generated by PPa not only cleaves the TK linkage and then triggers locoregional, controllable and on-demand CPT release, but also exhibits cytotoxic effects on tumor cells. Thus, CPT-mediated chemotherapy and PPa-induced photodynamic therapy lead to the combined and enhanced suppression of tumor growth. Accordingly, such laser-triggered, localized, controllable, and on-demand drug release systems may provide an alternative option for CPT formulations.

Full text of DOI:10.1002/adfm.202005918

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ROS-Responsive Camptothecin Prodrug Nanoparticles
for On-Demand Drug Release and Combination of
Chemotherapy and Photodynamic Therapy
Bingyang Chu, Ying Qu, Xinlong He, Ying Hao, Chengli Yang, Yun Yang, Danrong Hu,
Fangfang Wang, and Zhiyong Qian*
1. Introduction
Minimizing drug leakage in systemic circulation, synchronizing the in vivo
fate of multiple drugs, and precisely controlling tumor locoregional drug
Although many methods, such as sur-
gery, radiotherapy, chemotherapy, gene
release, remain challenging for nanomedicine-based cancer therapy. Here,
therapy and immunotherapy have been
a reactive oxygen species (ROS)-responsive camptothecin (CPT) prodrug
developed and applied for cancer therapy,
delivery system (MPEG-(TK-CPT)-PPa) is developed, in which CPT and
photosensitizer pyropheophorbide-a (PPa) are concurrently conjugated to
the same poly(ethylene glycol) methyl ether (MPEG) via ROS-responsive
thioketal (TK) and lipid linkage. The synthesized MPEG-(TK-CPT)-PPa
conjugate self-assembles to form nanoparticles (NPs) (43.6 0.8 nm) in
solution. The covalently conjugated prodrug prevents drug leakage during
systemic circulation and synchronizes the in vivo distribution of the two
drugs. The generated fluorescence signal of PPa helps precisely track and
locate the NPs at tumor sites. Under the guidance of imaging, a near-infrared
laser locally irradiates tumor tissue upon reaching the strongest fluorescence.
The ROS generated by PPa not only cleaves the TK linkage and then triggers
locoregional, controllable and on-demand CPT release, but also exhibits
cytotoxic effects on tumor cells. Thus, CPT-mediated chemotherapy and
PPa-induced photodynamic therapy lead to the combined and enhanced
suppression of tumor growth. Accordingly, such laser-triggered, localized,
controllable, and on-demand drug release systems may provide an alternative
option for CPT formulations.
cancer remains a global problem health
issue.[1] In the clinic, chemotherapy is still
one of the most commonly used method
to treat cancer.[2] However, some may
cause serious side effects. One reason
is that most chemotherapeutic agents
need to use toxic excipients to improve
their poor water solubility for intrave-
nous administration. Another reason is
that the formulations may lack tumor
targeting properties, making it unable to
distinguish tumor tissues from healthy
ones.[3] New technologies have been uti-
lized to try to reduce these side effects,
improve safety, and enhance treatment
efficacy, but it has remained challenging
to develop ideal formulations of chemo-
therapeutic agents.
Camptothecin (CPT) is an important
chemotherapeutic agent, even though
no clinical formulation has been used.
It inhibits tumor growth via inhibiting
topoisomerase I, leading to the inhibi-
tion of DNA synthesis and apoptosis of cancer cells.[4] How-
ever, the poor water solubility and instability of the lactone
ring have limited its clinical application.[5] Great efforts
have been taken to resolve this issue to better meet clinical
needs better.[6] Nanoparticle (NP) formulation has garnered
great attention, as it can improve water solubility and sta-
bility without using toxic excipients.[4b] Physical encapsula-
tion using nanocarriers and chemical conjugation are two
major methods used for NP formulation. However, for con-
ventional physical encapsulation, premature drug release or
inevitable drug leakage typically occur during systemic cir-
culation, which can cause side effects.[7] For chemical conju-
gation, drugs are covalently conjugated to the nanocarriers
in prodrug form via some biodegradable linkages, which can
keep it stable and prevent drug leakage in systemic circula-
tion. Thus, these chemically conjugated prodrug formula-
tions have gained extensive attention.[6b,8]
Dr. B. Chu, X. He, Dr. Y. Hao, Dr. C. Yang, Dr. Y. Yang. Dr. D. Hu,
Prof. Z. Qian
State Key Laboratory of Biotherapy and Cancer Center
West China Hospital
Sichuan University
Collaborative Innovation Center for Biotherapy
Chengdu, Sichuan 610041, P. R. China
E-mail: zhiyongqian@scu.edu.cn
Dr. Y. Qu, F. Wang
Department of Hematology and Research Laboratory of Hematology
State Key Laboratory of Biotherapy
West China Hospital
Sichuan University
Collaborative Innovation Center for Biotherapy
Chengdu, Sichuan 610041, P. R. China
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adfm.202005918.
DOI: 10.1002/adfm.202005918
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Figure 1. Schematic illustration of the MPEG-(TK-CPT)-PPa NPs for photo-triggered ROS-activatable CPT prodrug release and local tumor therapy.
Based on the tumor microenvironment, various sensitive
prodrug delivery systems have been developed to achieve
tumor-specific drug release using the abovementioned strat-
egies for chemically conjugating prodrugs.[9] Among these,
pH,[10] redox,[11] and enzyme[12] sensitive prodrug delivery
systems have been widely explored based on the properties
of lower pH value, higher glutathione concentration, and
higher expression of matrix metalloproteinase in tumor tis-
sues than in normal tissues. These prodrug systems display
better biosafety and lower toxicity than conventional sys-
tems. However, the potential risk of off-target effects still
exists, because these intrinsic signals are not wholly owned
by the tumor, which are just higher or lower than those in
normal tissues.[13] With the progression of precision medi-
cine, prodrug system with higher tumor-specific is urgently
needed.
