Molecular Self-Assembly of Bioorthogonal Aptamer-Prodrug Conjugate Micelles for Hydrogen Peroxide and pH-Independent Cancer Chemodynamic Therapy

Article abstract of DOI:10.1021/jacs.9b10755

Chemodynamic therapy (CDT) has demonstrated new possibilities for selective and logical cancer intervention by specific manipulation of dysregulated tumorous 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.

Full text of DOI:10.1021/jacs.9b10755

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Article
Molecular Self-assembly of Bioorthogonal Aptamer-
Prodrug Conju-gate Micelles for Hydrogen Peroxide
and pH-Independent Cancer Chemodynamic Therapy
Wenjing Xuan, yinghao xia, Ting Li, Linlin Wang, Yanlan Liu, and Weihong Tan
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b10755 • Publication Date (Web): 20 Dec 2019
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Molecular Self-assembly of Bioorthogonal Aptamer-Prodrug Conju-
gate Micelles for Hydrogen Peroxide and pH-Independent Cancer
Chemodynamic Therapy
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＃a
＃a
Wenjing Xuan , Yinghao Xia , Ting Lia, Linlin Wanga, Yanlan Liu*a and Weihong Tan*a,b,c
aMolecular 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.
bInstitute of Cancer and Basic Medicine (IBMC), Chinese Academy of Sciences; The Cancer Hospital of the University of
Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China.
cFoundation for Applied Molecular Evolution, 13709 Progress Boulevard, Alachua, FL 32615, USA.
KEYWORDS: Aptamer-prodrug conjugate, bioorthogonal chemistry, self-assembly, free radicals, chemodynamic therapy
ABSTRACT: Chemodynamic therapy (CDT) has demonstrated new possibilities for selective and logical cancer intervention by
specific manipulation of dysregulated tumorous free radical homeostasis. Current CDT methods largely rely on conversion of endog-
enous 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 conjugates (ApDC): aptamer-
prodrug conjugate (ApPdC) micelle for improved and cancer-targeted CDT. The hydrophobic prodrug base 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 neither H2O2 nor pH, yet concurrently with dimin-
ished 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.
yet more powerful treatment option than traditional therapies
against a myriad of cancers.9
INTRODUCTION
Free radicals, a group of reactive chemical substrates generated
in virtually all types of cells, are indispensable managers in var-
ious biological processes at low levels.1 Indeed, the free radical
theory has been known for more than fifty years. However, it is
only the past decade that extensive studies have been carried out
to explore their crucial roles in the development of diseases in-
cluding 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 meta-
bolic 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,
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 exogenous low-reaction H2O2 into
cancer-killing hydroxyl radicals (.OH) by transitional metals.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 chemi-
cal reaction kinetics. It is well known that high Fenton and Ha-
ber-Weiss reaction rates require very low pH values (pH 2-4),9,
15 making them hard to fully utilize under tumor microenviron-
ments (TME). The other derives from the heterogeneity of ex-
ogenous H2O2 levels among tumors, leading to insufficient and
dramatically decreased therapeutic benefit by the consumption
of H2O2.8 What's worse, cancer cells upregulate antioxidant de-
fense (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
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Scheme 1. Schematic illustration of bioorthogonal ApPdC micelles for self-circulation and in-situ amplified generation of toxic free radi-
cal in cancer cells.
ideal chemodynamic medication should at least satisfy the fol-
lowing features: (i) tumor-targeted accumulation for negligible
influence on normal redox signaling; (ii) facile and spatially
controlled chemical reactions for site-directed release of delete-
rious free radicals; (iii) less dependence on tumorous ROS for
toxic free radical formation; (iv) concurrent depletion of can-
cerous antioxidant defense, and (v) good biocompatibility, bio-
degradability, and quality control. However, to the best of our
knowledge, a therapeutic methodology capable of meeting all
of these criteria remains elusive.
on tumorous acidity or H2O2. Moreover, hemin reduction-in-
duced GSH depletion can further decrease the antioxidative
ability of cancer cells, thus exerting synergistic therapeutic ef-
fects. Third, the produced heme (Fe2+) can lead to generation of
more labile Fe2+ ions in cancer cells, which, in turn, participate
in next-cycle prodrug activation in ApPdC micelles, thus offer-
ing a “vicious” circle for accumulation of C-based free radicals.
Besides, ApDC was automatically synthesized with well-con-
trolled conjugation site and density of prodrug bases via phos-
phoramidite 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-re-
lated molecular mechanisms.
