Article

Cite This: J. Am. Chem. Soc. 2020, 142, 937−944 pubs.acs.org/JACS

Molecular Self-Assembly of Bioorthogonal Aptamer-Prodrug

Conjugate Micelles for Hydrogen Peroxide and pH-Independent
Cancer Chemodynamic Therapy
Wenjing Xuan,†,∥ Yinghao Xia,†,∥ Ting Li,† Linlin Wang,† Yanlan Liu,*,† and Weihong Tan*,†,‡,§
†
Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College
of Chemistry and Chemical Engineering, Aptamer Engineering Center of Hunan Province, Hunan University, Changsha 410082,
China
‡
Institute of Cancer and Basic Medicine (IBMC), Chinese Academy of Sciences, The Cancer Hospital of the University of Chinese
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Academy of Sciences, Hangzhou, Zhejiang 310022, China

§
Foundation for Applied Molecular Evolution, 13709 Progress Boulevard, Alachua, Florida 32615, United States
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*
S Supporting Information

ABSTRACT: Chemodynamic therapy (CDT) has demon-

strated new possibilities for selective and logical cancer
intervention by specific manipulation of dysregulated tumor-
ous free radical homeostasis. Current CDT methods largely
rely on conversion of endogenous hydrogen peroxide (H2O2)
into highly toxic hydroxyl radicals via classical Fenton or
Haber−Weiss chemistry. However, their anticancer efficacies
are greatly limited by the requirement of strong acidity for
efficient chemical reactions, insufficient tumorous H2O2, and
upregulated antioxidant defense to counteract free radical-
caused oxidative damage. Here, we present a new concept
whereby bioorthogonal chemistry and prodrug are combined to create a new type of aptamer drug conjugate (ApDC): aptamer-
prodrug conjugate (ApPdC) micelle for improved and cancer-targeted CDT. The hydrophobic prodrug bases can not only
promote self-assembly of aptamers but also act as free radical generators via bioorthogonal chemistry. In depth mechanistic
studies reveal that, unlike traditional CDT systems, ApPdC micelles enable in situ activation and self-cycling generation of toxic
C-centered free radicals in cancer cells through cascading bioorthogonal reactions, with no dependence on either H2O2 or pH,
yet concurrently with diminished cancerous antioxidation by GSH depletion for a synergistic CDT effect. We expect this work
to provide new insights into the design of targeted cancer therapies and studies of free radical-related molecular mechanisms.

■ INTRODUCTION
Free radicals, a group of reactive chemical substrates generated
in virtually all types of cells, are indispensable managers in
various biological processes at low levels.1 Indeed, the free
radical theory has been known for more than 50 years.
However, it is only in the past decade that extensive studies
have been carried out to explore their crucial roles in the
development of diseases including cancer.2 Unlike normal cells,
cancer cells generally overproduce free radicals, mainly
hydrogen peroxide (H2O2), throughout their lifetime to drive
rapid proliferation, metastasis, and other events for tumor
development, resulting from metabolic reprogramming.3,4
However, free radical is a double-edged sword,5 such that
crossing the threshold of free radical levels will cause
irreversible oxidative damage and eventually cell death.6,7
Ironically, high levels of inherent free radicals in cancer cells
also make them much more vulnerable to additional oxidative
assault than normal cells.8 Therefore, chemodynamic therapy
(CDT) involving specific manipulation of dysregulated redox
homeostasis in cancer cells is rapidly emerging as a novel, yet
more powerful treatment option than traditional therapies
against a myriad of cancers.9
To date, multiple efforts have been devoted to developing
various CDT strategies for cancer treatment. In essence, these
approaches rely substantially on classical Fenton or Haber−
Weiss reactions to convert endogenous low-reaction H2O2 into
cancer-killing hydroxyl radicals (•OH) by transitional met-
als.10−14 However, two key bottlenecks act as limiting factors
in these processes that significantly impact the otherwise
