A redox-activatable biopolymer-based micelle for sequentially enhanced mitochondria-targeted photodynamic therapy and hypoxia-dependent chemotherapy

Article abstract of DOI:10.1039/d0cc03667f

A tumor redox-activatable micellar nanoplatform based on the naturally occurring biomacromolecule hyaluronic acid (HA) was developed for complementary photodynamic/chemotherapy against CD44-positive tumors. Here HA was first conjugated with l-carnitine (Lc)-modified zinc phthalocyanine (ZnPc) via disulfide linkage and then co-assembled with tirapazamine (TPZ) to afford the physiologically stable micellar nanostructure. The mitochondria-targeted photodynamic activity of ZnPc-Lc could efficiently activate the mitochondrial apoptosis cascade and deplete the oxygen in the tumor intracellular environment to amplify the hypoxia-dependent cytotoxic effect of TPZ.

Full text of DOI:10.1039/d0cc03667f

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A redox-activatable biopolymer-based micelle for the sequentially
enhanced mitochondria-targeted photodynamic therapy and
hypoxia-dependent chemotherapy
Received 00th January 20xx,
Accepted 00th January 20xx
Yanan Li†a, Linawati Sutrisno†b, Yanhua Houc, Yang Feia, Chengcheng Xuea, Yan Hub, Menghuan Li
*a, Zhong Luo*a
DOI: 10.1039/x0xx00000x
A tumor redox-activatable micellar nanoplatform based on the strategies for tumor treatment to improve the tumor treatment
natural-occurring biomacromolecule hyaluronic acid (HA) was outcome. Typically, tirapazamine (TPZ) is an emerging
developed for the complementary photodynamic/chemotherapy bioreductive prodrug that could be converted into cytotoxic
against CD44-positive tumors. Here HA was first conjugated with L- radicals in hypoxic tumor intracellular environment,7, 8 which is
10
carnitine (Lc)-modified zinc phthalocyanine (ZnPc) via disulfide a universal feature for many tumor indications.9, TPZ is a
linkage and then co-assembled with tirapazamine (TPZ) to afford benzotriazine compound that could be metabolized by both
the physiologically-stable micellular nanostructure. The one-and two-electron reductases in tumor cells under hypoxic
mitochondrial-targeted photodynamic activity of ZnPc-Lc could conditions and form highly reactive radicals, which would then
efficiently activate the mitochondrial apoptosis cascade and react with tumor DNA to cause single-strand breaks, double-
deplete the oxygen in the tumor intracellular environment to strand breaks and chromosome aberrations, leading to potent
amplify the hypoxia-dependent cytotoxic effect of TPZ.
tumor inhibition effect.11 Nevertheless, the efficacy of TPZ may
be compromised by the spatial heterogeneity of oxygen
availability in tumor tissues.12 To overcome these obstacles, TPZ
is usually combined with photodynamic therapy (PDT), which
could consume the intracellular oxygen to produce cytotoxic
reactive oxygen species (ROS).13 However, the biological half-
life of ROS is usually very low, which may attenuate the
antitumor activity of PDT in vivo.14 Therefore, new modification
and release mechanisms are urgently needed to coordinate the
therapeutic activity of PDT and TPZ to enhance the eventual
treatment outcome.
Here, we report a redox-activatable micellar nanoplatform
based on HA to deliver L-carnitine (Lc)-modified zinc
phthalocyanine (ZnPc) and TPZ to CD44-positive tumor cells for
complementary mitochondria-targeted PDT and TPZ treatment
(Scheme 1).15 Specifically, the ZnPc-Lc photosensitizers were
first conjugated onto the HA backbone via disulfide bond and
then self-assembled with TPZ molecules to afford the
physiologically stable micelles, which demonstrated long blood
circulation time and were effectively taken in by CD44-positive
tumor cells via HA-CD44 interaction. The disulfide bond could
then be cleaved by the excess glutathione (GSH) in tumor
intracellular environment and detach the ZnPc-Lc, which would
destabilize the micelles and release the TPZ. Due to the high
positive charge and lipophilicity of ZnPc-Lc, it would home to
the negatively-charged mitochondrial membrane and initiate
the mitochondrial apoptosis cascade via the in-situ NIR-
activated PDT. Meanwhile, the mitochondria-targeted PDT
would also consume the oxygen in the tumor cytosol and
facilitate the activation of TPZ in a synergistic manner. It’s
anticipated that
Recently, there’s an upsurge in developing antitumor
nanotherapeutics with natural-occurring biopolymers such as
proteins, peptides and polysaccharides, which may offer unique
properties over their synthetic counterparts including high
biocompatibility, biodegradability and low immunogenicity. 1, 2
Hyaluronic acid (HA) is a promising candidate of those
biopolymers and holds potential for clinical translation.3,
4
Specifically, HA is
a hydrophilic linear macromolecular
polysaccharide composed of glucuronic acid and N-
acetylglucosamine. It’s one of the major components in the
extracellular matrix (ECM) of both healthy and malignant
tissues and has intrinsic biocompatibility and biodegradability.
