Rational Design of Efficient Organic Phototherapeutic Agents via Perturbation Theory for Enhancing Anticancer Therapeutics

Article abstract of DOI:10.1002/cmdc.201900302

The development of efficient phototherapeutic agents (PTA) through rational and specific principles exhibits great potential to the biomedical field. In this study, a facile and rational strategy was used to design PTA through perturbation theory. Accordi

Full text of DOI:10.1002/cmdc.201900302

Accepted Article
Title: Rational Design of Efficient Organic Phototherapeutic Agents via
Perturbation Theory for Enhancing Anticancer Therapeutics
Authors: Yunjian Xu, Menglong Zhao, Licai Wu, Feiyang Li, Mingdang
Li, Mingjuan Xie, Shujuan Liu, Wei Huang, and Qiang Zhao
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To be cited as: ChemMedChem 10.1002/cmdc.201900302
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10.1002/cmdc.201900302
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Rational Design of Efficient Organic Phototherapeutic Agents via
Perturbation Theory for Enhancing Anticancer Therapeutics
Yunjian Xu,[a] Menglong Zhao,[a] Licai Wu,[a] Feiyang Li,[a] Mingdang Li,[a] Mingjuan Xie,[a] Shujuan
Liu,*[a] Wei Huang*[a,b] and Qiang Zhao*[a]
[a]
Y. Xu, Dr. M. Zhao, L. Wu, F. Li, M. Li, Prof. S. Liu, Prof. W. Huang and Prof. Q. Zhao
Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors
Institute of Advanced Materials (IAM) Nanjing University of Posts and Telecommunications
Nanjing, 210023 (P. R. China)
E-mail: iamsjliu@njupt.edu.cn
provost@nwpu.edu.cn
iamqzhao@njupt.edu.cn
[b]
Prof. W. Huang
Shaanxi Institute of Flexible Electronics (SIFE)
Northwestern Polytechnical University
Xi'an 710072 (P.R. China)
Supporting information for this article is given via a link at the end of the document.
Abstract: The development of efficient phototherapeutic agents
(PTA) through rational and specific principles exhibits great
significance to the biomedical field. Herein, a facile and rational
strategy has been presented to design PTA via perturbation theory.
According to the theory, through the rational optimization of donor–
acceptor structure, heavy atom number and their functionalization
position, which can effectively decrease energy gap between the
singlet and triplet states and increase spin–orbit coupling constant,
the enhanced intersystem crossing rate for singlet oxygen
generation and nonradiative transition for photothermal conversion
efficiency can be realized simultaneously. Finally, the efficient PTA
have been obtained, which exhibited excellent performance on
multimode imaging guided synergetic photodynamic/photothermal
therapy. Thus, this study expounds the intrinsic mechanism of
organic PTA and guides the rational design of more excellent
organic PTA via perturbation theory.
however, are based on trial and error discovery. To date, there
were only few works about PTA reported following specific
design principles.[7] Thereby,
a facile and rational design
strategy to develop highly efficient organic PTA was necessary.
Herein, we establish a rational strategy to design highly
efficient PTA via perturbation theory. According to the theory,
the intersystem crossing (ISC) rate constant (kISC) was shown as
below:[8]
̂
⟨ | | ⟩ (ΔΕ
)
(1)
̂
where ⟨ | | ⟩ and EST were the spin–orbit coupling (SOC)
matrix element and the energy gap between the singlet and
triplet states, respectively. Namely, large SOC constant and
narrow EST contributed to the enhanced ISC process.[9] Besides,
̂
heavy atom effect can effectively increase ⟨ | | ⟩ for
improving kISC and quench fluorescence for enhancing
nonradiative conversion,[10] which help to increase singlet
oxygen generation and photothermal conversion performance.
Photo-induced therapy (PT), including photothermal therapy and
photodynamic therapy, has attracted extensive research
interests owing to its non-invasiveness, highly spatiotemporal
resolution and easy manipulation.[1] For PT, it is a key issue to
develop highly efficient phototherapeutic agents (PTA) that can
effectively kill tumor cells.[2] To date, a large number of PTA
based on inorganic materials have been investigated for efficient
cancer treatment by utilizing their excellent stability, high singlet
oxygen generation and photothermal conversion performance.[3]
When compared to inorganic materials, organic PTA possesses
many advantages, such as inherent biodegradability, low toxicity
and flexible design.[4] Therefore, the development of highly
efficient organic PTA is a good alternative approach for PT.
