De Novo Design of Phototheranostic Sensitizers Based on Structure-Inherent Targeting for Enhanced Cancer Ablation

Article abstract of DOI:10.1021/jacs.8b09117

Structure-inherent targeting (SIT) agents are of particular importance for clinical precision medicine; however, there still exists a great lack of SIT phototheranostics for simultaneous cancer diagnosis and targeted photodynamic therapy (PDT). Herein, fo

Full text of DOI:10.1021/jacs.8b09117

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Article
De Novo Design of Phototheranostic Sensitizers based on
Structure-inherent Targeting” for Enhanced Cancer Ablation
“
Mingle Li, Saran Long, Yao Kang, Lianying Guo, Jingyun Wang, Jiangli Fan, Jianjun Du, and Xiaojun Peng
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09117 • Publication Date (Web): 31 Oct 2018
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De Novo Design of Phototheranostic Sensitizers based on
“
Structure-inherent Targeting” for Enhanced Cancer Ablation
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Mingle Li, Saran Long, Yao Kang, Lianying Guo, Jingyun Wang, Jiangli Fan, Jianjun Du,
and Xiaojun Peng*,†
†
†
†
‡
ǀǀ
†
†
†
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China
‡
Department of Pathophysiology, Dalian Medical University, Dalian 116044, China
ǀǀ
Department School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China
ABSTRACT: Structure-inherent targeting (SIT) agents are of particular importance for clinical precision medicine; however, there
still exists a great lack of SIT phototheranostics for simultaneous cancer diagnosis and targeted photodynamic therapy (PDT).
Herein, for the first time, we propose a “one-for-all” strategy by using Forster Resonance Energy Transfer (FRET) mechanism to
construct such omnipotent SIT phototheranostics. Of note, this novel tactic can not only endow conventional sensitizers with highly
effective native tumor targeting potency, but also simultaneously improve their photosensitization activities, resulting in
dramatically boosted therapeutic index. After intravenous injection of the prepared SIT theranostic, the neoplastic sites are
distinctly “lighted up” and distinguished from neighboring tissues, showing a NIR signal-to-background ratio value as high as 12.5.
More importantly, benefiting from the FRET effect, markedly amplified light-harvesting ability and
demonstrated. Better still, other favorable features are also simultaneously achieved, including specific mitochondria anchoring,
augmented cellular uptake (> 13-fold), as well as ideal biocompatibility, all of which allows orders-of-magnitude promotion in
anticancer efficiency both in vitro (> 56-fold) and in vivo (> 16-fold). We believe this “one-for-all” SIT platform will provide a new
idea for future cancer precision therapy.
1
2
O production are
As an ideal alternative, structure-inherent targeting (SIT),23
namely, the inherent chemical structures of compounds own
native targeting potency for interested lesions, offers a new
opportunity to realize targeting delivery. This SIT strategy
eliminates the need of conjugations. Over the years, SIT
molecules have been widely used in Fluorescence-Assisted
Resection and Exploration (FLARE) imaging,
been known as excellent contrast agents to visualize sensitive
endogenous tissues such as thyroid gland and cartilage.
INTRODUCTION
The problem of severe side effects of current “one-size-fits-all”
diagnostic and therapeutic protocols in clinical cancer
treatment stimulates the development of “precision medicine”
2
3
with the purpose of maximizing therapeutic index while
1,2
minimizing
untoward
sufferings.
As
a
typical
2
4-26
and have
photon-triggered therapeutic modality, photodynamic therapy
3-6
(PDT) has attracted increasing attention in recent years,
2
7-30
while several factors seriously compromise the PDT clinical
practice, especially the poor selectivity towards malignant
However, up to now, their application markedly focus on
bio-imaging, rare SIT theranostics combining cancer precision
diagnosis and targeted therapy in one component have been
designed and utilized for cancer PDT. And also, accesses to
obtain SIT agents are overwhelmingly concentrated on
7-9
tissues. To achieve accurate PDT, current strategies mainly
rely on nanoparticle-mediated delivery systems based on
enhanced permeability and retention (EPR) effects,
10,11
and
12
biological conjugation with target ligands (e.g., transferrin,
2
6,30
combinatorial screening,
which is time consuming,
13
3
14
peptide cRGD,
biotin, and trastuzumab ). However,
laborious, and inefficient. Undoubtedly, seeking a more smart
and facile approach to construct potent SIT theranostics will
provide huge benefits for clinical translation
growing clinical and experimental data suggest that even in the
ideal case only a relatively small fraction of injected biologics
can finally reach their parenchymal targets,
15,16
such as 1 to 10
and ~0.7% for
15,17,18
Notably, for better cancer PDT, besides selectivity to
carcinomas, superior optical and physicochemical properties
parts per 100,000 for antibody conjugates
19,20
nanoparticles.
