Superoxide Radical Photogenerator with Amplification Effect: Surmounting the Achilles' Heels of Photodynamic Oncotherapy

Article abstract of DOI:10.1021/jacs.8b13141

Strong oxygen dependence, poor tumor targeting, and limited treatment depth have been considered as the "Achilles' heels" facing the clinical usage of photodynamic therapy (PDT). Different from common approaches, here, we propose an innovative tactic by u

Full text of DOI:10.1021/jacs.8b13141

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Article
Superoxide Radical Photogenerator with Amplification Effect:
Surmounting the Achilles’ heels of Photodynamic Oncotherapy
Mingle Li, Tao Xiong, Jianjun Du, Ruisong Tian, Ming Xiao, Lianying Guo, Saran Long,
Jiangli Fan, Wen Sun, Kun Shao, Xiangzhi Song, James W. Foley, and Xiaojun Peng
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13141 • Publication Date (Web): 17 Jan 2019
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Superoxide Radical Photogenerator with Amplification Effect:
Surmounting the Achilles’ heels of Photodynamic Oncotherapy
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,
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⊥
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†
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‡
†
†
Mingle Li, Tao Xiong, Jianjun Du,
Ruisong Tian, Ming Xiao, Lianying Guo, Saran Long,
Jiangli Fan,
,
,
, ,
⊥
⊥
⊥
†
†
ǀǀ
§
* †
Wen Sun,
Kun Shao,
Xiangzhi Song, James W. Foley, and Xiaojun Peng
†State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China
‡
Department of Pathophysiology, Dalian Medical University, Dalian 116044, China
ǀǀ
College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China
§
Rowland Institute at Harvard, Harvard University, Cambridge, Massachusetts 02142, United States
⊥Research Institute of Dalian University of Technology in Shenzhen, Shenzhen 518057, China
ABSTRACT: Strong oxygen dependence, poor tumor targeting, and limited treatment depth have been considered as the “Achilles’
heels” facing the clinical usage of photodynamic therapy (PDT). Different from common approaches, here, we propose an
innovative tactic by using photon-initiated dyad cationic superoxide radical (O2−•) generator (ENBOS) featuring “0 + 1 > 1”
amplification effect to simultaneously overcome these drawbacks. In particular, by taking advantage of the Förster resonance
energy transfer theory, the energy donor successfully endows ENBOS with significantly enhanced NIR absorbance and photon
−•
utility, which in turn lead to ENBOS more easily activated and generating more O2 in deep tissues, thus dramatically intensifies
the type I PDT against hypoxic deep tumors. Moreover, benefiting from the dyad cationic feature, ENBOS achieves superior
“structure-inherent targeting” abilities with the signal-to-background ratio as high as 25.2 at 48 h post intravenous injection,
offering opportunities for accurate imaging-guided tumor treatment. Meanwhile, the intratumoral accumulation and retention
performance are also markedly improved (> 120 h). Based on these unique merits, ENBOS selectively inhibits the deep-seated
hypoxic tumor proliferation at a low light-dose irradiation. Therefore, this delicate design may open up new horizons and cause a
paradigm change for PDT in future cancer therapy.