Herein, we report, for the first time, the introduction of
bioorthogonal chemistry and prodrug design to create a novel
aptamer drug conjugates (ApDC): aptamer-prodrug conjugate
(ApPdC) micelle capable of TME dual-targeting and self-circu-
lation for in-situ amplified generation of toxic free radicals in
cancer cells (Scheme 1). The engineered ApPdC micelles com-
prise 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, multi-compo-
nent supramolecular structure, thus simultaneously assuring ro-
bust tumor reorganization, remarkably improved drug loading
capacity, and minimized undesirable leakage of active com-
pounds. Second, ApPdC micelles are inactive under physiolog-
ical conditions, whereas they elicit cascading bioorthogonal re-
actions upon receptor-mediated uptake by cancer cells. Specif-
ically, the loaded hemin (Fe3+) would undergo reduction by in-
tracellular GSH into heme (Fe2+) to trigger “T” activation to di-
rectly release toxic C-based free radicals, with no dependence
RESULTS AND DISCUSSION
Synthesis and molecular assembly of ApPdC
To obtain the Fe2+-activatable ApPdC, the adamantane-stabi-
lized tetraoxane monomer (“T”) was first synthesized as the ar-
tificial 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 charac-
terization by NMR and ESI-MS spectrometry (Figure S1-S9),
“T” was then attached to the 5’-end of a 26-nucleotide DNA
aptamer (AS1411), a model aptamer that specifically recog-
nizes nucleolin overexpressed in many cancers,18 using the
DNA solid phase synthesizer. Notably, our previous study re-
vealed that the introduction of intermolecular G-quadruplexes
could help decrease nonspecific membrane insertion of DNA
micelles resulting from disruption by serum albumin.19 There-
fore, 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
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performance liquid chromatography (HPLC) and characteriza-
high drug loading capacity and synergistic therapy can be real-
ized. As a proof-of-concept study, Ap-6G-2“T” and Ap-6G-3“T”
with two and three artificial prodrug bases were synthesized us-
ing the DNA synthesizer (Figure S10B,C). Compared to Ap-
6G-“T”, Ap-6G-2“T” and Ap-6G-3“T” showed further de-
creased electrophoretic mobility rates (Figure S13), because of
the increased molecular weight and hydrophobicity. Further-
more, the size and surface charge for Ap-6G-2“T” and Ap-6G-
3“T” gradually increased, as confirmed by AFM, DLS and Z-
potential analysis (Figure S14-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.
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tion 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 mon-
odisperse 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 aggre-
gation, as determined by dynamic light scattering (DLS) and Z-
potential measurements (Figure 1C,D and Figure S11).
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Figure 2. Agarose gel analysis and quantification of the degrada-
tion rates of the free aptamer and different ApPdC micelles in FBS
(A, B) or nucleases (C,D).
Serum and nuclease stability
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”.
Since adequate serum tolerance is a prerequisite and critical fac-
tor 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 mi-
celles 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). Impres-
sively, the stability was significantly improved upon conjuga-
tion with the prodrug base under the same condition, with more
than 70% of conjugates remaining, thus revealing their higher
resistance to serum-mediated degradation.
Aside from promoting the molecular self-assembly of ap-
tamers, 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 mi-
celles to yield Ap-6G-H-“T” through hydrophobic interactions
between hemin and “T”, as well as complex with the G-quad-
ruplexes21 in Ap-6G-“T”. Interestingly, loading of hemin had a
negligible impact on the structure, as evidenced by similar mor-
phology and size 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. Fur-
thermore, UV-Vis absorption provided additional evidence for
the successful loading of hemin (Figure S12).
To better elucidate the significance of prodrug bases on
aptamer stabilization, we also compared the anti-degradation
performance of ApPdC micelles with free aptamers against nu-
cleases. Similar to the serum stability experiments, ApPdC mi-
celles could effectively protect aptamers from nuclease-regu-
lated degradation. No, or slight, aptamer degradation was de-
tected 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.
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,
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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
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 S18).
These observations indicated that hemin-loaded ApPdC mi-
celles could be used as a Fe2+ self-provisioned platform for in-
situ and pH-independent activation in cancer cells.
Unlike normal cells, accumulated evidence has proven that can-
cer cells generally acquire large amounts of Fe2+ to maintain
rapid proliferation, growth, and metastasis by altering the ex-
pression of iron metabolism-related genes to increase iron up-
take and storage, as well as reduce iron export.20, 24 Such high
intracellular iron level also reveals a higher basal level of free
radicals in cancer cells vs. 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 ex-
pected that these Fe2+-activatable ApPdC micelles could act as
a general medication for targeted cancer treatment.