Generally speaking, an excellent organic PTA should exhibit
strong near-infrared (NIR) light capture capability, namely
intense NIR absorption for singlet oxygen generation,
photothermal and other conversion simultaneously.[5] Up to now,
cyanine, phthalocyanine, squaric acid, rhodamine and aza-
boron-dipyrromethene (aza-BODIPY) derivatives have been
used as photosensitizers (PSs) or photothermal agents for
efficient anticancer by utilizing heavy atom effects, photoinduced
electron transfer (PET) mechanism or combination of various
electronic donors and acceptors.[6] Most of these works,
Figure 1. (a) Chemical structures of aza-BODIPY dyes C-1 – C-4. (b) UV-vis
absorption spectra of C-1 – C-4 in CH2Cl2. The absorption intensity has been
normalized. (c) Temperature rise of samples in CH2Cl2 under irradiation (660
or 730 nm, 0.4 W cm-2) with time. The ambient temperature was 15 oC. (d) The
consumption of DPBF in its various mixtures under irradiation (660 or 730 nm,
0.4 W cm-2) with time.
1
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Thus, to validate the above strategy, aza-BODIPY
derivatives were chosen as the promising candidates of PTA,
owing to their high stability, long-wavelength absorption and
easily tunable structure.[11] Noteworthily, substitutes in different
positions of aza-BODIPY core exhibited considerable difference
on their photophysical properties,[12] which offered a great
chance to develop excellent PTA. With above consideration, we
have rationally designed and facilely synthesized an aza-
BODIPY architecture (C-4) with enhanced singlet oxygen
generation and photothermal conversion performance via
perturbation theory. C-4 was mainly composed of four parts
(Figure 1a), including aza-BODIPY core, thienyl rings, bromine
atoms and flexible alkyl chain. Thienyl rings with small steric
hindrance, as the rich electron donor, were conjugated at
position 3 and 5 of electron-deficient aza-BODIPY core to form
donor and acceptor (D-A) architecture for small EST and
enhanced NIR absorption,[13] as well as increased ISC process
then explored the singlet oxygen generation capacity of C-1, C-2,
C-3 and C-4 by monitoring the photo-oxidation of 1,3-
diphenylisobenzofuran (DPBF) under a 660 nm or 730 nm laser
irradiation. The introduction of sulphur and bromine atoms can
promote ISC process for enhanced singlet oxygen generation
capacity, especially, bromine atoms at 2 and 6 position of aza-
BODIPY core led to the best performance (Figure 1d and S2).
To further validate the positive influence of heavy atom effect
to promote photothermal conversion and singlet oxygen
generation performance, time-resolved photoluminescence
(TRPL) spectra were first measured. Under the same excitation
condition, C-2 (τ = 1.73 ns) and C-4 (τ = 0.82 ns) displayed the
weaker emission and shorter photoluminescence lifetimes than
C-1 (τ = 2.49 ns) and C-3 (τ = 3.09 ns), respectively (Figure 2a
and 2b). C-3 showed the longer photoluminescence lifetimes
than C-2. The result was attributed to the effective electron
transfer from thienyl rings to aza-BODIPY core caused by the
small steric hindrance and excellent electron-rich performance of
thienyl rings (Figure 2a and 2b). The above results collectively
validated the positive influence of heavy atom effect to enhance
photothermal conversion ability. The positive influence of heavy
atom effect to enhance singlet oxygen generation performance
was then explored by theory calculations. C-1, C-2, C-3 and C-4
presented the increased SOC constants as 0.14, 8.79, 57.81
and 99.16 cm-1 (Figure 2c), accompanying with a decreased
EST as 1.22, 1.20, 1.11 and 1.10 eV in turn, respectively,
suggesting the largest kISC of C-4 for the best singlet oxygen
generation performance according to the perturbation theory.
Noteworthily, the results of theory calculations were well
consistent with the experimental data. These principles provided
a guidance to develop highly efficient PTA for imaging-guided
tumor therapy (Figure 2d).
[14]
̂
by increasing ⟨ | | ⟩.