Recently, reactive oxygen species (ROS)-sensitive prodrug
delivery systems have been developed.[14] The thioketal (TK)
linkage is one important ROS cleavable linkage, but the intrinsic
ROS concentration in the body is too low to cleave it.[13] Photo-
sensitizer irradiated by laser can efficiently generate ROS to
cleave the TK linkage, which can markedly increase ROS levels,
resulting in significant damage to cells, a phenomenon known
as photodynamic therapy.[15] Utilizing this property, the ROS-
sensitive prodrug delivery system based on a photosensitizer
and TK linkage could achieve locoregional controllable intra-
tumoral drug release under local laser irradiation. Pei et al.
developed chlorin e6-loaded TK-linked polyphosphoester-
doxorubicin polymeric prodrug NPs.[14a] Yue et al. prepared zinc
phthalocyanine-loaded TK-linked CPT-polyethylene glycol con-
jugate and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-
N-[methoxy (polyethylene glycol)] hybrid NPs.[16] However, in
these reported ROS-sensitive systems, one common drawback
is that the photosensitizers are physically encapsulated in
prodrug nanocarriers, which inevitably leads to leakage during
blood circulation as that of the chemotherapeutic agents’
delivery via physical encapsulation.[14a,17] Photosensitizers may
induce severe phototoxicity to normal tissues.[18] Moreover, the
therapeutic efficacy may be reduced by the inadequate ROS
generation due to photosensitizer leakage. Thus, careful atten-
tions should be paid to this issue. Inspired by chemoprodrugs,
developing a chemotherapeutic agent and photosensitizer
dual prodrug codelivery system that allows dual drugs to
simultaneously and synchronously arrive at tumor sites is
necessary and significant.
Here, we reported an ROS-cleavable dual prodrug self-
assembly nanoparticle system for combined local-regional
chemotherapy and photodynamic therapy (Figure 1). Chemo-
therapeutic agent CPT was conjugated to poly(ethylene glycol)
methyl ether (MPEG) by a TK linkage, while photosensitizer
pyropheophorbide-a (PPa) was conjugated to the same MPEG
via lipid linkage. Hydrophobic CPT and PPa formed the inner
core of the NPs, and hydrophilic MPEG formed the outer
shell. The MPEG shell prolonged the blood circulation time of
the NPs. PPa not only generated ROS, but also endowed the
NPs with precise localization characteristics in vivo via direct
fluorescence imaging. Under the guidance of imaging, a laser
was locally irradiated on tumor tissues, and the subsequently
generated ROS by accumulated MPEG-(TK-CPT)-PPa NPs not
only was able to cleave the ROS-sensitive TK linkage to realize
locally and on-demand CPT release, but was also capable
of killing tumor cells to achieve photodynamic therapy. The
combination of chemotherapy and photodynamic therapy has
shown stronger antitumor activity than single therapy.
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For the conjugation of drugs to MPEG, MPEG was first reacted
with N-Boc-N′-Fmoc-Lysine (Figure S5, Supporting Information).
Then, the Fmoc group was deprotected and converted to free
amino groups in 20% piperidine/N,N-dimethylformamide solu-
tion. Then, CPT-TK-PNP was reacted with these amino groups
under the catalysis of triethylamine to obtain the MPEG-TK-CPT
conjugate. Subsequently, the Boc group was deprotected by tri-
fluoroacetic acid to form amino groups, which further reacted
with the carboxyl group of PPa to obtain the MPEG-(TK-CPT)-
PPa conjugate. From the 1H-NMR spectrum, it could be seen that
the spectrum contained the main characteristic proton peaks of
both CPT and PPa (Figure S6, Supporting Information). In addi-
tion, characteristic absorbance peaks of both CPT (253–270 nm)
and PPa (640–690 nm) were present in the ultraviolet visible
(UV–vis) spectra of MPEG-(TK-CPT)-PPa (Figure S11, Supporting
Information), which further confirmed the successful prepara-
tion of the MPEG-(TK-CPT)-PPa conjugate. The content of CPT
in the MPEG-(TK-CPT)-PPa conjugate was ≈10.1% according to
1H-NMR analysis and UV–vis spectra. The ROS-uncleavable conju-
gate, MPEG-(cc-CPT)-PPa (Figure S10, Supporting Information),
was also prepared by a similar method but using 1,6-hexanediol
to substitute thioketal linker. The control single prodrug conju-
gate without CPT, MPEG-PPa (Figure S7, Supporting Informa-
tion), was prepared via amidation reaction of the amino group
of MPEG-NH2 and the carboxyl group of PPa. Other single
prodrug conjugates, ROS-sensitive MPEG-TK-CPT (Figure S8,
Supporting Information) and ROS-unresponsive MPEG-cc-CPT
(Figure S9, Supporting Information), were directly synthesized
using MPEG-NH2 and CPT-TK-PNP or CPT-cc-PNP, respectively.
2. Results and Discussion
2.1. Synthesis and Characterization of Prodrug NPs
The MPEG-(TK-CPT)-PPa conjugate was synthesized through
multistep reactions that were mainly divided into two parts.
One part was the modification of CPT with ROS-sensitive TK,
the other part was the conjugation of both CPT-TK derivative
and PPa to MPEG. The synthetic scheme is shown in Figure 2.
The single prodrugs, such as MPEG-PPa, MPEG-TK-CPT, were
synthesized by directly conjugating PPa or CPT-TK deriva-
tive with MPEG (Figures S1 and S2, Supporting Information).
The ROS-unresponsive controls, such as MPEG-cc-CPT and
MPEG-(cc-CPT)-PPa conjugates, were synthesized in the same
approach using 1,6-hexanediol instead of ROS-responsive TK as
the linkage (Figures S3 and S4, Supporting Information).