Moreover, the multistep covalent
1
(e.g., relatively strong absorption and high O
2
quantum yield),
excellent
biocompatibility, and high cellular uptake are also required for
conjugations may alter the conformation, steric freedom, and
orientation of target ligands, inevitably compromising their
specific
subcellular
organelle-targeting,
21,22
binding abilities.
3
1-34
photosensitizers (PSs).
Unfortunately, most conventional
PSs can hardly reach these requirements. Taking
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porphyrin,37 and bodipy PSs as
phthalocyanine,
examples,
Inspired by these, we wonder if it was possible that cationic
chromophores simultaneously act as SIT sponsors to endow
common PSs with native tumor targeting ability, and as energy
donors to tailor the photosensitization activities of PSs through
FRET mechanism, as a result, markedly improving the
anticancer precision and efficiency. As a proof of concept, a
representative cationic rhodamine moiety (RDM) was used as
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38,39
they are usually hydrophobic and easy to
aggregate in physiological environments, leading to limited
membrane permeability and cell uptake. While for cyanine
40-43
44,45
chromophores
state (T energy levels or short
singlet-to-triplet intersystem crossing (ISC) are inefficient; this
and AIE gens,
due to the improper triplet
1
)
T
1
lifetimes, their
1
badly impedes the subsequent
O
2
generation. Although
energy donor to pair up with
a
PS example,
strategies including structural modification with spin
diiodo-distyryl-bodipy (BDP), to induce the FRET process. As
depicted in Figure 1, our strategy successfully endowed the
resultant SIT theranostic (RDM-BDP) with great tumor
targeting, showing a NIR signal-to-background ratio as high as
12.5. Importantly, due to the FRET effect, RDM-BDP
46
47,48
convertor or hydrophilic capping polymers,
as well as
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encapsulation within inorganic/metallic materials can mitigate
49-51
these problems,
potential limitations still remain, for
instance, low drug loading efficiency, accumulation mainly in
reticuloendothelial organs, unknown systemic toxicity of
1
achieved distinctly broader absorption and intensified
O
2
52,53
exogenous materials, and more.
Regarding these concerns,
production. Better still, multiple favorable properties were also
noted, including specific mitochondria anchoring, promoted
cellular uptake (> 13-fold), and ideal biocompatibility. Finally,
these merits successfully resulted in significantly enhanced
therapeutic response both in vitro and in vivo. As far as we
know, this is the first demonstration of “one-for-all”
FRET-based SIT theranostic for cancer precision therapy.
we are motivated to develop a "one-for-all" tool that can not
only solve these dilemmas, but also simultaneously achieve
SIT features in single one small molecule.
Figure 2. (a) Absorption spectra, (b) fluorescence spectra, (c)
fluorescence decay curves of RDM, BDP, and RDM-BDP.
Nanosecond time-resolved transient difference absorption spectra
of (d) RDM-BDP and (e) BDP in deaerated DCM after pulsed
excitation, λex = 532 nm. Decay traces of (f) RDM-BDP and (g)
BDP at 650 nm in DCM. (h) Photo-degradation curves of DPBF
in the presence of RDM-BDP (inset: BDP) under 550 nm
irradiation. (i) Comparison of DPBF decomposition rates at 415
nm as a function of irradiation time under panchromatic light
illumination.
Figure 1. Schematic illustration of FRET mechanism based SIT
1
phototheranostic (RDM-BDP) for amplified O
2
generation, native
tumor targeting, as well as light-triggered enhanced tumor PDT.
Herein, for the first time, we proposed an innovative
strategy for using Forster Resonance Energy Transfer (FRET)
mechanism to devise robust SIT phototheranostics for
significantly amplifying the therapeutic outcome. Previous
studies have revealed that cancer cells possess higher
magnitude of membrane potential than normal cells, and
compounds with permanently delocalized positive charge such
as rhodamine derivatives have larger degree of preferential
RESULTS AND DISCUSSION
Synthesis and Spectroscopic Properties. As detailed in
Scheme S1, RDM-BDP was synthesized and, to better
understand our concept, BDP lacking RDM unit was prepared
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4-56
accumulation and retention within tumors.