widespread anticancer applications of CDT. One is associated
with chemical reaction kinetics. It is well-known that high
Fenton and Haber−Weiss reaction rates require very low pH
values (pH 2−4),9,15 making them hard to fully utilize under
tumor microenvironments (TME). The other derives from the
heterogeneity of endogenous H2O2 levels among tumors,
leading to insufficient and dramatically decreased therapeutic
benefit by the consumption of H2O2.8 What is worse, cancer
Received: October 7, 2019
Published: December 20, 2019

© 2019 American Chemical Society 937 DOI: 10.1021/jacs.9b10755

J. Am. Chem. Soc. 2020, 142, 937−944
Journal of the American Chemical Society
Scheme 1. Schematic Illustration of Bioorthogonal ApPdC Micelles for Self-Circulation and In Situ-Amplified Generation of
Toxic Free Radical in Cancer Cells
cells upregulate antioxidant defense (mainly glutathione/GSH)
to adapt to free radical-caused oxidative damage, revealing
another challenge to improving therapeutic efficiency.16 For
maximized therapeutic effect and minimized safety concerns, it
is, therefore, conceivable that an ideal chemodynamic
medication should at least satisfy the following features: (i)
tumor-targeted accumulation for negligible influence on
normal redox signaling; (ii) facile and spatially controlled
chemical reactions for site-directed release of deleterious free
radicals; (iii) less dependence on tumorous ROS for toxic free
radical formation; (iv) concurrent depletion of cancerous
antioxidant defense; and (v) good biocompatibility, biodegrad-
ability, and quality control. However, to the best of our
knowledge, a therapeutic methodology capable of meeting all
of these criteria remains elusive.
Herein, we report, for the first time, the introduction of
bioorthogonal chemistry and prodrug design to create a novel
aptamer drug conjugate (ApDC): an aptamer-prodrug
conjugate (ApPdC) micelle capable of TME dual-targeting
and self-circulation for in situ-amplified generation of toxic free
radicals in cancer cells (Scheme 1). The engineered ApPdC
micelles comprise three moieties: aptamer as the tumor-
recognizing unit, Fe2+-activable tetraoxane (“T”) as the free
radical prodrug, and hemin as the TME-responsive Fe2+ source
for in situ activation of “T”. Several unique properties make
this methodology highly attractive for enhanced and cancer-
targeted CDT. First, unlike traditional micelles that typically
utilize nonfunctional lipids for self-assembly and subsequent
drug delivery,17 a single prodrug base in our design can
function as the hydrophobic tail to trigger self-assembly of
aptamers into a rigid, multicomponent supramolecular
structure, thus simultaneously assuring robust tumor reorgan-
ization, remarkably improved drug loading capacity, and
minimized undesirable leakage of active compounds. Second,
ApPdC micelles are inactive under physiological conditions,
whereas they elicit cascading bioorthogonal reactions upon
receptor-mediated uptake by cancer cells. Specifically, the
loaded hemin (Fe3+) would undergo reduction by intracellular
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GSH into heme (Fe2+) to trigger “T” activation to directly
release toxic C-based free radicals, with no dependence on
tumorous acidity or H2O2. Moreover, hemin reduction-
induced GSH depletion can further decrease the antioxidative
ability of cancer cells, thus exerting synergistic therapeutic
effects. Third, the produced heme (Fe2+) can lead to the
generation of more labile Fe2+ ions in cancer cells, which, in
turn, participate in next-cycle prodrug activation in ApPdC
micelles, thus offering a “vicious” circle for accumulation of C-
based free radicals. Besides, ApDC was automatically
synthesized with well-controlled conjugation site and density
of prodrug bases via phosphoramidite chemistry, enabling
precise and scalable synthesis favorable for future clinical
translation. With these merits, we believe that the combined
bioorthogonal chemistry and prodrug design can be extended
to other aptamers to create an arsenal of advanced ApDCs and
provide new insights into free radical-related molecular
mechanisms.