(e.g., antibodies, metabolic substrates, and small interfering
RNA),18-21 and nanotechnologies based on enhanced
INTRODUCTION
Photodynamic therapy (PDT),
a noninvasive treatment
permeability and retention (EPR) effects.22,23 To fight against
hypoxia, recently, Yoon and Liu have reviewed the facile
approaches for ameliorating anti-hypoxia efficiencies,24 such
as packaging O2 self-supplement materials including MnO2,10
catalase,25-27 or perfluorocarbon,28 with PSs in one “package”,
hypoxia responsive prodrugs,29,30 etc.. While for deep PDT,
utilizing smart excitation tools (e.g., X-ray,31 two photon,32,33
upconversion,34 and internal self-luminescence35,36) are the
most common patterns. Indeed, these approaches have
achieved significant advance, however, they can hardly
overcome all three challenges at the same time. Besides, owing
to above-mentioned strategies depend largely on the inorganic
nanocomposites, the substantial risk of unknown
immunotoxicities, high cost/benefits ratio, and limited
reproducibility further hamper their FDA approval and clinical
translation.37-39 Therefore, it is reasonable to envision that
developing organic small-molecule PSs capable of
simultaneously surmounting these Achilles’ heels may be of
more clinical benefits. Unfortunately, no such molecules have
modality, has garnered tremendous popularity in personalized
medicine, and in some cases it also has proven to be efficient
where clinical protocols, such as surgery, chemotherapy, and
radiotherapy, have failed.1-3 However, PDT as a first-line
therapy still suffers from several “Achilles’ heels”.4,5 For
instance, its effectiveness is hindered by the poor tumor
selectivity of most current available photosensitizers (PSs),
inevitably causing off target toxicity and prolonged cutaneous
photosensitivity.6-8 In addition, due to the rapid tumor
progression and insufficient O2 supply,9,10 the inherent tumor
hypoxia drastically decreases the anticancer outcome, because
the tumoricidal effect of traditional PDT is strongly
oxygen-tension dependent.11-14 Moreover, the limited
therapeutic depth further makes the treatment of deep-seated or
large tumors fundamentally difficult.5,15-17
In order to overcome these drawbacks, various methods are
being intensively explored. For example, the therapeutic
accuracy of PSs can be improved by cancer specific ligands
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been reported to date.
Recently, we
benzophenothiazine compounds are appealing PSs for
anticancer and antibacterial.40-48 In particular, they are greatly
ideal for hypoxic tumor treatment, because such molecules
is perfectly suitable for elevating the molecular absorbance,59,60
we thus speculated that dyad cationic O2 generator
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−•
and
others
have
shown
that
constructed by FRET might be an optimal choice to facilitate
the selective hypoxic deep PDT response. Along this line, a
typical NIR chromophore (Nile blue derivative, here denoted
as ENBO) was applied to pair with a benzophenothiazine
analog (energy acceptor, ENBS) to induce the FRET process
(Figure 1a). As expected, the energy donor ENBO, as an
absorbance “adjuvant”, successfully provided the prepared
predominantly undergo type
I mechanism to generate
superoxide radicals (O2−•), which can subsequently take part in
superoxide dismutase mediated disproportionation reactions,49
as well as Haber-Weiss/Fenton reactions50 to synergistically
augment the antihypoxia outcome in a low O2-dependent
manner.40 Unfortunately, owing to their limited intratumoral
accumulation/retention capacities (even if conjugated by
targeting ligands, e.g., biotin40), especially for intravenous
(i.v.) injection, the overall in vivo therapeutic indexes are not
satisfactory. Moreover, since the strong light absorption,
reflection and scattering profiles of body tissues, the light
fluence attenuates exponentially as the light penetrating more
deeply (Scheme 1).5,51 Thus, although benzophenothiazine
behaves NIR absorption within the “therapeutic window”, the
suboptimal absorbance significantly limits or even terminates
its application in deep tumors, because of PSs with weak
optical absorbance in deep tissues usually failing to be
activated (Scheme 1).
9
−•
dyad O2 generator (ENBOS) with significantly boosted NIR
absorbance, photon utility, and O2−• generation (> 2.2-fold). As
a result, once exposure to NIR light, ENBOS was more easily
triggered in hypoxic deep tissues through type I mechanism,
achieving an amplified effect of “0 + 1 > 1” both in vitro and
in vivo. Besides, due to the powerful SIT function, ENBOS
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acquired
accumulation/retention abilities (> 120 h), and could
specifically “light up” the tumor site with
markedly
improved
intratumoral
a
signal-to-background ratio (SBR) as high as 25.2, which thus
highlighted the advantages of our strategy in terms of precision
medicine. To our knowledge, this is the first report of
−•
molecular SIT O2 generator for simultaneous cancer
diagnosis/therapeutics and resolution of the “Achilles’ heels”
of traditional PDT.
Scheme 1. Concept Illustration That PS with Low ε Only
Enables Superficial Tumor-Confined Phototoxicity, Whereas
PS with High ε can Result in Deep-Seated Tumor Eradication.