For the preparation of ROS-sensitive CPT derivative, a mix-
ture of acetone and mercaptoacetic acid was saturated with dry
hydrogen chloride gas and reacted for 6 h to form TK with car-
boxyl groups (TK-COOH). Then the carboxyl-terminal groups
were reduced to hydroxy groups by the strong reducing agent
lithium aluminum hydride to obtain a hydroxyl-terminal ROS-
sensitive TK linker (TK-OH). Next, CPT was reacted with exces-
sive TK-OH to produce CPT-TK-OH, which was further reacted
with 4-nitrophenyl chloroformate to obtain CPT-TK-PNP, in
which 4-nitrophenyl carbonate ester (PNP) had high reactivity
with amino group. The successful preparation of TK and CPT-
1
TK-PNP was confirmed by H-NMR spectra and ESI-MS, as
seen in the Supporting Information.
Figure 2. Synthetic route of MPEG-(TK-CPT)-PPa conjugate.
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Figure 3. Preparation and physiochemical characterizations of the MPEG-(TK-CPT)-PPa NPs. A) Particle size distribution, B) zeta potential, C) TEM
image, and D) stability in PBS solutions with or without 10% FBS of MPEG-(TK-CPT)-PPa NPs, scale bar = 100 nm.
In the prepared MPEG-(TK-CPT)-PPa conjugate, both CPT
and PPa are water-insoluble, while MPEG is highly hydrophilic.
Thus, MPEG-(TK-CPT)-PPa conjugate showed distinctly amphi-
philic characteristics, which facilitated self-assembly to form
NPs in solution with CPT and PPa as the hydrophobic inner
core and MPEG as the outer shell. However, the preparation
method influenced on the formation of NPs. The samples pre-
pared by directly adding conjugates in water displayed two peaks
with a large polydispersity index (PDI) value of 0.531 0.155,
indicating the unstable colloids. The samples prepared by the
thin-film hydration could not easily dissolve in water. While the
samples prepared by dialysis methods showed a single peak
with a hydrodynamic diameter of 43.6 0.8 nm (Figure 3A)
saline (PBS) or simulated serum condition (PBS with 10%
fetal bovine serum (FBS)), and no precipitation or significant
changes in average size were observed for 7 days (Figure 3D),
which is beneficial to the long-term circulation in vivo.
2.2. ROS-Responsive Drug Release In Vitro
Drug release from the physical encapsulated drug delivery system
depends on many factors, such as the environment surrounding
the system, dissociation of the carrier, and diffusion of the encap-
sulated drug. The encapsulating capacity is usually not strong
enough; thus, these systems generally have drug burst release
behavior, which might cause severe side effects and insufficient
therapy efficacy. By contrast, the chemical conjugation prodrug
system mainly depends on breakage of the linked bond. It
remains stable until it reaches the specific triggered environment.
Our designed system was based on the ROS-cleavable chemical
linkage (TK). Under irradiation with the 660 nm laser, ROS was
generated by PPa, which induced the rapid breakage of the TK
bond, resulting in photo-triggered ROS-dependent drug release.
To confirm the ROS-dependent CPT release, the gen-
eration of ROS was first studied using 9,10-anthracenediyl-
bis(methylene)dimalonic acid (ABDA) as an ROS probe. The
UV–vis spectra of free CPT, PPa, and MPEG-(TK-CPT)-PPa
were shown in Figure 4A, and the UV–vis spectra changes of
ABDA solutions with 1 × 10−6 m PPa or MPEG-(TK-CPT)-PPa
conjugate before and after 660 nm laser irradiations for dif-
ferent time were shown in Figure 4B,C, respectively. As shown
in Figure 4B,C, both free PPa and MPEG-(TK-CPT)-PPa had
gradually decreased absorbance at 379 nm with increasing
irradiation time, indicating the generation of ROS. In addition,
and a PDI value of 0.178
0.018, indicating the formation
of stable NPs. The zeta potential was nearly neutral (−5.0
1.3 mV) (Figure 3B). The size and zeta potential of other sam-
ples are shown in Table S1 of the Supporting Information. All
samples had very small PDI values, indicating the good disper-
sion in solutions. The average sizes of single prodrug NPs were
larger than that of dual prodrug NPs, which may be due to the
stronger hydrophobic interaction of dual prodrugs, resulting in
tighter core. Transmission electron microscopy (TEM) images
showed that MPEG-(TK-CPT)-PPa NPs had uniform and mono-
disperse spherical morphology (Figure 3C) and no aggregate
was found, indicating their stability in solution. The statistical
mean sizes (28.0 3.3 nm) were slightly smaller than that of
dynamic light scattering (DLS), possibly due to the dehydra-
tion and shrinkage effect of the hydrophilic PEG shell layer
during the drying process of the TEM sample preparation. A
similar phenomenon has also been reported in previous studies
on PEG-based amphiphilic polymeric NPs.[19] The MPEG-(TK-
CPT)-PPa NPs also showed high stability in phosphate-buffered
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Figure 4. ROS-generation and photo-triggered CPT prodrug release. A) UV–vis spectra of PPa, CPT, and MPEG-(TK-CPT)-PPa conjugate in DMSO at
10 × 10−6 m. UV–vis spectra of ABDA with 1 × 10−6 m B) PPa or C) MPEG-(TK-CPT)-PPa conjugate in DMSO before and after 660 nm laser irradiations for
different time. D) The quantified absorbance changes at 379 nm in the UV–vis spectra of (B) and (C). E) HPLC examination of ROS-responsive CPT release
from the MPEG-(TK-CPT)-PPa prodrug NPs. F) In vitro CPT release profiles of MPEG-(cc-CPT)-PPa and MPEG-(TK-CPT)-PPa NPs in PBS at 37 °C. The
black arrow indicated the 660 nm laser irradiation at 4 h for laser-treated groups. H2O2 and Fe2+ were used as extrinsic ROS sources via Fenton reaction.
the decrease of absorbance at 379 nm of MPEG-(TK-CPT)-PPa
was slightly higher than that of free PPa (Figure 4D), indicating
that MPEG-(TK-CPT)-PPa not only maintained the photosensi-
tive property of PPa, but also enhanced the capability of ROS
generation. This might be due to the fact that the chemical con-
jugation of PPa to MPEG increased the photostability of PPa.
Next, CPT release under laser irradiation was analyzed by
high performance liquid chromatography (HPLC) (Figure 4E).