After reduction, the prodrug base “T” would be activated
by heme (Fe2+)/free Fe2+ to release toxic radicals via single elec-
tron transfer (Figure 3A,B). The entire activation process in-
volves the formation of Fe2+-peroxy bridge complexes, fol-
lowed by single electron transfer from the metal to the anti-
bonding orbital σ* of the peroxy bridge. As a result, the perox-
ide bond can be cleaved, triggering the generation of oxy-radi-
cals via bioorthogonal chemistry. The produced oxy-radicals
are not stable, and they undergo rearrangements via C-C β scis-
sion into C-centered radicals.31 In addition to direct injury, these
radicals could stimulate excessive oxidative damage to cancer
cells. In order to verify this activation mechanism, we first car-
ried 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 gen-
erated C-centered radicals to give TEMPO adducts, as identi-
fied by EI-MS (Figure S19). Furthermore, TEMPO by itself is
a stable nitroxide radical-containing compound, and it thus pro-
duces strong signals by electron spin resonance (ESR) spectros-
copy. However, the ESR signals can be decreased upon oxida-
tion 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 acti-
vated “T”.
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 activa-
tion 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 coordi-
nated 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 ab-
sence vs. presence of “T” was first recorded using dithionite as
the reducing agent.30 In the absence of “T”, a shift in the absorb-
ance peak from 400 to 418 nm occurred when hemin was re-
duced to heme by dithionite (Figure S17A). On the other hand,
“T” did not react with hemin, as indicated by its negligible in-
fluence on the absorption characteristics of hemin. When dithi-
onite was added into the mixture of “T” and hemin, nevertheless,
the peak centered at 418 nm dropped, accompanied by the ap-
pearance of a new peak at around 475 nm, suggesting the reac-
tion between heme and “T”. Similar absorption changes were
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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.
To directly confirm the formation of C-centered radicals
during the activation process of ApPdC micelles, we also per-
formed 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 col-
lected for DEPMPO alone, DEPMPO + Fe2+/hemin, or hemin +
dithionite groups. Conversely, when hemin-loaded ApPdC mi-
celles were added into the solution containing hemin + dithio-
nite/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 af-
ter Fe2+ activation. It is also worth noting that H2O2 was not re-
quired 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 (Fig-
ure S21), indicating that ApPdC micelles can be used for H2O2-
and pH-independent cancer CDT.
nontargeted micelles, which was presumably ascribed to non-
specific membrane insertion caused by the enhanced hydropho-
bicity of the tail.19,34 Therefore, Ap-6G-“T” and Ap-6G-2“T”
were studied for the following characterizations, unless other-
wise indicated.
Impressively, the encapsulation of hemin did not have any
effect on AS1411-mediated nucleolin recognition ability of Ap-
PdC micelles. Ap-6G-H-2“T” demonstrated stronger fluores-
cence and a relatively higher shift in flow cytometry 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 was gradu-
ally increased, with effective transportation from 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 ex-
periments also revealed no or negligible overlay between Ap-
6G-“T”/Ap-6G-2“T” and cell membrane 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 lo-
calization in lysosomes compared to the free aptamer group
(Figure 4C), primarily ascribable to the micellar structures. In-
terestingly, aside from lysosomes, we also found that part of
ApPdC micelles accumulated in mitochondria, as identified by
the colocalization of fluorescence between FITC and Mito-
Tracker (Figure S25). Since mitochondria are known as vital
organelles with numerous biosynthetic, bioenergetic, and regu-
latory functions, essentially contributing to dysregulated iron
Cancer cell recognition and in-situ generation of toxic free
radicals
After having revealed the activation mechanism of ApPdC mi-
celles, we then investigated their targeting capability and inter-
nalization in cancer cells. In this study, HepG2, a liver hepato-
cellular cell line characterized by upregulation of nucleolin,33
was used as the cancer model. For the purpose of effective can-
cer therapy and minimized off-target effect, we first examined
the impact of the number of prodrug “T” motifs on the cell tar-
geting 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 (Fig-
ure S22). As the number of “T” increased to three, nonspecific
cell binding of ApPdC micelles to HepG2 cells became appar-
ent, but no significant binding difference between targeted and
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and redox homeostasis in cancer cells,38 it is thus not surprising
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 signifi-
cant cytotoxicity could be detected for free “T” only at the con-
centration up to 50 μM (Figure S31B). These findings indicated
the contribution of ApPdC micelles to the enhanced cellular up-
take of “T”. On the other hand, we also prepared another ap-
tamer-based micelle (Figure S10D) using a conventional lipid
to exclude the micellar structure-mediated enhanced uptake and
subsequent cytotoxicity of AS1411, given its intrinsic therapeu-
tic effect.18 Nevertheless, no noticeable toxicity was detected,
and the cell survival remained almost 90%, even at the highest
tested concentration (Figure S31C).