Remarkably, HA molecules contain many hydroxyl, carboxyl and
N-acetyl groups for the facile coupling of various therapeutic
substances. Meanwhile, HA could provide long circulation time
and enhanced safety due to its high hydrophilicity and low
immunogenicity. More importantly, HA could bind to the CD44
glycoproteins overexpressed on many types of tumor cells,
leading to improved therapeutic index and lower systemic
toxicity.5, 6 These properties make HA particularly relevant for
tumor-targeted drug delivery.
In addition to biopolymer-based drug delivery technologies,
scientists are also searching for new therapeutic agents and
a. School of Life Science, Chongqing University, Chongqing 400044, China
b. Key Laboratory of Biorheological Science and Technology, Ministry of Education,
College of Bioengineering, Chongqing University, Chongqing 400044, China
c. Chongqing Engineering Research Center of Pharmaceutical Sciences. Chongqing
Medical and Pharmaceutical College, Chongqing 401331, P. R. China.
† These authors contributed equally to this paper.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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suggesting the high stability of the micelles in boVdieywfAlurtiicdlesOanlninde
beneficial to enhance the tumor specific DacOcI:u1m0.u10la39ti/oDn0.CC03667F
The redox-sensitivity of the micelle under GSH stimulation
was then investigated via Uv-vis spectroscopy. As shown in
Figure 1d, minimal TPZ leakage (nearly 10%) was observed in
the control group within 50 hours, suggesting the excellent
stability of the micelles. In contrast, the final TPZ release ratio
reached 70% under 10 mM of GSH. It was also observed that the
amount of the TPZ release was positively correlated with the
incubation time, indicating that GSH could be exploited as a
redox trigger to regulate the release patterns of TPZ. We also
monitored the morphological changes of the micelles before
and after the GSH treatment via TEM and found that the
micelles have been disintegrated into small fragments after
incubation in the GSH-containing solution for 24 h (Figure 1e),
further validating with the proposed drug release mechanism
based on the GSH-triggered micelle destabilization. Considering
the high level of GSH inside tumor cells, this feature is beneficial
for preventing the undesirable activation of the drug-loaded
micelles in healthy cells and tissues.16
We then monitored the targeting effect of the micelles by
comparing their uptake by CD44-positive 4T1 cells and CD44-
negative HUVECs (healthy primary endothelial cells). FITC was
used to label the ZnPc moieties to indicate their cellular
distribution patterns. Only negligible FITC fluorescence was
found in HUVECs incubated with micelles for 2 h (Figure 2a and
Scheme 1. Schematic illustration of the molecular structure of HA-S-S-ZnPc-
Lc@TPZ micelles and mitochondria-targeted PDT and hypoxically-enhanced TPZ
chemotherapy.
this study may offer new avenues to suppress tumor
proliferation with reduced adverse effects.