Figure 1. Chemical structures of (a) ENBOS, (b) ENBS, and (c)
ENBO. (d) Absorption and (e) emission spectra of ENBO, ENBS,
and ENBOS. (f) Photophysical data. a ε value at 660 nm.
Motivated by above concerns, herein, for the first time, we
described a fundamentally different approach by using Förster
resonance energy transfer (FRET) theory to fabricate
RESULTS AND DISCUSSION
−•
photon-initiated “structure-inherent targeting (SIT)” O2
Synthesis and Spectroscopic Characterization. The SIT
O2−• generator (ENBOS) and its analogue without FRET effect
(ENBS, reference compound) were designed and synthesized
(Figure S1). The proposed structures were confirmed by
HRMS, 1H NMR and 13C NMR (Figures S2-S10). As shown in
Figure 1d, compared with bare ENBS, ENBOS presented a
obviously broader and stronger NIR absorption band ranging
from 600-750 nm due to the spectra overlap of other two units,
and meanwhile, the extinction coefficient (ε) of ENBOS at 660
nm was increased from 45100 to 71000 M-1cm-1(Figure 1f),
generator with encouraging “0 + 1 > 1” effect to realize
substantially intensified selective ablation of deep-seated
hypoxic tumors. It is well documented that compounds
featuring lipophilic cationic chemical structures possess
apparent self-recognition function toward neoplasms because
malignant tumors usually develop higher degree of membrane
potential than normal tissues,52,53 also termed as SIT,53-55 which
has shown immense clinic values in biological imaging and
diagnosis in recent years.56-58 Additionally, FRET mechanism
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which indicated that the NIR absorbance and concomitant
Specifically, the energy donor ENBO displayed an intense
emission at 684 nm; however, upon coupling, this donor signal
was completely quenched in ENBOS, suggesting the
reasonable construction of intramolecular FRET action.
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photon utility of ENBOS have been significantly improved by
the FRET effect. Then, the efficient energy transfer was
ascertained by fluorescence emission spectra (Figure 1e).
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Figure 2. (a) Fluorescence spectra of DHR123 for O2 detection. (b) The fluorescence intensity of DHR123 at 526 nm after different
−•
treatments (c) Comparison of O2 generation as a function of irradiation time. (d) The fluorescence intensity of Singlet Oxygen Sensor
Green (SOSG) for 1O2 detection. (e) Illustration of enhanced O2−• generation due to FRET effect for ENBOS. (f) Relative intracellular O2
level generated by ENBOS upon light irradiation. (g) Intracellular hypoxia confirmation using ROS-ID as the anaerobic probe and O2
imaging under normoxic (21% O2) and hypoxic (2% O2) environments. *P < 0.05, ***P < 0.001, and ****P < 0.0001.
−•
−•
−•
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FRET-Amplified
O2
Generation
and
the
and hypoxic environments using the in vitro O2 fluorescent
Photosensitization Mechanism Confirmation for ENBOS.
Since strong optical absorption of PS can facilitate its photon
utilization such that generates more ROS, we then assessed the
indicator dihydroethidium (DHE),62 and the corresponding
detection mechanism was displayed in Figure S14. For 4T1
cells treated with ENBOS followed by NIR light irradiation in
−•
FRET-amplified PDT performance of ENBOS by using the
normoxia (21% O2), brightly red signals attributing to O2
−•
O2
probe,
dihydrorhodamine
123
(DHR123).61
were noted (Figure S15), and the average fluorescence
intensity increased from 94 to 2429 as the rise of light-dose
from 1.2 to 48 J/cm2 (Figure 2f), suggesting ENBOS could
Monochromatic light of 660 nm was used as the irradiation
source in this study. Because of the absence of intersystem
crossing (ISC) process, ENBO did not produce measurable
O2−•, which was similar to the results of DHR123 alone (Figure
2a). Surprisingly, under light irradiation, ENBOS substantially
increased the fluorescence intensity of DHR123 by 4.8 times,
but ENBS without energy transfer only showed limited O2
signals (Figures 2a, b), indicating better O2−• generation ability
of ENBOS than ENBS. Remarkably, ENBOS also led to a
faster O2 generation rate in comparison with ENBS (Figure
−•
highly efficiently induce the O2 formation in vitro. And also,
−•
the intracellular O2 was almost completely scavenged by the
radical scavenger Vitamin C (Vc), further confirming the
robust O2−• induction capacity of ENBOS.