■ RESULTS AND DISCUSSION
Synthesis and Molecular Assembly of ApPdC. To
obtain the Fe2+-activatable ApPdC, the adamantane-stabilized
tetraoxane monomer (“T”) was first synthesized as the artificial
prodrug base (Figure 1A and Scheme S1), in which the
hydrophobic adamantane segment and hydrogen-bond donor
amide group are essential for micelle formation. After
characterization by NMR and ESI−MS spectrometry (Figures
S1−S9), “T” was then attached to the 5′-end of a 26-
nucleotide DNA aptamer (AS1411), a model aptamer that
specifically recognizes nucleolin overexpressed in many
cancers,18 using the DNA solid-phase synthesizer. Notably,
our previous study revealed that the introduction of
intermolecular G-quadruplexes could help decrease nonspecific
membrane insertion of DNA micelles resulting from disruption
by serum albumin.19 Therefore, six G bases were also
incorporated as the linker between “T” and AS1411 to yield
ApPdC (hereinafter denoted as Ap-6G-“T”), followed by
purification using reversed-phase high performance liquid
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Figure 1. (A) Schematic illustration of the synthesis of “T”. (B) Gel
electrophoresis analysis of (1) free AS1411, (2) Ap-6G-“T”, and (3)
Ap-6G-H-“T”. (C,D) DLS analysis, AFM imaging, and Z-potential of
Ap-6G-“T” and Ap-6G-H-“T”.
chromatography (HPLC) and characterization by mass
spectrometry (Figure S10A). As noted above, the strong
hydrophobicity of “T” enables it to function as a lipid-like tail
to promote self-assembly of Ap-6G-“T” into micelles. Gel
electrophoresis analysis demonstrated bright and compact
bands for free AS1411, whereas a smeared band was observed
for Ap-6G-“T” (Figure 1B), due to the elevated molecular
weight and micellar structure-induced weaker electrophoretic
mobility. This characterization was further confirmed by
atomic force microscopy (AFM) imaging, showing uniform
and monodisperse spherical structures, with an average size of
32.4 nm. Moreover, the Ap-6G-“T” micelles were stable and
negatively charged under physiological conditions without
obvious aggregation, as determined by dynamic light scattering
(DLS) and Z-potential measurements (Figure 1C,D and
Figure S11).
Aside from promoting the molecular self-assembly of
aptamers, the strongly hydrophobic “T” motifs, along with
the secondary structure of DNA sequences, enable ApPdC
micelles to act as a vehicle for simultaneous delivery of other
cargos for combined cancer therapy. Considering the roles of
heme in the iron metabolism in cancer cells,20 hemin, the
oxidized form of heme with Fe3+ in the coordinated center, was
loaded within micelles to yield Ap-6G-H-“T” through hydro-
phobic interactions between hemin and “T”, as well as complex
with the G-quadruplexes21 in Ap-6G-“T”. Interestingly, loading
of hemin had a negligible impact on the structure, as evidenced
by similar morphology and size as compared to hemin-free Ap-
6G-“T” micelles. Only a positive shift of the surface charge was
detected (Figure 1D), presumably attributable to the positive
Fe3+ in hemin. Furthermore, UV−vis absorption provided
additional evidence for the successful loading of hemin (Figure
S12).
Another key advantage of the ApPdC micellar strategy is
precise control over the number and type of drug motifs that
are automatically conjugated with the aptamer at predesignated
sites using modular phosphoramidite chemistry. Therefore,
high drug loading capacity and synergistic therapy can be
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realized. As a proof-of-concept study, Ap-6G-2“T” and Ap-6G-
3“T” with two and three artificial prodrug bases were
synthesized using the DNA synthesizer (Figure S10B,C). As
compared to Ap-6G-“T”, Ap-6G-2“T” and Ap-6G-3“T”
showed further decreased electrophoretic mobility rates
(Figure S13), because of the increased molecular weight and
hydrophobicity. Furthermore, the size and surface charge for
Ap-6G-2“T” and Ap-6G-3“T” gradually increased, as con-
firmed by AFM, DLS, and Z-potential analysis (Figures S14
and S15). Of note, both Ap-6G-2“T” and Ap-6G-3“T” showed
a stronger tendency to self-assemble into compacted micellar
structures, as revealed by the decreased critical micelle
concentration (CMC) value (Figure S16); the CMC values
were comparable to, or even lower than, those of many classic
lipids,22 further revealing the feasibility of using the artificial
prodrug base as a hydrophobic tail to construct multifunctional
DNA micelles.