In addition,
FRET theory has been extensively applied in triplet PSs
development aiming at enlarging the photon utility and
1
as control. All structures were fully characterized by H NMR,
1
37,57
13
concomitant
O
2
generation,
because for most existing
C NMR, and ESI-MS (Figure S1-S12). From the UV-Vis
photoactive agents, there are only one major absorption profile,
absorbance spectra in Figure 2a, it could be seen that both
RDM-BDP and BDP displayed a typical Q band in the range
5
7
leading to their light harvesting capacity disappointed.
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of 600-700 nm, whereas RDM-BDP possessed an additional
time points after incubation with BDP or RDM-BDP. Subcellular
colocalization images of (c) BDP and (d) RDM-BDP with
Hoechst 33342 (Hoechst), MitoTracker Green (MTG), and
LysoTracker Green (LTG) in 4T1 cells, R is the correlation
coefficient.
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intense absorption peak centered at 557 nm which belonged to
the RDM moiety. That was to say RDM-BDP achieved a much
broader absorption, suggesting a stronger light-harvesting
ability as compare to free BDP. Upon 568 nm excitation, the
fluorescence of RDM-BDP at 575 nm was completely
quenched, showing 129-fold weaker than that of RDM (Figure
Cellular Uptake and Subcellular Colocalization. Then,
we proceeded to investigate whether our strategy enabled to
improve the cellular uptake. As displayed in Figure 3a,
RDM-BDP could rapidly internalize the tumor cells, and the
fluorescence intensity of RDM-BDP in cells after 150 min
incubation was 13-fold larger than that of BDP (Figure 3b),
revealing noticeably improved cellular uptake was obtained for
RDM-BDP. This might be attributed to RDM unit to a certain
extent because of larger membrane potential of cancer cell
allowing more preferential transport and retention of lipophilic
2
b). Meanwhile, the fluorescence lifetime of RDM-BDP was
also shorter than that of RDM, further proving that efficient
energy transfer had taken place in RDM-BDP (Figure 2c).
Moreover, the nanosecond time-resolved transient difference
absorption spectra demonstrated that the transient features of
RDM-BDP were similar to that of BDP (Figure 2d-2g),
indicating FRET effect did not interfere with the distribution
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of T , and the T of RDM-BDP was predominantly distributed
on the energy acceptor unit BDP, but not on the RDM moiety.
1O
Generation Ability. To determine whether the
54
2
cationic agents. Another item worth noting was that,
1
broadband absorption facilitates to enhance the O
2
generation,
compared to the typical lysosome localization of BDP
(Pearson’s coefficient 0.82), the red signals of RDM-BDP was
nicely coincident with the green fluorescence of mitochondrial
tracking dye (Pearson’s coefficient 0.88) (Figure 3c and 3d).
Since intracellular mitochondria are more susceptible to toxic
we firstly used 550 nm and 660 nm as the representative
wavelengths of energy donor and acceptor in the following
studies, respectively. The measurement mechanism of DPBF
1
for O
2
was illustrated in Figure S13. As indicated by the
1
DPBF decolorization curves, irradiation of RDM-BDP and
O than other organelles, and mitochondria targeted PSs have
2
1
43,58,59
BDP with 660 nm light source leaded to a comparable O
2
more interesting to trigger cell apoptosis,
we speculated
generation (Figure S14). However, when they were exposed to
50 nm light, the decrease of DPBF absorbance at 415 nm
that the specific mitochondrial anchoring together with
distinctly improved cellular uptake of RDM-BDP would
markedly maximized the PDT outcome.
5
caused by RDM-BDP was 59% in 105 s, while there was
almost no decrease for BDP (Figure 2h), suggesting much
1
better
O
2
generation capacity of RDM-BDP than BDP.
1
According to the decay curves of DPBF, the O
2
yield of
RDM-BDP was 9-fold higher than that of BDP (Figure S15).
To further understand our concept, a panchromatic light source
1
(
400-700 nm) was used for photoexcitation. As expect, the O
2
generation of RDM-BDP was greatly better than that of BDP
(
Figure 2i and S16). Moreover, the phosphorescence emission
1
spectrum around 1270 nm validated the O
2
production again
(Figure S17). Hence, FRET effect clearly manipulated the
photophysical properties of RDM-BDP, giving rise to an
1
elevated photon utilization as well as boosted O
2
generation,
which were critically important for killing tumor cells during
PDT.