−•
To better mimic the hypoxic state of solid tumors, 4T1 cells
were firstly cultured under a severe hypoxic atmosphere (2%
O2) for longer than 8 h within an incubator chamber
(MIC-101, Billups-rothenberg). Afterwards, the intracellular
hypoxia was determined by using the anaerobic probe
ROS-ID.63 As illustrated in Figure 2g, the ROS-ID
fluorescence intensity in hypoxia-treated cells indeed showed
31-fold increment relative to normoxia (21% O2), thus
suggesting the successful construction of in vitro hypoxic
model. Importantly, even under severe hypoxic condition (2%
−•
2c). These results fully validated that the intramolecular FRET
effect indeed promoted the NIR photon utilization, thereby
greatly boosting the O2 production of ENBOS, which was
−•
critically important for robust PDT actions. On the other hand,
under the same conditions, we did not found obvious singlet
oxygen (1O2) (Figures 2d and S11) and hydroxyl radical (OH•)
generation (Figure S12), revealing that ENBOS predominantly
underwent the type I mechanism, also known as the electron
transfer photochemical process (Figure 2e). The excellent
photostability of ENBOS also allowed us to further use it for
various biomedical applications (Figure S13).
−•
O2), ENBOS still had satisfactory O2 level as indicated by
the bright DHE signals, meaning that ENBOS was capable of
surmounting the tumor hypoxia, because widely perceived
−•
mechanism holds that O2 can act as ROS initiator to convert
into other highly cytotoxic oxidants, such as OH•, through
intracellular cascade reactions (e.g. Haber-Weiss reaction and
Fenton reaction)50 to synergistically augment the antihypoxia
efficiency in an O2 reuse manner, just like evidenced in our
−•
Light-Induced Intracellular O2 Generation under
Normoxia (21% O2) and Hypoxia (2% O2). Next, we
evaluated the O2 formation in living cells under normoxic
−•
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light dose- and ENBOS concentration-dependent manner
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previous work.40
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(Figure 4a). In particular, the in vitro anticancer efficiency of
our ENBOS even was superior to the clinical PS, Ce6 (Figure
S22), behaving a half maximal inhibitory concentration (IC50)
Amplification Effect Enables ENBOS to Be More Easily
Activated in Simulated Hypoxic Deep-Tissue Settings. To
investigate whether the enhanced NIR absorbance could lead
to ENBOS more easily activated in deep-seated hypoxic tumor
of merely 0.033 M, 32-fold lower than that of Ce6 (IC50
1.064 M). To our gratification, because of the ideal O2
=
−•
−•
cells, we compared ENBOS- and ENBS-mediated O2
generation potency in hypoxic environments, ENBOS
successfully led to potent photodamage toward hypoxic cancer
cells (Figure S23). Corresponding IC50 values have been
summarized in Table S1.
generation in a simulated deep-tissue setting (Figure S16). As
control experiments, when 4T1 cells were directly exposed to
NIR light, no appreciable difference in DHE signals was
noticed for ENBOS and ENBS (Figure 3a). In sharp contrast,
once a piece of 5 mm pork tissue was placed between cells and
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It was noteworthy that, even the light source was blocked by
5 mm pork slice, ENBOS still promised an excellent PDT
response, however, the anticancer efficacy of ENBS was
−•
light source, the cellular O2 generation induced by ENBS
dramatically decreased by 92% and was barely detectable,
whereas the DHE signals still remained at an ideal level in
ENBOS-loaded cells (Figure 3b). These data unambiguously
proved that, with the help of non-PS ENBO as the absorbance
enhancer, ENBOS enabled to be more easily triggered in
internal tissues, yielding the effect of “0 + 1 >1” to amplify the
deep PDT efficiency in essence.
−•
sharply impaired due to its inability to produce O2 in
deep-tissues (Figure 4b). In detail, the inhibition rate was only
reduced by 23%, 8%, and 2% at ENBOS concentration of
0.08, 0.16, and 0.32 M, respectively; whereas the
phototherapy outcome was drastically decreased with 47%,
53%, and 39% for the same three ENBS dose, respectively.