Serum and Nuclease Stability. Because adequate serum
tolerance is a prerequisite and critical factor in determining
whether a newly developed anticancer drug can be used for in
vivo applications,23 the stability of ApPdC micelles was then
examined. To do this, different ApPdC micelles consisting of
Ap-6G-“T”, Ap-6G-2“T”, or Ap-6G-3“T” were first analyzed in
90% phosphate-buffered serum solutions at 37 °C. As a
reference, “T”-free aptamer was also evaluated. Free aptamers
were found to rapidly degrade with less than 40% of aptamers
left after 48 h of incubation (Figure 2A,B). Impressively, the
Figure 2. Agarose gel analysis and quantification of the degradation
rates of the free aptamer and different ApPdC micelles in FBS (A,B)
or nucleases (C,D).
stability was significantly improved upon conjugation with the
prodrug base under the same condition, with more than 70%
of conjugates remaining, thus revealing their higher resistance
to serum-mediated degradation.
To better elucidate the significance of prodrug bases on
aptamer stabilization, we also compared the antidegradation
performance of ApPdC micelles with free aptamers against
nucleases. Similar to the serum stability experiments, ApPdC
micelles could effectively protect aptamers from nuclease-
regulated degradation. No, or slight, aptamer degradation was
detected for Ap-6G-“T”, Ap-6G-2“T”, and Ap-6G-3“T”
micelles, even after 12 h of incubation with nucleases (Figure
2C,D). Conversely, free aptamers demonstrated a fast
degradation after 2 h. Taken together, these findings clearly
indicated the high stability and high potential of ApPdC
micelles for improved pharmacokinetic profiles and enhanced
bioavailability.
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Figure 3. Activation mechanism of the prodrug base “T” mediated by (A) free Fe2+ or (B) heme (Fe2+). ESR measurements of free radical
generation by “T” under activation of (C) free Fe2+ or (D) heme (Fe2+) produced by dithionite-induced reduction of hemin, using DEPMPO as
the spin-trapping agent.
Activation Mechanisms. Unlike normal cells, accumu-
lated evidence has proven that cancer cells generally acquire
large amounts of Fe2+ to maintain rapid proliferation, growth,
and metastasis by altering the expression of iron metabolism-
related genes to increase iron uptake and storage, as well as
reduce iron export.20,24 Such a high intracellular iron level also
reveals a higher basal level of free radicals in cancer cells versus
normal cells, given iron-mediated Fenton chemistry. However,
cancer cells are more sensitive to changes in free radicals and/
or intracellular Fe2+ levels. Thus, redox homeostasis and Fe2+
in cancer cells represent promising targets for specific cancer
treatment.25−28 To this end, we expected that these Fe2+-
activatable ApPdC micelles could act as a general medication
for targeted cancer treatment.
Prior to cellular investigations, a series of mechanistic studies
were carried out to understand whether Fe2+ is necessary for
activation of ApPdC micelles and the corresponding activation
process for the proposed in situ-amplified generation of toxic
free radicals. Of note, selective codelivery of hemin with
artificial prodrug bases was introduced in our design to allow
Fe2+ self-supply for efficient prodrug activation, as the
coordinated Fe3+ in hemin can be reduced into Fe2+-
coordinated heme by intracellular reducing agents (e.g.,
GSH) in cancer cells.29 To confirm our hypothesis, UV−vis
absorption of hemin in the absence versus presence of “T” was
first recorded using dithionite as the reducing agent.30 In the
absence of “T”, a shift in the absorbance peak from 400 to 418
nm occurred when hemin was reduced to heme by dithionite
(Figure S17A). On the other hand, “T” did not react with
hemin, as indicated by its negligible influence on the
absorption characteristics of hemin. When dithionite was
added into the mixture of “T” and hemin, nevertheless, the
peak centered at 418 nm dropped, accompanied by the
appearance of a new peak at around 475 nm, suggesting the
reaction between heme and “T”. Similar absorption changes
were observed in the case of Ap-6G-H-“T” (Figure S17B). It is
also noteworthy that rapid and effective prodrug activation
could be achieved at different TME-relevant pH values (Figure
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S18). These observations indicated that hemin-loaded ApPdC
micelles could be used as a Fe2+ self-provisioned platform for
in situ and pH-independent activation in cancer cells.