A peak of MPEG-(TK-CPT)-PPa NPs after laser irradiation with
the same elution time as free CPT was observed, indicating
that the parent CPT had been released. To further confirm the
ROS-induced CPT release, we used an extrinsic ROS source
(50 × 10−3 m H2O2 and 1.0 × 10−6 m Fe2+) as the control group.
H2O2 reacting with Fe2+ can produce highly reactive hydroxyl
radicals (·OH), which can also efficiently cleave the TK bond
from the ROS produced by photosensitizer.[13] The parent CPT
was also found after treatment with this extrinsic ROS. Notably,
ROS induced CPT release and did not affect the activity of CPT.
Thereafter, the in vitro laser-triggered drug release behavior
was studied (Figure 4F). It was found that no matter with or
without the laser, CPT release from MPEG-(cc-CPT)-PPa NPs
was very low throughout the whole 48 h experimental period
because of the absence of ROS-sensitive linkages. CPT release
from MPEG-(TK-CPT)-PPa NPs without laser illumination was
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also very low (about 7% at 48 h). Regarding with MPEG-(TK-
CPT)-PPa NPs with laser illumination, it was observed that the
released CPT in the first 4 h without laser irradiation was negli-
gible, but subsequently the released CPT showed a significantly
increased behavior (21.0% at 12 h and 40.4% at 48 h) after 4 h.
TK linkage can be quickly cleaved under photosensitizer and
laser.[14b] However, the CPT release did not reach up to 100%;
it may be because that the poor solubility of CPT limited the
amount of CPT in release medium, and some CPT precipita-
tion could be found at the bottom of the dialysis bags during the
experiment. But in vivo, it would not be a problem because the
released CPT in tumor tissues could directly act on tumor cells
and kill them. The drugs were covalently conjugated to carriers
and there were no other stimulus responsive groups except the
ROS-sensitive group. So, the CPT release may be only affected
by the ROS, not by other stimuli. The CPT release in simulated
release medium may reflect the release in tumor environment.
We also evaluated the release behavior under the existing ROS
environment (adding 50 × 10−3 m H2O2 and 1.0 × 10−6 m Fe2+ into
the release media). The CPT release showed a fast and gradu-
ally increased release behavior after adding extrinsic ROS source.
These results showed that our prodrug system indeed was an
ROS-sensitive on-demand and controllable drug delivery system
without drug burst release phenomenon, which are favorable
characteristics for reducing side effects and improving the safety.
2.3. In Vitro ROS Generation and Cytotoxicity Assays
Considering the good ROS-responsive CPT release behavior
under laser illumination in solution, we next studied ROS gen-
eration in HCT116 cells using 2,7-dichlorodihydro fluorescein
Figure 5. In vitro cellular uptake and ROS generation after different treatments (G1: MPEG-(TK-CPT)-PPa, G2: MPEG-(TK-CPT)-PPa + Laser, G3: MPEG-
(TK-CPT)-PPa + Vitamin C + Laser, 660 nm laser with power of 100 mW cm−2 for 5 min). A) CLSM images of CPT and PPa distribution and intracellular
generated ROS, blue, red, and green signal were respectively represented CPT, PPa, and ROS (scale bars = 50 µm). B) Flow cytometric curves of ROS in
different groups. C) The quantitative analysis of mean fluorescence intensity of ROS in different groups, **: p < 0.01 (vs MPEG-(TK-CPT)-PPa + Laser).
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diacetate (DCFH-DA) as the ROS indicator. Nonfluorescent
DCFH could be oxidized to green fluorescent 2′,7′-dichlorofluo-
rescein (DCF) by ROS, so more green fluorescence indicated
higher ROS level. Due to the overlap of the fluorescence of CPT
and nuclear dye, such as DAPI or Hoechst, the cell nucleus was
not stained with dye to avoid interference with confocal laser
scanning microscopy (CLSM) examination. From the CLSM
images (Figure 5A), it was seen that MPEG-(TK-CPT)-PPa could
be efficiently internalized into HCT116 cells, and the blue fluo-
rescence of CPT and the red fluorescence of PPa was mainly dis-
tributed in the cytoplasm. Almost no green signal was observed
in the group treated with MPEG-(TK-CPT)-PPa NPs without
laser. By contrast, after irradiation by laser, significantly enhanced
green fluorescence was found in the MPEG-(TK-CPT)-PPa NPs
with laser group, indicating ROS generation. We also determined
whether it still had ROS after adding an ROS quencher, vitamin
c (Vc). It could be observed that the green fluorescence signifi-
cantly decreased under laser irradiation. The ROS level was
semiquantitatively analyzed by flow cytometry (Figure 5B,C).
Upon laser illumination, the ROS level markedly increased com-
pared to the group without laser (3.6-fold lower than the former),
whereas it decreased after the addition of Vc (2.6-fold lower).
Inspired by photo-triggered intracellular prodrug release prop-
erties, the cytotoxicity of different treatment groups of human
colon cancer HCT116 cells was evaluated (Figure 6A; Figure S12,
Supporting Information). The cell viability of single laser-treated
group was higher than 85% even if the irradiating power and time
were increased by twofold (Figure S12, Supporting Information),
indicating that single laser irradiation had almost no effect on
the growth of cells. While MPEG-PPa NPs also had no inhibitory
Figure 6. A) In vitro cytotoxicity of various formulations or free CPT with or without 660 nm laser irradiations (100 mW cm−2, 5 min). The IC50 values
were shown in brackets. B) Flow cytometric analysis of HCT-116 cell apoptosis induced by different treatments, G1: PBS, G2: MPEG-(TK-CPT)-PPa, G3:
MPEG-(TK-CPT)-PPa + Laser, G4: MPEG-(TK-CPT)-PPa + Vitamin C + Laser.