We first immobilized ZnPc onto the HA backbone via thiol-
disulfide exchange (Figure S1), during which ZnPc reacted with
3-(2-pyridyldithio) propanoic acid via carbodiimide reaction and
was then grafted to the thiolated HA at a loading ratio of around
8% (wt/wt) (Figure S3). Lc moieties were then conjugated to
1
ZnPc via carbodiimide reaction and confirmed using H NMR
spectra, UV-vis spectroscopy and zeta potential (Figure S2 and
Figure S3). HA-S-S-ZnPc-Lc would then co-assemble with TPZ via
an oil-in-water emulsion approach to form micelles (Figure S1)
with a TPZ loading ratio of around 4% (Figure S3). The micelles
have a spherical shape with distinct boundaries (Figure 1e), and
their average diameter is around 200 nm (Figure 1c), which is
optimal for passive targeting. Zeta potential analysis indicated
that the surface charge of the HA biopolymer has increased
slightly after the ZnPc conjugation from -28.0 mV to -20.0 mV
due to the amine groups in ZnPc and the surface charge of HA-
S-S-ZnPc-Lc (-16.0 mV) was even higher than HA-S-S-ZnPc due
to the highly positively charged Lc groups (Figure S3).
Meanwhile, Uv-vis absorption results showed that a new peak
emerged at around 270 nm for HA-S-S-ZnPc-Lc@TPZ micelles
compared to pristine HA-S-S-ZnPc-Lc, suggesting successful TPZ
incorporation (Figure 1a). The HA-S-S-ZnPc-Lc has a CMC
(critical micelle concentration) of 0.045 mg/mL (Figure 1b),
Figure 1. (a) The UV-vis absorption spectra of the HA-S-S-ZnPc-Lc and HA-S-S-ZnPc-
Lc@TPZ. Inserted image shows the enhanced absorption around 270 nm due to
TPZ incorporation. (b) The critical micelle concentration of HA-S-S-ZnPc-Lc. (c) Size
distribution of HA-S-S-ZnPc-Lc@TPZ micelles. (d) Release profile of TPZ after
incubation with and without GSH (n=6). (e) Morphological changes of the HA-S-S-
ZnPc-Lc@TPZ micelles after incubation with 10 mM GSH for 24 h.
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Figure S4), while micelle-treated 4T1 cells showed evidently
stronger FITC fluorescence. Meanwhile, the FITC fluorescence
in the HA-pretreated 4T1 cells remained at a low level
comparable to HUVECs due to the saturation of the membrane
CD44. Statistical analysis further revealed that the total FITC
fluorescence intensity in the biopolymer-treated 4T1 cells was
more than two-fold higher than the CD44-negative HUVECs
(Figure S5). These results implied that HA-S-S-ZnPc-Lc@TPZ
micelles could be efficiently endocytosed by 4T1 tumor cells
due to HA-CD44 interaction, thus potentiating tumor-specific
drug deposition while sparing healthy cells. Furthermore, we
extended the incubation time to 24 h and observed the cellular
distribution patterns of the micelles after endocytic uptake. As
depicted in Figure 2b and Figure S6, the red fluorescence
(mitotracker) in the HA-S-S-ZnPc-Lc@TPZ group has almost
overlapped with the green fluorescence (FITC-labeled ZnPc-Lc)
with a colocalization ratio of 0.71, while those in the HA-S-S-
ZnPc@TPZ group were still largely separated (colocalization
ratio: 0.3), indicating that the Lc-modified ZnPc has been
attached to the mitochondrial membrane.
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DOI: 10.1039/D0CC03667F
Figure 2. (a) CLSM images of 4T1 cells after incubation with HA-S-S-ZnPc-Lc-FITC.
(a1) CLSM images of CD44-blocked 4T1 cells after incubation with HA-S-S-ZnPc-Lc-
FITC. The cells were pretreated with HA (5 mg/mL) for competitive CD44 binding.
(a2) CLSM images of HUVECs cells treated with HA-S-S-ZnPc-Lc-FITC. Scale bars: 20
μm. (b) CLSM images showing tumor mitochondria (red fluorescence) and the
intercellular distribution of FITC-labeled biopolymers (green fluorescence) after
treatment with PBS (b), HA-S-S-ZnPc-FITC (b1) and HA-S-S-ZnPc-Lc-FITC (b2). CLSM
images of 4T1 cells stained with JC-1 to assess the mitochondrial damage after
treatment with PBS (c), HA-S-S-ZnPc+NIR (c1) and HA-S-S-ZnPc-Lc+NIR (c2). (d)
CLSM images on the changes in the intracellular ROS and hypoxia levels in 4T1
cells after treatments with PBS (d1), ROS inducer (d2), HA-S-S-ZnPc-Lc (d3),
PBS+NIR (d4), hypoxia inducer (d5), HA-S-S-ZnPc-Lc + NIR (d6). The ROS inducer
and hypoxia inducer were added as positive control. (e) Live/dead cell imaging of
4T1 cells after treatment with (I) PBS, (II) TPZ, (III) HA-S-S-ZnPc-Lc and (IV) HA-S-S-
ZnPc-Lc@TPZ micelles with or without laser irradiation.