After reduction, the prodrug base “T” would be activated by
heme (Fe2+)/free Fe2+ to release toxic radicals via single
electron transfer (Figure 3A,B). The entire activation process
involves the formation of Fe2+-peroxy bridge complexes,
followed by single electron transfer from the metal to the
anti-bonding orbital σ* of the peroxy bridge. As a result, the
peroxide bond can be cleaved, triggering the generation of oxy-
radicals via bioorthogonal chemistry. The produced oxy-
radicals are not stable, and they undergo rearrangements via
C−C β scission into C-centered radicals.31 In addition to
direct injury, these radicals could stimulate excessive oxidative
damage to cancer cells. To verify this activation mechanism, we
first carried out spin-trapping experiments to explore the
formation of C-centered radical intermediates in the reaction
between “T” and Fe2+, using (2,2,6,6-tetramethylpiperidin-1-
yl)oxyl (TEMPO) as the radical marker. TEMPO can capture
the generated C-centered radicals to give TEMPO adducts, as
identified by ESI−MS (Figure S19). Furthermore, TEMPO by
itself is a stable nitroxide radical-containing compound, and it
thus produces strong signals by electron spin resonance (ESR)
spectroscopy. However, the ESR signals can be decreased upon
oxidation by other reactive free radicals. As shown in Figure
S20, the ESR amplitude of TEMPO was significantly decreased
by “T” in the presence of either Fe2+ or hemin-dithionite
mixture, providing additional support for free radical formation
by activated “T”.
To directly confirm the formation of C-centered radicals
during the activation process of ApPdC micelles, we also
performed ESR analysis using 5-diethoxyphosphoryl-5-methyl-
1-pyrroline N-oxide (DEPMPO) as the spin-trap agent.32
Unlike TEMPO, DEPMPO shows no ESR signals, but can
react with the highly reactive O- or C-centered free radicals to
form stable paramagnetic adducts, coupled with the generation
of strong ESR signals. As shown in Figure 3C,D, no ESR signal
was collected for DEPMPO alone, DEPMPO + Fe2+/hemin, or
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Figure 4. Flow cytometry analysis (A) and confocal fluorescence imaging (B) of the binding capability of different ApPdCs and nontargeted
micelles in HepG2 cells. (C) Lysosomal colocalization of free aptamer and ApPdC micelles.
hemin + dithionite groups. Conversely, when hemin-loaded
ApPdC micelles were added into the solution containing
hemin + dithionite/Fe2+, strong ESR signals from the
paramagnetic adduct of DEPMPO were detected at the same
conditions, suggesting the ability of ApPdC micelles to
produce free radicals, but only after Fe2+ activation. It is also
worth noting that H2O2 was not required for the generation of
C-centered free radicals in such Fe2+-mediated bioorthogonal
chemistry. Moreover, the pH value of the reaction system has
no obvious influence on the generation efficacy of free radicals,
as confirmed by ESR (Figure S21), indicating that ApPdC
micelles can be used for H2O2- and pH-independent cancer
CDT.
Cancer Cell Recognition and In Situ Generation of
Toxic Free Radicals. After having revealed the activation
mechanism of ApPdC micelles, we then investigated their
targeting capability and internalization in cancer cells. In this
study, HepG2, a liver hepatocellular cell line characterized by
upregulation of nucleolin,33 was used as the cancer model. For
the purpose of effective cancer therapy and minimized off-
target effect, we first examined the impact of the number of
prodrug “T” motifs on the cell targeting behavior of ApPdC
micelles. After respective incubation of HepG2 cells with
ApPdC micelles containing one to five “T” bases within a
single ApPdC strand, as well as ApPdC micelles with a random
DNA sequence, the aptamer-mediated nucleolin recognition
was investigated by flow cytometry analysis (Figure S22). As
the number of “T” bases increased to three, nonspecific cell
binding of ApPdC micelles to HepG2 cells became apparent,
but no significant binding difference between targeted and
nontargeted micelles was found, which was presumably
ascribed to nonspecific membrane insertion caused by the
enhanced hydrophobicity of the tail.19,34 Therefore, Ap-6G-“T”
and Ap-6G-2“T” were studied for the following character-
izations, unless otherwise indicated.