Similar results were also observed in Live/Dead cell
co-staining assay (Figure 4c). These findings were consistent
−•
with the data from intracellular O2 detection in a simulated
deep-tissue, clearly verifying the superior “0 + 1 >1” effect of
our ENBOS in treating large or deep-seated hypoxic tumors.
−•
Figure 3. Relative O2 generation in 4T1 cells after ENBOS- or
ENBS-mediated PDT treatment (48 J/cm2), with excitation light
(a) unblocked or (b) blocked with a piece of 5 mm pork tissue.
***P < 0.001.
In Vitro O2−•-Mediated PDT Evaluation under Hypoxia
and Normoxia. The ENBOS-induced photodynamic cancer
cell killing was first studied by fluorescence imaging. As
shown in Figure S17, owing to the dyad cationic structural
property, ENBOS efficiently penetrated into 4T1 cells, and
meanwhile, the NIR fluorescence staining was matched well
with the commercial lysosome dyes (LysoTracker green),
indicating the specific lysosome anchoring of ENBOS (Figure
S18). Upon light irradiation, as evidenced by the AO
Figure 4. (a) Light- and ENBOS-dose dependent viabilities in
4T1 cells. (b) In vitro anticancer efficiency of ENBS and ENBOS
with light source blocked by a piece of 5 mm pork tissue under
hypoxia (2% O2) (48 J/cm2). (g) Live/Dead cell co-staining assays
using Calcein AM and Propidium Iodide as fluorescence probes.
**P < 0.01 and ***P < 0.001.
In Vivo Xenograft Tumor-Targeting Accumulation and
Imaging. Since specific tumor targeting capability is of
paramount importance for precise PDT, we then investigated
the SIT potency of ENBOS by i.v. injection (Figure 5a).
Evidently, the 4T1 tumors could be clearly visualized from
neighboring tissues with a SBR value as high as 25.2 at 48 h
post-injection (Figure 5b). Of note, such high SBR was greatly
encouraging and relative rare because a SBR value more than
2.5 is generally regarded as preferential accumulation in
essence.66 24 h after administration, the maximal tumor signal
−•
staining,64,65 the formed O2 severely disrupted the lysosome
integrities (Figure S19) and subsequently resulted in serious
cancer cell apoptosis and death (Figure S20). Then the in vitro
PDT efficiency was further explored in details by standard
methyl thiazolyltetrazolium (MTT) and live/dead cell
co-staining assays. Obviously, negligible cytotoxicity of
ENBOS was observed in the absence of light (Figure S21),
however, under NIR light irradiation, ENBOS induced severe
damage to tumor cells, and the cell viability exhibited both
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was attained, and even 120 h later, the tumor site still could be
clearly distinguished. In contrast, nearly no ENBS signals
were found during the observation course (Figures 5c and
also, the targeting potency of ENBOS was obviously better
than the previously reported biotin-conjugated O2 generator
which could merely maintained at tumor for 24 h,40 thereby
highlighting the advantages of our SIT strategy in terms of
tumor-targeting and imaging-guided precision therapy.
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S24),
revealing
the
more
durable
intratumoral
accumulation/retention ability of ENBOS than ENBS. And
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Figure 5. (a) In vivo real time fluorescence imaging of 4T1 tumor-bearing mice after i.v. injection of ENBOS. (b) Fluorescence change of
ENBOS in the tumor and adjacent muscle tissues. (c) Relative fluorescence intensity of ENBOS and ENBS in 4T1 tumors at different
time points. (d) Immunofluorescence imaging of tumor slice. (e) Schematic illustration of ENBOS-mediated PDT in simulated hypoxic
deep tumors. (f) Relative tumor volume, (g) average tumor weights, and (h) representative photographs from different groups. Data were
expressed as mean ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001 determined by Student’s t test.