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effects on the growth of cancer cell (Figure 6A). But after com-
bined application of laser and MPEG-PPa NPs, the inhibitory
effect was significantly increased with an IC50 of 1.0 × 10−6 m (com-
pared to >100 × 10−6 m of that without laser, p < 0.01), suggesting
that the powerful photocytotoxicity was generated by PPa under
laser irradiation. The IC50 value of free CPT was 0.87 × 10−6 m,
while the IC50 value of the combined application of CPT and
MPEG-PPa NPs with laser decreased to 0.064 × 10−6 m. The
combination index was calculated to be 0.14, verifying the high
synergistic therapeutic effect between CPT and MPEG-PPa with
laser. When there was no laser irradiation, MPEG-CPT or MPEG-
CPT-PPa NPs with or without ROS-sensitive linkage showed very
limited inhibition of the proliferation activity of cancer cells. But
after laser irradiation, the IC50 value of MPEG-(TK-CPT)-PPa
NPs treated group significantly decreased from 16.7 × 10−6 to
0.12 × 10−6 m, which was lower than that of MPEG-(cc-CPT)-PPa
(0.86 × 10−6 m) and MPEG-PPa. While the IC50 values of the latter
two were no significant difference. This was mainly due to that
the generated ROS by PPa under laser irradiation not only trig-
gered CPT release, but also exerted a photodynamic therapeutic
effect, thereby playing a synergistic therapeutic effect. In addi-
tion, unlike the pH or redox-responsive prodrug system, drugs
also could be released from the prodrug formulations in cells
due to the intracellular stimuli, in this TK-based ROS-responsive
prodrug system, the prodrug was almost kept stable due to the
inadequate ROS level, and had no effect on cells without laser.
Once irradiated by laser, they showed powerful antitumor activity,
indicating the high controllability of the ROS-responsive system.
Furthermore, the cell apoptosis ratios, including early apop-
tosis and late apoptosis, were also evaluated by flow cytometry
using Annexin V-FITC and propidium iodide (PI) apoptosis
detection kits. As shown in Figure 6B, the apoptosis ratio induced
by MPEG-(TK-CPT)-PPa NPs in the absence of laser was 24.3%,
but after laser irradiation the apoptosis ratio increased to 74.8%,
which was about 3.1-fold higher than the former. However, the
apoptosis ratio significantly decreased to 32.8% after adding the
ROS quencher (Vc). It could be concluded that the enhanced
anticancer efficacy of MPEG-(TK-CPT)-PPa under laser illumi-
nation was attributed to the combined effects of laser-irradiated
CPT release and photodynamic effect of generated ROS.
2.4. In Vivo Biodistribution
The biodistribution and drug delivery to the tumor of MPEG-
(TK-CPT)-PPa NPs were studied in HCT116 tumor-bearing nude
mice. Interestingly, due to the near-infrared (NIR) fluorescent
characteristics of PPa, it could be directly used as a probe for
near infrared fluorescence imaging in vivo instead of physical
encapsulating with other dyes. In addition, the NPs could be
Figure 7. In vivo biodistribution of MPEG-(TK-CPT)-PPa NPs evaluated by near infrared fluorescence imaging and frozen sections. A) Fluorescence
images of HCT116 tumor-bearing nude mice postintravenous injection of different formulations. B)The ex vivo images of the main organs and tumors at
24 h postinjection. C) Semiquantitative analysis of the mean fluorescence intensity in main organs and tumors at 24 h postinjection, *: p < 0.05. D) Fluo-
rescence images of frozen section tissues after 4 h postinjection of MPEG-(TK-CPT)-PPa NPs and another 5 min laser irradiation (100 mW cm−2), scale
bar = 1000 µm.
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precisely tracked and located without off-targeting due to the
covalently linked of PPa to carrier. Fluorescence imaging was
performed on an in vivo imaging system (IVIS) to monitor the
NPs distribution in vivo. At the predetermined time points, the
images were collected. As shown in Figure 7A, there was almost
no gradual accumulated fluorescence signal observed for the
mice treated with free PPa, indicating the poor accumulation
in tumor tissues and quick clearance from the body. For the
MPEG-(TK-CPT)-PPa NPs, the fluorescence signal was exten-
sively distributed throughout the whole body at 1 h postinjec-
tion in mouse. As time increased, it gradually decreased in other
parts of the body, while tumor tissue displayed a tendency to first
increase and then decrease, indicating a certain tumor targeting
ability. At about 4 h postinjection, the fluorescence signal inten-
sity at the tumor site was the strongest, indicating the maximum
accumulation of MPEG-(TK-CPT)-PPa NPs at that moment. The
subsequent decrease might be due to the systemic clearance of
NPs. In addition, the ex vivo main organs and tumors were also
collected to analyze the distribution at the end of the last time
point (24 h postinjection). As shown in Figure 7B,C, the fluores-
cence signal in the heart, lung, and kidney of both groups was
very weak, but was still slightly higher in the NPs group than
that in the free PPa group. For the liver and tumor tissues in
the MPEG-(TK-CPT)-PPa NPs group, the signal was significantly
higher (2.5-fold and 4.3-fold) compared to the free PPa group.
Considering the same amount of PPa, the increased fluorescence
signal during the whole circulation and in ex vivo tumor tissue
further verified the long circulation effect and tumor targeting
ability of MPEG-(TK-CPT)-PPa NPs.
In addition, after 4 h postinjection of MPEG-(TK-CPT)-PPa
NPs and another 5 min laser irradiation, the main organs and
tumor of the mouse were harvested for the frozen tissue slices.
As shown in Figure 7D, both blue fluorescence of CPT and pink
fluorescence of PPa were observed in main organs and tumors.
The blue or pink signal overlapped well in the heart, liver,
spleen, lung, and kidney. However, the interesting thing was
that the blue and pink fluorescence signal did not completely
overlap in tumor tissues. The blue fluorescence signal was dis-
tributed throughout the whole tumor, while the pink signal was
mainly localized in the inner of tumor, which might be due to
two reasons. One reason might be that the released CPT from
NPs quickly diffused over the whole tumor, while the chemical
linked PPa to MPEG limited the diffusion of PPa. Another pos-
sible reason was that laser irradiation led to the inactivation or
quenching of the outer PPa. In view of the above results, it could
be concluded that MPEG-(TK-CPT)-PPa could highly accumu-
late in the tumor tissues and diffuse over the whole tumor.