Figure 4. (a) Cell viability of 4T1 cells against various light- and
RDM-BDP-dose. (b) Cell viability of 4T1 cells treated with BDP
or RDM-BDP at various doses under 550/660 nm light irradiation
2
(
12 J/cm ). (c) PDT efficacy of RDM-BDP and Ce6 in 4T1 cells
2
after 660 nm illumination (12 J/cm ). (d) Calcein AM and
Propidium Iodide co-staining fluorescence imaging in 4T1 cells.
In Vitro Light-induced Cellular Toxicity Evaluation. As
expected, RDM-BDP indeed displayed a superior inhibition of
cell growth in a concentration- and light dose-dependent
manner (Figure 4a), whereas no toxicity was observed in the
2
absence of irradiation (Figure S18). With 12 J/cm light
irradiation (660 nm), the half maximal inhibitory concentration
(IC50) of RDM-BDP was merely 0.036 M, but the data was as
high as 2.02 M for BDP, suggesting greatly enhanced PDT
potency of RDM-BDP by about 56-fold (Figure 4b).
Interestingly, when 4T1 cells were exposed to 550 nm light
source, almost no cells were killed by BDP in all doses, in
Figure 3. (a) The cellular uptake of RDM-BDP and BDP in 4T1
cells. (b) Intracellular average fluorescence intensity at different
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contrast, RDM-BDP still resulted in a striking photoactive cell localized in cellular mitochondria, we envisioned that the
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damage with an 81-fold enhancement (IC50 = 0.055 M for
RDM-BDP vs. IC50 = 4.45 M for BDP). Similarly, such
encouraging results were also demonstrated in panchromatic
light-treated cells (Figure S19), revealing the enhanced
light-harvesting potency resulting from FRET effect indeed
improved the PDT outcome in essence. In fact, RDM-BDP
even was much more outstanding than the widely used clinical
PS (Ce6) which has an IC50 value of ca. 1.8 M, 50-fold
higher than RDM-BDP (Figure 4c). To the best of our
knowledge, RDM-BDP demonstrated the lowest IC50 value of
generated
O
2
would severely disrupt the mitochondria
integrity and initiate cell apoptosis. Remarkably, the JC-1
staining for mitochondria membrane potential showed that, no
matter upon 660 nm or 550 nm irradiation, RDM-BDP leaded
to severe mitochondrial depolarization as depicted by the
increase of green fluorescence signals (Figure 5b, c),
subsequently resulting in serious apoptotic cell death (Figure
5d). Similarly, with the addition of NaN
efficiently prevented from the fate of death, suggesting the cell
2
death was really induced by generated O . Contrarily, BDP
only caused slightly lysosome dysfunction (Figure S20) and
early cell apoptosis (Figure S21).
3
, 4T1 cells were
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bodipy-based PSs reported to date in cellular PDT study. The
live/dead cell co-staining data in Figure 4d intuitively
demonstrated the robust PDT ability of RDM-BDP again, as
revealed by the intense homogeneous red fluorescence for
dead cells. All of these results confirmed that our strategy
enabled to greatly improve the in vitro therapeutic action.
Figure 6. (a) In vivo fluorescence imaging of 4T1 tumor-bearing
BALB/c mice after intravenous injection of BDP and RDM-BDP.
(b) Normalized fluorescence intensity of BDP and RDM-BDP in
tumor sites at different time points. (c) The ex vivo tumor
fluorescence imaging after 36 h injection of RDM-BDP and BDP.
(d) Relative fluorescence change in the tumor and adjacent muscle
tissues for RDM-BDP-administrated mice.
Figure 5. (a) Evaluation of 1
O generation in 4T1 cells. (b)
2
Mitochondrial membrane potential assay for RDM-BDP-mediated
photodamage of mitochondria. (c) The JC-1 monomer to
aggregate (green/red) ratio. (d) Confocal fluorescence images of
Annexin V-FITC/PI stained 4T1 cells.
Therapeutic Mechanism of RDM-BDP-mediated PDT.
To obtain insights into the potential therapeutic mechanism
1
down to subcellular level, the cellular O
2
was evaluated by
using the ROS probe 2, 7-dichlorodihydrofluorescein diacetate
DCFH-DA) (Figure 5a). Compared to control group, in the
(
presence of 660 nm illumination, the RDM-BDP-treated 4T1
cells displayed obviously green fluorescence, indicating an
effective ROS generation. Contrarily, relatively dim ROS
signals were observed in BDP-treated cells mainly because of
its limited cellular uptake. Notably, with 550 nm light
irradiation, for RDM-BDP, the ROS generation was also 16
times better than BDP, matching well with the MTT results.