Photodynamic Anticancer efficiencies of ENBOS on
Deep-Seated Hypoxic Tumors. In order to explore the
advantages of ENBOS in overcoming the deficiencies of PDT,
we examined the anticancer efficacy of ENBOS in 4T1
tumor-bearing Balb/c mice by i.v. injection. The
immunofluorescence imaging of hypoxia inducible factor
(HIF-1) and tumor blood vasculatures (CD-31) indicated that
the tumors were in a severely hypoxic state (Figure 5d).11
Then, as a proof of principle experiment, the subcutaneous
tumor was covered by 5 mm pork tissue so as to simulate the
hypoxic tumors which locate deeply inside body, and followed
by NIR light exposure (0.1 W/cm2, 15 min) to conduct PDT
treatment (Figure 5e). Similar to control group (PBS alone),
neither ENBOS injection by itself or irradiation alone could
delay the tumor growth during the study period, showing 
15-fold increment (Figure 5f). For tumors exposed directly to
NIR light, ENBOS exhibited prominent tumor suppression
with the tumor growth inhibition (TGI) rate as high as 84%.
Surprisingly, even in the case of pork tissue blocking, the TGI
rate remained up to 68%, which highlighted the predominant
targeted PDT effect of ENBOS on hypoxic deep tumors. The
average tumor weights (Figure 5g) and corresponding tumor
photographs (Figure 5h) validated the accurate antitumor
results of ENBOS again.
NIR light irradiation, despite ENBS showing some extent
tumor inhibition, the TGI rate was lower than 30%. However,
nearly complete tumor regression was presented in mice
injected with ENBOS (Figure 6b), and the tumor weigh was
only 1/3 of ENBS-treated tumors (Figure 6c). This optimal
therapeutic efficacy was mainly attributed to the more robust
−•
O2 production of ENBOS in deep-tissues, thanks to its
significantly enhanced NIR absorbance resulting from FRET
construction. Furthermore, as shown in the T2-weighted MRI
in vivo on day 15 after PDT treatment, much better tumor
suppression was found for ENBOS (Figure 6d). The
histological analysis also revealed that the destruction depth of
tumor by ENBOS was greater than ENBS, as indicated by the
shallower color of staining region for apoptotic and necrotic
cancer cells (Figure 6e). During the course of therapy, all mice
did not have noticeable abnormal body weight changes (Figure
S25), and no obvious destructive cell necrosis or inflammation
lesions in all major organs including heart, liver, spleen, lung
and kidneys were observed (Figure S26), thereby implying the
high biocompatibility and safety of ENBOS for biomedical
applications.
CONCLUSION
In summary, by using FRET mechanism, we have developed
Subsequently, to more intuitively evidence the superb
potency of ENBOS for deep-seated hypoxic tumors, we
evaluated the in vivo therapeutic efficacy through intratumoral
(i.t.) administration of ENBOS and ENBS so as to ensure the
same PS dosage within the tumors (Figure 6a). Under blocked
−•
an appealing dyad SIT O2 generator to amplify the selective
anticancer performance against deep-seated hypoxic tumors, as
a result, successfully addressing the Achilles’ heels of
photodynamic therapy with an encouraging amplification
effect of “0 + 1 > 1”. Owing to the outstanding SIT feature and
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low oxygen sensitivity, ENBOS could spontaneously target to
and work well within the hypoxic compartment of malignant
tissues through type photoreactions. Meanwhile, the
Corresponding Author
*pengxj@dlut.edu.cn
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I
Notes
enhanced NIR absorbance and photon utility enabled ENBOS
to be more easily triggered in deep tissues, thereby ensuring
the PDT efficiency against hypoxic deep tumors. Better still,
other unique merits were also achieved, such as significantly
enhanced intratumoral accumulation/retention ability (> 120 h)
as well as excellent SBR value (> 25.2), which are critically
important for precise anticancer actions. More importantly,
this facile molecule design method was also suitable for
improving the photosensitization activities of other photoactive
agents, such as BODIPYs, phthalocyanines, and porphyrins.
As such, we postulated that this work would cause a paradigm
change of hypoxic deep PDT, and might be of great clinical
benefits for future cancer precision therapy.
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
We thank the National Natural Science Foundation of China
(Project Nos. 21421005, 21576037) and the NSFC-Liaoning
United Fund (Project No. U1608222) for financial support.
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