2.5. In Vivo Antitumor Effect
Encouraged by the excellent in vitro cytotoxicity, good accumu-
lation in the tumor, and photo-triggered drug release of MPEG-
(TK-CPT)-PPa NPs, we further evaluated the in vivo antitumor
Figure 8. In vivo antitumor efficacy in HCT116-tumor-bearing BALB/c nude mice. A) Tumor growth profiles and B) body weight of mice treated with
different formulations with or without laser irradiation (100 mW cm−2, 5 min). G1: PBS, G2: PBS + Laser, G3: MPEG-PPa + Laser, G4: MPEG-(cc-CPT),
G5: MPEG-(TK-CPT), G6: MPEG-(cc-CPT)-PPa, G7: MPEG-(TK-CPT)-PPa, G8: MPEG-(cc-CPT)-PPa + Laser, G9: MPEG-(TK-CPT)-PPa + Laser. Data
are presented as the mean SD, n = 5. *: p < 0.05; **: p < 0.01; ***: p < 0.001 (vs MPEG-(TK-CPT)-PPa + Laser). C) Photographs of the tumors with
different treatments at the end of antitumor study.
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therapeutic efficacy on HCT116-tumor bearing nude mice. The
mice were intravenously injected into different formulations at
the CPT dosage of 10 mg kg−1. Based on the in vivo distribu-
tion result, the laser was irradiated at 4 h postinjection of NPs
when the maximum accumulation of NPs occurred. The tumor
volume curves and tumor images are shown in Figure 8A,C.
The mice treated with PBS with or without laser showed rap-
idly increased tumor volumes, while MPEG-(TK-CPT)-PPa with
laser irradiation displayed the greatest tumor inhibitory effect
and antitumor efficacy. Although MPEG-TK-CPT, MPEG-(cc-
CPT)-PPa and MPEG-(TK-CPT)-PPa without laser showed very
weak anticancer activity in the in vitro cytotoxicity assay, they
exhibited the inhibition of tumor growth in vivo compared to
PBS control groups, which might be caused by the slow release
of CPT via the degradation of ester linkage bond in tumor tis-
sues. The MPEG-PPa NPs with laser irradiation also shrunk
the tumor volumes due to the phototoxicity, but the inhibitory
effect was lower than that of MPEG-(TK-CPT)-PPa NPs with
laser, which was mainly due to the combined therapeutic effect
of the quickly released CPT and the generated ROS, resulting
in the improved antitumor efficiency of the combination of
photodynamic therapy and chemotherapy.
In addition, the biosafety of each treatment group was
investigated by observing the general condition of the mice,
and monitoring their body weights, and analyzing the hema-
toxylin and eosin (H&E)-stained tissue sections of the main
organs. During the entire experimental period, the behavior
and movement of all tested mice were no abnormal. All mice
Figure 9. H&E staining of the main organs (heart, liver, spleen, lung, and kidney) after different treatments, scale bar = 100 µm. G1: PBS, G2: PBS +
Laser, G3: MPEG-PPa + Laser, G4: MPEG-(cc-CPT), G5: MPEG-(TK-CPT), G6: MPEG-(cc-CPT)-PPa, G7: MPEG-(TK-CPT)-PPa, G8: MPEG-(cc-CPT)-PPa +
Laser, G9: MPEG-(TK-CPT)-PPa + Laser.
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were sensitive to sound, light, and other stimuli. The feces
were regular in shape and normal in color without any mucus
or blood. In addition, all mice did not display any significant
body weight reduction over the whole experiment period
(Figure 8B), indicating the excellent biocompatibility of these
chemical conjugation prodrug formulations. There were also
no observable changes in tissue morphology, hemorrhage, and
inflammatory cell infiltration in the H&E-stained sections of
the heart, liver, spleen, lung, and kidney of all treated groups
(Figure 9). The results were totally different from our previous
observed in mice treated with free CPT, which showed severe
adverse effects including extremely painful condition of mice,
dramatic body weight loss, death, and obvious hemorrhage and
inflammatory cell infiltration in the liver and kidney tissues.[6b]
Overall, the above results confirmed the excellent biosafety of
such photoactivated ROS-sensitive prodrug systems.
The histopathological characteristics of tumor tissues were
examined by H&E staining (Figure 10). Compared with the PBS
with or without laser group, the tumor cells in the treatment
groups showed increased shrinking cells and nuclear pyknosis,
especially the group treating with MPEG-(TK-CPT)-PPa with
laser. The proliferation and apoptosis of tumor tissues were also
inspected by the immunofluorescence staining of Ki-67 and
terminal deoxynucleotidyl transferase-mediated nick-end labe-
ling (TUNEL) (Figure 10), respectively. It was observed that the
most efficient suppression of proliferation cells (Ki-67 positive
cells, red color) and the greatest percentage of apoptosis cells
(green fluorescence) were induced by the treatment of MPEG-
(TK-CPT)-PPa with laser compared with other groups, which
further verified the excellent antitumor efficacy of MPEG-(TK-
CPT)-PPa NPs with laser.