Impressively, the encapsulation of hemin did not have any
effect on the AS1411-mediated nucleolin recognition ability of
ApPdC micelles. Ap-6G-H-2“T” demonstrated stronger
fluorescence and a relatively higher shift in flow cytometry as
compared to Ap-6G-H-“T” (Figure 4A,B), due to receptor-
and rigid spherical structure-mediated cellular uptake.35 As
time passed, cellular uptake of ApPdC micelles by HepG2 cells
gradually increased, with effective transportation from
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membrane into cytosome at the 4 h time interval (Figure
S23). In sharp contrast, no noticeable internalization was
detected for those nontargeted micelles. In addition, cell
membrane staining experiments also revealed no or negligible
overlay between Ap-6G-H-“T”/Ap-6G-H-2“T” and cell mem-
brane staining dye at the same time point (Figure S24).
It is well-known that lysosomes are compartments that can
accumulate high levels of redox and iron, thus becoming more
vulnerable to further oxidative assault induced by exogenous
agents.36 They also play an important role in regulating cell
apoptosis or necrosis.37 In this regard, we then asked whether
Fe2+-activated ApPdC micelles would accumulate in lysosomes
to amplify the oxidative stress and trigger efficient cancer cell
apoptosis. Specifically, HepG2 cells were incubated with free
AS1411, Ap-6G-“T”, or Ap-6G-2“T” labeled with FITC, and
their lysosomal colocalization was investigated by lysosomal
staining with LysoTracker Red. As expected, both Ap-6G-“T”
and Ap-6G-2“T” demonstrated higher cellular uptake and
localization in lysosomes as compared to the free aptamer
group (Figure 4C), primarily ascribable to the micellar
structures. Interestingly, aside from lysosomes, we also found
that part of ApPdC micelles accumulated in mitochondria, as
identified by the colocalization of fluorescence between FITC
and MitoTracker (Figure S25). Because mitochondria are
known as vital organelles with numerous biosynthetic,
bioenergetic, and regulatory functions, essentially contributing
to dysregulated iron and redox homeostasis in cancer cells,38 it
is thus not surprising to see the mitochondrial accumulation of
Fe2+-activated ApPdC micelles. We therefore expect that such
high accumulation in redox-related subcellular compartments
could allow effective activation of ApPdC micelles and
subsequently enhanced anticancer efficiency.
Having confirmed receptor-mediated cancer cell recognition
and internalization of ApPdC micelles, we then assessed
whether they could be used for site-directed release of toxic
free radicals in cancer cells. HepG2 cells were treated with
hemin, R-6G-“T”, Ap-6G-“T”, or Ap-6G-H-“T”, and the
intracellular free radical level was monitored using 2′,7′-
dichlorofluoroscein diacetate (DCFH-DA) as the indicator. As
shown in Figure 5, very weak fluorescence was noticed for
control cells and the cells treated with free hemin, indicating
the low levels of intracellular free radicals. As a comparison,
when cells were treated with R-6G-“T”, they showed increased
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Figure 5. Flow cytometry analysis (A) and confocal fluorescence
imaging (B) of the free radical levels in HepG2 cells with different
treatments.
amounts of free radicals, as a result of “T” activation by
intracellular Fe2+ and subsequent bioorthogonal reactions,39
similar to the case of free “T” base (Figure S26). Such
activation was further enhanced in cells treated with Ap-6G-
“T”, benefiting from the increased receptor-mediated cellular
uptake. More impressively, the strongest green fluorescence
was observed for Ap-6G-H-“T”-incubated cells, suggesting a
further enhanced level of free radicals. Notably, no obvious
difference in intracellular free radical accumulation was noticed
in ApPdC micelle-treated HepG2 cells under different pH
values (Figure S27), further confirming the independence of
ApPdC micelles from the strong acidic environment for
effective chemical reaction. We speculate that the mechanisms
underlying the significantly elevated free radical production by
ApPdC micelles may be attributable to two potential processes.