Moreover, the ROS induced by RDM-BDP was completed
Figure 7. (a) Relative tumor volume changes in tumor-bearing
mice during the treatments (660 nm irradiation). (b)
1
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scavenged by NaN
implying the produced ROS was
3
(a widely accepted O
2
scavenger), further
. Since RDM-BDP
1
O
2
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Representative photos of mice at the 14th day. (c) Relative tumor
volume in the mice after different treatments (550 nm irradiation).
d) Survival curves of mice in different groups during the 60 day
evaluation. (e, f) Tumor weight, (g) H&E staining of tumor slices,
h) representative tumor images from different groups of
there was no substantial damage or distinct inflammation
lesions in all normal organs including heart, liver, spleen, lung,
and kidney (Figure S23), suggesting the low in vivo toxicities
of various treatments in this work and that RDM-BDP was
suitable for in vivo application.
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(
(
tumor-bearing mice.
CONCLUSION
Structure-Inherent Targeting Ability of RDM-BDP. To
evaluate the SIT performance of RDM-BDP, the BALB/c mice
bearing a subcutaneous 4T1 tumor was intravenously injected
with 8 nmol RDM-BDP, followed by in vivo fluorescence
imaging at different time points. Encouragingly, very
appealing spontaneous tumor targeting was displayed (Figure
In summary, we have developed a novel approach based on
intramolecular FRET mechanism to design SIT theranostic for
precise light-triggered tumor treatment. In particular,
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RDM-BDP operated based on FRET mechanism and
1
significantly elevated the light-harvesting ability as well as O
2
generation. In vivo tumor fluorescence imaging study clearly
confirmed that RDM-BDP could spontaneously accumulate
within tumor site and exhibited a fairly high SBR value,
enabling the tumor and its margins easily visualized and
proving the potential for precise imaging-guided tumor PDT.
Furthermore, RDM-BDP could specifically target to
mitochondria, and substantially enhanced the internalization in
cancer cells. These features thereby markedly maximized the
PDT effect on tumors. More importantly, this unconventional
strategy can be readily adopted for improving the therapeutic
performance of other existing photoactive agents, such as
phthalocyanines and porphyrins. We believe that our
FRET-based SIT PS development platform will provide
considerable advantages for cancer diagnostic and therapeutic.
6
a). Only 1 hour after injection, the tumor site could be clearly
“
lighted up” and distinguished from neighboring tissues. With
time increasing, the fluorescence intensity at tumor site was
gradually enhanced (Figure 6b), reaching the maximum at 6 h
post-injection (p.i.), while nearly no fluorescence signals were
observed in BDP-injected mice. The representative ex vivo
tumor image further demonstrated the more efficient
tumor-specific uptake and retention of RDM-BDP than BDP
(
(
Figure 6c). Significantly, RDM-BDP exhibited a SBR value
calculated from the difference in fluorescence intensity
between tumor and surrounding muscle) up to 12.5 (Figure 6d),
whereas previously, such SBR value in a tumor more than 2.5
2
7-30
was regarded as substantially preferential accumulation,
highlighting the advantages of our SIT platform in terms of
tumor targeting and diagnose, and suggesting that RDM-BDP
enabled to confine the PDT on tumor regions so as to reduce
the side effects on normal tissues.
ASSOCIATED CONTENT
Supporting Information. Detailed experimental conditions
and methods, Scheme S1 and Figures S1-S23.
In Vivo Anticancer Effect on Tumor Growth and
Biosafety Evaluation. For in vivo cancer therapy (Figure 7a),
tumors in the mice treated with PBS followed by 660 nm
irradiation increased rapidly with an ca. 14 fold increment,
similar to that treated with PBS alone, excluding the influence
of NIR irradiation on tumor inhibition. Owing to the negligible
dark toxicity, free RDM-BDP and BDP also showed no tumor
suppression. After 660 nm irradiation, RDM-BDP resulted in
an extraordinary tumor regression with no tumor relapse;
however, only slight tumor inhibition was observed for mice
administrated with BDP. Meanwhile, the tumor sizes of BDP
irradiation mice were about 16 times larger than that of
RDM-BDP irradiation mice at the end of observation period.
Besides, the thorough tumor eradication further proved the
robust in vivo PDT efficacy of RDM-BDP (Figure 7b). For
AUTHOR INFORMATION
Corresponding Author
*
pengxj@dlut.edu.cn
Notes
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
This work was supported by National Science Foundation of
China (project 21421005, 21576037, and U1608222).
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