3. Conclusion
In summary, we successfully designed and prepared an ROS-
responsive MPEG-(TK-CPT)-PPa prodrug delivery system
for on-demand drug release. The CPT was covalently grafted
to the MPEG via the ROS-sensitive TK linkage, while the
PPa was conjugated to MPEG via lipid linkage. The prepared
MPEG-(TK-CPT)-PPa conjugate could self-assemble to form
nanoparticles. Compared to the conventional physical encapsu-
lated drug delivery system, our chemical conjugation prodrug
system could reduce the premature drug release and deliver
two drugs to the tumor site synchronously. In addition, both
chemotherapeutic agent and photosensitizer were inactive
without chemotoxicity and phototoxicity in the systemic circu-
lation, making it significantly decrease the side effects to the
Figure 10. H&E, Ki-67, and TUNEL staining of tumor after different treat-
ments, scale bar = 100 µm. G1: PBS, G2: PBS + Laser, G3: MPEG-PPa +
Laser, G4: MPEG-(cc-CPT), G5: MPEG-(TK-CPT), G6: MPEG-(cc-CPT)-
PPa, G7: MPEG-(TK-CPT)-PPa, G8: MPEG-(cc-CPT)-PPa + Laser, G9:
MPEG-(TK-CPT)-PPa + Laser.
healthy tissues. Moreover, under the guidance of NIR imaging, 4. Experimental Section
the laser could be precisely applied. Furthermore, after locore-
Materials: CPT was purchased from Dalian Meilun Biotechnology
gional laser irradiation on the tumor site, the accumulated PPa
generated ROS, which rapidly cleaved the ROS-sensitive TK
linkage, resulting in the on-demand release of CPT. Finally,
MPEG-(TK-CPT)-PPa NPs displayed improved and synergistic
tumor therapy efficacy due to the combination of the photo-
dynamic therapy and chemotherapy. Overall, these NIR laser-
triggered MPEG-(TK-CPT)-PPa prodrugs with low systemic
toxicity and superior combined therapy efficacy may offer a
good strategy for drug delivery and controllable local therapy.
Co., Ltd. (Dalian, China). PPa was obtained from Shanghai Yuanye
Biotechnology Co., Ltd. (Shanghai, China). 3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT), DCFH-DA, and ABDA
was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s
modified Eagle’s medium (DMEM), FBS, trypsin, and Annexin V-FITC
Apoptosis Detection Kit were obtained from Thermo-Fisher Scientific
(Waltham, MA, USA). Other reagents were analytic reagent grade and
used without further purification.
Synthesis of the Conjugates: The detailed experimental procedures and
characterizations of MPEG-(TK-CPT)-PPa conjugate, single prodrug controls
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(MPEG-PPa, MPEG-TK-CPT), and the ROS-unresponsive controls (MPEG-
cc-CPT, MPEG-(cc-CPT)-PPa) are shown in the Supporting Information.
Characterization: NMR spectra were recorded via Bruker Avance III
400MHz NMR spectrometer (Bremen, Germany). The size and zeta
potential of the prepared nanoparticles were measured by DLS (Nano-ZS
90, Malvern Panalytical, UK). The morphology of the prepared samples was
examined using TEM (Tecnai G2 F20 S-TWIN, USA). The UV–vis spectra
were measured by a UV-2600 spectrophotometer (Shimadzu, Japan).
The concentration of CPT was measured by HPLC (Agilent 1260, Agilent
Technologies, USA) with an eluent of methanol and water (80/20, v/v).
Preparation of Nanoparticles: The MPEG-(TK-CPT)-PPa NPs were
prepared by a dialysis method. Briefly, MPEG-(TK-CPT)-PPa (10.0 mg)
was dissolved in dimethyl sulfoxide (DMSO) (1.0 mL), and then added
dropwise into 9.0 mL of deionized water. After light-shield stirring for
6 h, the solution was transferred to a dialysis bag (molecular weight
cut-off/MWCO = 2000 Da) and dialyzed against deionized water for 24 h.
Next, the NPs were filtered using a 0.22 µm filter (Millipore, Burlington,
MA, USA) to remove any undissolved substances for the further use.
MPEG-PPa, MPEG-TK-CPT, MPEG-cc-CPT, and MPEG-(cc-CPT)-PPa NPs
were prepared using similar methods.
In Vitro Generation of ROS: The ROS generation of NPs was monitored
by using ABDA as an ROS indicator. Briefly, the ABDA solution with an
absorbance intensity of 0.5 at 379 nm was first prepared. Then, 100 µL
NPs solution or free PPa was added into the above ABDA solution to
obtain a final PPa concentration of 10 × 10−6 m. Thereafter, the obtained
solutions were irradiated by a 660 nm laser with a power of 100 mV cm−2
for 5 min. The UV–vis spectra of the solutions were recorded every
minute. The generated ROS were reflected by the decreased absorption
of ABDA at 379 nm.
that, these cells were cultured for another 24 h. For the groups without
laser, the cells were directly cultured for 48 h. Thereafter, the medium of
each well was replaced with 100 µL fresh medium, and then added into
20 µL freshly prepared MTT solution (5 mg mL−1). After another 4 h,
the media were removed and 150 µL DMSO was added to dissolve the
formed formazan crystal. The absorbances at 570 nm were measured by
a microplate reader. The cell viability was determined as the ratio of the
absorbance of sample to that of the control.
In Vitro Apoptosis Assay: HCT 116 cells in the logarithmic phase were
seeded into 12 wells with a cell density of 5 × 104 cells per well, and
cultured for 24 h at 37 °C. The cells were treated with MPEG-(TK-CPT)
NPs with or without Vc. After being cultured for 4 h, the cells were
washed with PBS to remove the extracellular NPs. Then, the cells were
irradiated by a 660 nm laser with a photo-intensity of 100 mW cm−2 for
5 min. Subsequently, the cells were cultured for another 12 h at 37 °C.
After that, the cells were digested by trypsin without EDTA, collected,
washed with PBS, and then stained by PI and Annexin V-FITC solutions
for 15 min according to the guideline of the apoptosis kit. The
percentages of apoptosis were measured by flow cytometry.
Animal and Tumor Models: All BALB/c nude mice (6 weeks old, female)
were purchased from Beijing HFK Bioscience Co. Ltd. (China) and fed in
the SPF-class laboratory animal room. All procedures were performed
following the guidelines and the protocol approved by the Institutional
Animal Care and Treatment Committee of Sichuan University. The
xenograft tumor modes were established through subcutaneously
injecting HCT116 cells suspension (1 × 106 cells) into the right flank of
the mice. When the tumor volumes reached 500–1000 mm3, the mice
were used for the biodistribution and near-infrared fluorescence imaging
studies, while the mice with tumor volumes of 100–200 mm3 were used
for antitumor treatment.