First, after internalization, the loaded hemin (Fe3+) in ApPdC
Figure 6. (A,B) Cell viability of HepG2 cells treated with Ap-6G-“T”, Ap-6G-H-“T”, and the corresponding nontargeted samples. (C) Calcein-AM
and propidium iodide (PI) costaining of HepG2 cells alone and HepG2 cells treated with Ap-6G-“T”, Ap-6G-H-“T”, or the corresponding
nontargeted samples. (D,E) Cell viabilities of HepG2 cells treated with Ap-6G-2“T”, Ap-6G-H-2“T”, or the corresponding nontargeted samples.
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micelles will be reduced into heme (Fe2+) by GSH in cancer
cells, as confirmed by the decreased intracellular GSH content
upon treatment with ApPdC micelles (Figure S28). Along with
the inherent Fe2+ in cancer cells, this will trigger the
aforementioned cascade reaction of “T” more efficiently than
will the hemin-free samples. Second, the free radical-induced
stress could activate intracellular heme oxygenase in cancer
cells to degrade the as-produced heme and release free Fe2+,40
as identified by increased intracellular iron level in ApPdC
micelle-treated cells (Figure S29), which could, in turn,
participate in the next-cycle prodrug activation to release
more toxic C-centered radicals. In addition to direct free
radical-induced injury, the elevated free Fe2+ may also
potentially accelerate cancer cell death via ferroptotic path-
way-mediated lysosomal permeabilization,8,41 as identified by
the loss of lysosomal membrane integrity (Figure S30).
Meanwhile, intracellular GSH depletion caused by hemin
reduction could significantly diminish cancerous antioxidation,
leading to an improved cancer-killing effect of the resulting
toxic C-centered radicals.
Synergistic Cytotoxicity in Cancer Cells. Given the
promising specific recognition and site-selective activation
mechanisms of ApPdC micelles, we proceeded to examine
their targeted anticancer efficiency. For this purpose, HepG2
cells were treated with Ap-6G-“T” or nontargeted R-6G-“T”
for 48 h, and cell viability was examined using the CCK-8
assay. To better illustrate its potential for specific cancer
therapy, the cytotoxicity of free “T” in HepG2 cells was also
investigated. As depicted in Figure 6A, Ap-6G-“T” showed a
dose-dependent inhibition of cell proliferation and growth with
an IC50 value of 18.84 μM. In contrast, neither R-6G-“T” nor
free AS1411 showed obvious toxicity to the HepG2 cells at the
same tested concentrations (Figure S31A). Moreover, a
significant cytotoxicity could be detected for free “T” only at
the concentration up to 50 μM (Figure S31B). These findings
indicated the contribution of ApPdC micelles to the enhanced
cellular uptake of “T”. On the other hand, we also prepared
another aptamer-based micelle (Figure S10D) using a
conventional lipid to exclude the micellar structure-mediated
enhanced uptake and subsequent cytotoxicity of AS1411, given
its intrinsic therapeutic effect.18 Nevertheless, no noticeable
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toxicity was detected, and the cell survival remained almost
90%, even at the highest tested concentration (Figure S31C).
To validate the potential of ApPdC micelles for synergistic
cancer therapy, the cytotoxicity of hemin-loaded ApPdC
micelles was measured. Obviously, Ap-6G-H-“T” inhibited
cell proliferation and growth more efficiently than hemin-free
Ap-6G-“T”, and the IC50 value was decreased to 10.42 μM at
the same conditions (Figure 6B). However, no significant
toxicity was seen in the control cells treated with either free
hemin or nontargeted R-6G-H-“T” (Figure S31D). Such an
amplified cytotoxicity effect was directly visualized by Calcein-
AM/PI costaining. As shown in Figure 6C, Ap-6G-H-“T” was
among the most effective in killing cancer cells, and most cells
were dead upon 48 h incubation, as evidenced by the strongest
red fluorescence among different experimental groups. In
contrast, negligible cell death was detected for cells treated
with PBS, R-6G-“T”, or R-6G-H-“T”, which was very
consistent with the results from CCK-8 assay. Moreover, the
cell-killing effect could be augmented as the number of “T”
bases increased (Figure 6D,E). As compared to mono “T”-
based ApPdC micelles, the IC50 value was decreased by 2.2-
and 2.7-fold for Ap-6G-2“T” (6.88 μM) and Ap-6G-H-2“T”
(4.79 μM), respectively. No cells survived at 20 μM. Notably,
Ap-6G-H-2“T” was among the most powerful in inhibiting cell
survival and proliferation, and almost 90% of HepG2 cells were
killed at 10 μM, clearly suggesting its high potential for
efficient selective cancer therapy.