survival rate of HA-S-S-ZnPc/HA-S-S-ZnPc-Lc-treated cells
remained the same to the control group, indicating the
excellent biocompatibility of the micelles. In comparison, the
cell viability in the HA-S-S-ZnPc group dropped to 48.4% after
NIR treatment, which was attributed to the ZnPc-mediated PDT.
The survival rate was even lower in the HA-S-S-ZnPc-Lc+NIR
group at 38.5%, which indicates that modifying ZnPc with Lc
moieties could enhance the overall efficacy of PDT. The most
pronounce tumor inhibition effect was observed in the HA-S-S-
ZnPc@TPZ+NIR group, which clearly demonstrated the superior
antitumor efficacy by combining mitochondria-targeted PDT
and the hypoxia-augmented TPZ chemotherapy. Markedly, the
tumor inhibition efficiency of HA-S-S-ZnPc@TPZ and HA-S-S-
ZnPc-Lc@TPZ is statistically different at lower TPZ
concentrations, but the difference became non-significant
when the equivalent TPZ concentration reached 10 μg/mL due
to the diminished antitumor susceptibility at larger
nanoformulation concentrations.17 The MTT results were also
supported by the flow cytometry analysis and live/dead cell
staining (Figure S11 and Figure 2e).
The antitumor efficacy of the micelles was evaluated on 4T1
tumor-bearing mice. It was demonstrated that more than 30%
of the injected micelles accumulated in tumors (Figure 3a,
Figure S13) thanks to the size-mediated passive targeting and
HA-mediated active targeting. Meanwhile, no apparent drug
deposition was detected in the central nervous system due to
the protecting effect of the blood brain barrier therein (Figure
S12). The micelles also showed prolonged blood retention
capability and were almost eliminated from the body after 96 h
of incubation (Figure S12), thus providing balanced treatment
and safety performance. Next, we investigated the changes in
tumor sizes to evaluate the treatment response. As shown in
Figure 3b, tumors in the PBS and PBS+NIR groups grew rapidly
due to the lack of effective treatment. Meanwhile, tumors in
the TPZ+NIR group showed negligible growth inhibition due to
the low cytotoxicity of TPZ under normoxic conditions. In
comparison, significant tumor suppression was observed in the
Besides, we also explored the PDT-induced ROS generation
and cellular deoxygenation. It was observed all groups showed
negligible ROS generation without NIR and the oxygen levels
remained normal, indicated by the minimal green and red
fluorescence in the intracellular environment (Figure 2d and
Figure S7). Contrastingly, high level of intracellular ROS has
been observed in HA-S-S-ZnPc-Lc and HA-S-S-ZnPc-Lc@TPZ
groups under NIR irradiation (720 nm, 200 mW/cm2, 5 min),
accompanied by increasing local hypoxia. The results support
our hypothesis that the ZnPc-mediated PDT could generate
large amount of cytotoxic ROS by consuming the cytosolic
oxygen, thus creating synergies with the co-delivered TPZ.