In Vivo Biodistribution Studies: The in vivo biodistribution of NPs
was studied by near-infrared fluorescence imaging and frozen tissue
slices, respectively. Briefly, free PPa or MPEG-(TK-CPT)-PPa NPs were
intravenously injected into the tumor-bearing mice. At the predetermined
time points, the mice were anesthetized by isoflurane and the NIR
signals of PPa were collected by an IVIS Spectrum (Perkin-Elmer, USA)
with an excitation wavelength of 640 nm and emission wavelength of
710 nm. At 24 h, the mice were sacrificed. Tumor and main organs were
harvested and washed with PBS, and then imaged.
For the frozen tissue slices, after 4 h postinjection of MPEG-(TK-
CPT)-PPa NPs, the tumor tissue was irradiated by a 660 nm laser for
5 min (100 mW cm−2). Thereafter, the mouse was sacrificed. The tumors
and main organs were collected, washed with PBS, and fixed by an
embedding medium. After that, the tissue slices were prepared by a
frozen section machine, and then scanned using a Pannoramic Scanner
to obtain the images of drug distribution.
In Vivo Antitumor Efficacy: The tumor-bearing mice were randomly
divided into 9 groups (6 mice per group), and intravenously injected
with 100 µL of PBS, MPEG-PPa, MPEG-TK-CPT, MPEG-cc-CPT, MPEG-
(cc-CPT)-PPa, and MPEG-(TK-CPT)-PPa NPs with the dosage of CPT
(10 mg kg−1) and PPa (15 mg kg−1). The samples were administrated
every two days for a total of three times. After 4 h postinjection, the
tumors of laser irradiation groups were irradiated by a 660 nm laser
with a power intensity of 100 mW cm−2 for 5 min. The tumor size and
mice weight were measured every two days. The tumor volumes were
calculated by the (length × width2)/2. The mice were euthanized at day
21 after treatment.
Immunohistochemical Analysis: On the day following the last
treatment, one mouse of each group was sacrificed, and the main organs
were collected, fixed in 10% wt neutral formalin solutions, embedded in
paraffin, and sectioned. H&E were used to evaluate the morphology of
organs. Ki-67 and TUNEL immunohistochemical stains were conducted
to analyze the proliferation and apoptosis of tumor tissues, respectively.
Statistical Analysis: All results were presented as means standard
error of the mean. Statistical analysis was evaluated by Student’s t-test
for two groups, and one-way analysis of variance (ANOVA) for multiple
groups using Graphpad Prism 7.0 software. The threshold for statistical
significance was p < 0.05.
In Vitro Photo-Triggered CPT Release: The solutions of NPs (1 mg mL−1)
were placed in dialysis bags (MWCO, 2000 Da) and then immersed into
the PBS buffers containing 0.5% tween 80 (pH = 7.4, 10 mL). The release
was conducted at 37 °C with gentle shaking (100 rpm). At predetermined
time points, the released media were withdrawn and replaced with fresh
warmed PBS. To evaluate the photo-triggered release behavior of CPT,
the sample solutions were irradiated with 660 nm laser with a power
intensity of 100 mW cm−2 for 5 min at 4 h. The samples without any
laser illumination were used as the negative control group. A sustaining
extrinsic ROS source (adding 50 × 10−3 m H2O2 and 1.0 × 10−6 m Fe2+
to the release medium) was used as the positive control groups. The
collected release media was lyophilized, redissolved with methanol, and
then analyzed by HPLC to determine the content of CPT, the release was
conducted three times.
Cellular Uptake and Intracellular ROS Levels: Human HCT116 colon
cancer cells were cultured in DMEM containing 10% FBS and 1%
antibiotics at cell incubator (37 °C, 5% CO2). The cells in the logarithmic
phase were seeded into glass bottom cell culture dishes with a cell
density of 1 × 105 cells per well, and cultured for 24 h at 37 °C. Then,
the cells were treated with MPEG-(TK-CPT)-PPa NPs with a PPa
concentration of 0.5 × 10−6 m for 4 h. Another group was additionally
added 0.5 × 10−3 m ROS scavenger, Vc, to evaluate the contribution of
laser-induced ROS. Thereafter, the cells were washed three times with
PBS, added DCFH-DA (10 × 10−6 m) into the wells, and irradiated by a
660 nm laser with a photo-intensity of 100 mW cm−2 for 5 min. After
fresh medium was added and they were cultured for another 2 h, the
cells were washed with PBS. The cells were further fixed by the 10%
neutral formalin solution and washed with PBS for CLSM to observe the
cellular uptake and intracellular ROS generation (DAPI channel, GFP
channel and Cy 5.5 Channel were chosen for the fluorescence of CPT,
DCF, and PPa, respectively). In addition, for the quantitative analysis of
the generated ROS levels by flow cytometry, the cells were trypsinized by
trypsin, washed, and finally suspended in PBS.
In Vitro Cytotoxicity Studies: HCT116 cells in the logarithmic phase were
seeded into 96-well plates (5 × 103 cells per well). After being cultured for
24 h, each well was added into 100 µL fresh media containing different
concentrations of drug or NPs. After cultured for another 24 h, the
media were replaced with fresh media. For the laser treated group, each
well was irradiated by a 660 nm laser for 5 min at 100 mW cm−2. After
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Acknowledgements
This work was supported by the National Natural Science Foundation
of China (31930067, 31771096, 31700869, 31700868, and 31525009),
Science and Technology Project of Sichuan Province (2018FZ0030 and
2020YFS0201), China Postdoctoral Science Foundation (2018M633367
and 2018T110977), International Visiting Program for Excellent Young
Scholars of SCU, Post-Doc Research Project, West China Hospital,
Sichuan University (2018HXBH030), Scientific Research Foundation
of the Health and Family Planning Commission of Sichuan Province
(17PJ057 and 17PJ559), and 1·3·5 project for disciplines of excellence,
West China Hospital, Sichuan University (ZYGD18002).
Conflict of Interest
The authors declare no conflict of interest.
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