Finally, we also compared the cytotoxicity of ApPdC
micelles in HepG2 cells in the presence versus the absence
of the intracellular iron blocker42 to further support Fe2+-
modulated in situ activation mechanisms underlying the potent
synergistic in vitro anticancer efficacy of ApPdC micelles. As
expected, the preblocking of iron in HepG2 cells significantly
prevented cell death caused by ApPdC micelles, as compared
to the control groups at the same dose (Figure S32),
highlighting the crucial role of Fe2+ in the activation of
prodrug bases.
■
CONCLUSION
In summary, we have successfully synthesized a novel targeted
anticancer drug by combining prodrug and bioorthogonal
chemistry to target dysregulated redox homeostasis in cancer
cells for enhanced and cancer-targeted chemodynamic therapy.
Comprehensive mechanistic studies were performed to
improve our fundamental understanding of cancer cell-specific
activation and therapeutic activity of the engineered ApPdC
micelles. Our findings revealed that prodrug bases in ApPdC
micelles could be activated by intracellular Fe2+ to trigger
cascading bioorthogonal reactions for in situ-amplified
generation of toxic C-centered radicals. Moreover, the strong
hydrophobic prodrug bases allow loading of hemin in ApPdC
micelles. The loaded hemin could be reduced into heme by the
elevated GSH inside cancer cells, thus offering a Fe2+ self-
supplied platform to promote sufficient bioorthogonal
reactions. Notably, such a free radical generation process is
advantageous over traditional chemodynamic therapies, with-
out reliance on strong acidic pH or endogenous H2O2, yet with
concurrently diminished cancerous antioxidation by depleting
GSH. Using AS1411 as a model aptamer, the potential of
ApPdC micelles for specifically modulating free radicals in
cancer cells has been systemically evaluated. Our data showed
that ApPdC micelles could specifically recognize cancer cells
via receptor-mediated targeting, with enhanced cellular uptake
943
Article
as compared to nontargeted micelles. After endocytosis,
ApPdC micelles could produce toxic radicals more efficiently
than nontargeted micelles, contributing to significant oxidative
damage to cancer cells and enhanced antiproliferation effect in
cancer cells. More impressively, such anticancer effect could be
augmented by codelivery of hemin or increasing the number of
prodrug bases in the ApPdC strand. While our findings
demonstrated promising therapeutic profiles, further studies
will still be needed, such as in vivo pharmacokinetics and
anticancer efficacy of ApPdC micelles, to better illustrate their
potential for cancer treatment. Furthermore, even though
ApPdC micelles are designed to target the unique cancer cell
metabolism for the purpose of minimized side effects, more
investigations will be required in the future to evaluate the
long-term safety concerns to normal cells or tissues. However,
because dysregulated iron metabolism and vulnerability to
oxidative stress represent the common features among cancers,
this study can lay the foundation for developing novel effective
ApPdCs to specifically treat a myriad of advanced cancers.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.9b10755.
Experimental details and supporting results for synthesis;
NMR and ESI−MS characterization of the “T” base and
“T”-containing ApPdC; AFM, DLS, and CMC determi-
nation of different ApPdC micelles; UV−vis analysis of
hemin encapsulation and reduction; ESR measurement
of “T” activation; confocal fluorescence imaging of the
cellular uptake, free radical generation, and membrane
integrity of lysosomes; and cytotoxicity studies of
different samples in HepG2 cells (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*ylliu@hnu.edu.cn
*tan@hnu.edu.cn
ORCID
Weihong Tan: 0000-0002-8066-1524
Author Contributions
∥
W.X. and Y.X. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We are thankful for financial support from the National
Natural Science Foundation of China (NSFC21827811,
61527806, and 21877031) and the Science and Technology
Innovation Program of Hunan Province (2017XK2103 and
2018RS3043).
■
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