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 anti-
cancer efficiency.
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Having confirmed receptor-mediated cancer cell recogni-
tion 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’-dichlorofluoro-
scein diacetate (DCFH-DA) as the indicator. As shown in Fig-
ure 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 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 re-
ceptor-mediated cellular uptake. More impressively, the strong-
est green fluorescence was observed for Ap-6G-H-“T”-incu-
bated cells, suggesting a further enhanced level of free radicals.
Notably, no obvious difference in intracellular free radical ac-
cumulation was noticed in ApPdC micelle-treated HepG2 cells
under different pH values (Figure S27), further confirming the
independency of ApPdC micelles on strong acidic environment
for effective chemical reaction. We speculate that the mecha-
nisms underlying the significantly elevated free radical produc-
tion by ApPdC micelles may be attributable to two potential
processes. First, after internalization, the loaded hemin (Fe3+) in
ApPdC 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
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 iden-
tified 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-cen-
tered radicals. In addition to direct free radical-induced injury,
the elevated free Fe2+ may also potentially accelerate cancer cell
death via ferroptotic pathway-mediated lysosomal permeabili-
zation,8,41 as identified by the loss of lysosomal membrane in-
tegrity (Figure S30). Meanwhile, intracellular GSH depletion
caused by hemin reduction could significantly diminish cancer-
ous antioxidation, leading to improved cancer-killing effect of
the resulting toxic C-centered radicals.
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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.
To validate the potential of ApPdC micelles for synergistic
cancer therapy, the cytotoxicity of hemin-loaded ApPdC mi-
celles 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 cy-
totoxicity effect was directly visualized by Calcein-AM/PI cos-
taining. 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 fluores-
cence among different experimental groups. In contrast, negli-
gible 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” increased (Figure 6D,E). Com-
pared 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.
Synergistic cytotoxicity in cancer cells
Given the promising specific recognition and site-selective ac-
tivation mechanisms of ApPdC micelles, we proceeded to ex-
amine 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
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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.
Finally, we also compared the cytotoxicity of ApPdC mi-
celles in HepG2 cells in the presence vs. absence of the intra-
cellular 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.
oxidative damage to cancer cells and enhanced anti-prolifera-
tion 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 in-
vestigations will be required in the future to evaluate the long-
term safety concerns to normal cells or tissues. However, since
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.
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 activa-
tion 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 pro-
mote sufficient bioorthogonal reactions. Notably, such free rad-
ical generation process is advantageous over traditional chemo-
dynamic therapies, without reliance on strong acidic pH or en-
dogenous H2O2, yet with concurrently diminished cancerous
antioxidation by depleting GSH. Using AS1411 as a model ap-
tamer, the potential of ApPdC micelles for specifically modu-
lating free radicals in cancer cells has been systemically evalu-
ated. Our data showed that ApPdC micelles could specifically
recognize cancer cells via receptor-mediated targeting, with en-
hanced cellular uptake compared to nontargeted micelles. After
endocytosis, ApPdC micelles could produce toxic radicals more
efficiently than nontargeted micelles, contributing to significant
ASSOCIATED CONTENT
Supporting Information. Experimental details and supporting re-
sults 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 en-
capsulation and reduction; ESR measurement of “T” activation;
confocal fluorescence imaging of the cellular uptake, free radical
generation and membrane integrity of lysosomes; cytotoxicity
studies of different samples in HepG2 cells. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*ylliu@hnu.edu.cn
*tan@hnu.edu.cn
Author Contributions
W. J. Xuan and Y. H. Xia contributed equally to this work.
Notes
The authors declare no competing financial interest.
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Dissociation of an Intermolecular G-Quadruplex. ACS Nano 2017, 11,
12087-12093.
ACKNOWLEDGMENT
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Financial support from the National Natural Science Foundation of
China (NSFC21827811, 61527806, and 21877031), Science and
Technology Innovation Program of Hunan Province (2017XK2103
and 2018RS3043).
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