Subsequently, we monitored the photodynamic damage of the
micelles on tumor mitochondria by investigating the changes in
the mitochondrial transmembrane potential (ΔΨm) via JC-1
staining, during which the normal and damaged mitochondria
were stained red or green, respectively. The HA-S-S-ZnPc-
Lc+NIR group showed strong green fluorescence with almost no
red fluorescence (Figure 2c and Figure S8), evidently suggesting
the severe mitochondrial damage thereof. In comparison, the
HA-S-S-ZnPc+NIR group only showed modest green
fluorescence, which blended with the strong red fluorescence
of undamaged mitochondria and led to a yellowish fluorescence
pattern. The modest mitochondrial damage in the HA-S-S-
ZnPc+NIR group could be explained by the dissipation of ROS by
the reductive species in tumor cytosol. We also measured the
cytosolic levels of cytochrome c (cyt c) using western blot assay
and found that the intracellular cyt c levels in HA-S-S-ZnPc-
Lc+NIR and HA-S-S-ZnPc-Lc@TPZ+NIR groups have increased by
almost three-fold compared to the control group (Figure S10),
indicating the severe mitochondrial damage thereof. These
results again confirmed the mitochondria-targeting capability
of the micelles as well as its augmenting effect on PDT efficacy.
We further investigated the antitumor efficacy of the
micelles against CD44-positive tumors in vitro. Only slight
growth inhibition has been observed in non-NIR groups due to
the lack of effective treatment (Figure S9). Meanwhile, the
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J. Name., 2013, 00, 1-3 | 3
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enhanced PDT/chemotherapy of the biopolymer-based micelle
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DOI: 10.1039/D0CC03667F
is favorable for the treatment against a variety of tumor
indications, which may also facilitate the development of more
advanced nanotherapeutics for combinational tumor therapy.
Conflicts of interest
There are no conflicts to declare.
Notes and references
‡ This work was financially supported by National Key R&D
Program of China(2017YFB0702603, 2016YFC1100300), National
Natural Science Foundation of China (11832008, 51773023),
Fundamental Research Funds for the Central Universities
(2020CDJQY-A075, 2020CDJYGZL009), Returning Overseas
Scholar Innovation Program (CX2018062), Chongqing
Outstanding Young Talent Supporting Program(CQYC201905072)
and Natural Science Foundation of Chongqing Municipal
Government (cstc2018jcyjAX0368) and Chongqing Research
Program of Basic Research and Frontier Technology (Grant No.
cstc2018jcyjAX05 80).
Figure 3. (a) Changes in the systemic distribution of (I) Cy5 and (II) Cy5-labeled
micelles in tumor-bearing mice after tail vein injection. Black dash circles indicate
the tumor region. Images on the right show the organ distribution of the Cy5-
labeled micelles at 24 h post injection. (b) Visual comparison of the sizes of
extracted tumor after 21-day treatment with PBS (I), PBS+NIR (II), TPZ+NIR (III),
HA-S-S-ZnPc-Lc+NIR (IV) and HA-S-S-ZnPc-Lc@TPZ+NIR (V). (c) Quantitative
analysis on tumor volumes throughout the 21-day treatment period.
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tissue samples (Figure S15), in which the HA-S-S-ZnPc-Lc@TPZ + NIR
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apparent changes have been observed in the mouse body weight and
histological analysis of major organs in the biopolymer-treated
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and Figure S16). The in vivo results demonstrated that the
biopolymer-based micelles could effectively deliver ZnPc-Lc and TPZ
into CD44-positive tumors in a highly targeted manner and
efficiently eliminate the tumor cells through the combinational
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Conclusions
In summary, we developed a redox-activatable micellar
nanoplatform based on a natural polysaccharide hyaluronic acid
for the tumor-targeted delivery of mitochondria-targeted
photosensitizers (ZnPc-Lc) and tirapazamine (TPZ), leading to
complementarily enhanced PDT and chemotherapy. The
micelles could be disintegrated by the excess GSH in tumor cells
and release ZnPc-Lc and TPZ into the tumor cytosol. The
positively charged Lc moiety in the lipophilic ZnPc-Lc molecules
would then guide them to the negatively charged mitochondria
and damage the mitochondrial membrane via NIR-actuated
ROS generation, which could not only enhance the cytotoxic
effect of the short-lived ROS, but also consume the oxygen in
the tumor cytosol to amplify the antitumor efficacy of TPZ. The
high biocompatibility, delivery efficiency and sequentially
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This work reports a biopolymer-based micellar nanoplatform with redox-sensiDtiOvIi:t1y0.1f0o3r9/D0CC03667F
sequentially-enhanced mitochondria-targeted photodynamic therapy and hypoxia-amplified
chemotherapy against CD44-positive tumors.