Bromine atoms were included to
further enhance ISC and nonradiative transition process.[15]
Flexible alkyl chains were introduced to improve the solubility of
rigid aza-BODIPY.[16] For comparison, other three aza-BODIPY
dyes (C-1 – C-3, Figure 1a) containing different substituents
(phenyl rings, brominated phenyl rings or brominated thienyl
rings) were also designed and synthesized. The detailed
synthetic routes and corresponding chemical structures of as-
prepared aza-BODIPY dyes (C-1 – C-4) can be found in
Supporting Information. Their structures have been fully
characterized by NMR and MALDI-TOF-MS spectra.
C-2 showed the red-shifted and enhanced NIR absorption
(λabs. = 677 nm, 95400 L mol-1 cm-1) for narrower EST compared
to C-1 (λabs. = 665 nm, 78600 L mol-1 cm-1) in CH2Cl2 (Figure 1b
and Table S1), owing to the role of bromine atoms to change
the highest occupied molecular orbital of aza-BODIPY
structure.[12, 17] C-3 (λabs. = 741 nm, 167300 L mol-1 cm-1) and C-4
(λabs. = 732 nm, 117800 L mol-1 cm-1) exhibited further red-
shifted and enhanced NIR absorption for further decreased EST
because of the strong electron donating ability and small steric
hindrance of thienyl rings (Figure 1b and Table S1).[18] The
narrower EST helped to the enhanced kISC for singlet oxygen
generation. The large molar extinction coefficient manifested
their strong light capture ability for promising singlet oxygen
generation, photothermal and other conversion. C-1, C-2, C-3
and C-4 also showed sharp NIR emission peaks in the region of
675 – 825 nm, and their maximal emission peaks were at 698
nm, 712 nm, 760 nm and 766 nm, respectively (Figure S1 and
Table S1), indicating their potential bioimaging performance in
vivo. Their corresponding fluorescence quantum yields were
0.126, 0.049, 0.048 and 0.006 in turn (Table S1), indicating that
the heavy atom effect could effectively quench fluorescence to
increase nonradiative decay for photothermal conversion.
To demonstrate the enhanced photothermal conversion of C-
4 via heavy atom effect, we investigated the photothermal
performance of C-1, C-2, C-3 and C-4 by recording their
temperature change under a 660 or 730 nm laser irradiation. C-4,
C-3, C-2 and C-1 showed the decreased temperature changes
as 23.1 C, 22.6 C, 18.2 C and 13.4 oC, respectively (Figure
1c), indicating the positive influence of heavy atom effect on
photothermal conversion ability of aza-BODIPY core. The
introduction of heavy atom (Br or I) was an effective method to
increase ISC process for efficient singlet oxygen generation as
reported by Eisenberg, Akkaya and their coworkers.[19-24] We
o
o
o
Figure 2. (a) Emission spectra of C-1, C-2, C-3 and C-4 in CH2Cl2 (10 μM) (λ
= 650 nm). (b) Time-resolved photoluminescence spectra of C-1, C-2, C-3 and
C-4 in CH2Cl2 (10 μM). (c) Energy level diagrams and SOC coefficients (ε) for
C-1
− C-4 were obtained by theoretical calculations, respectively. (d)
Proposed mechanism for PT enhancement.
2
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C-4 with the best photothermal conversion capacity and
singlet oxygen generation performance was used for the
biological application in vivo. Water-soluble and biocompatible
C-4 nanoparticles (C-4 NPs) with 500 μM were obtained via the
relationship of concentration-dependent absorption of C-4 in
CH2Cl2 according to previous report (Figure S3).[25] They
exhibited spherical architecture with hydrodynamic diameter of
91.3 nm (Figure S4a) in PBS (pH 7.4), indicating their promising
tumor passive targeting capacity by enhanced permeation
retentioneffect (EPR) effect.[26] C-4 NPs exhibited absorption
with a maximal peak and a shoulder peak at 695 nm and 747
nm (Figure S4b), respectively, and dual emission (Figure S4b),
which resulted from the aggregation of C-4 in micelle,[13]
suggesting their promising fluorescence imaging (FI)
performance. Meanwhile, concentration-dependent PA and FI
amplitudes of C-4 NPs provided possibility for quantitative
analysis of the distribution of C-4 NPs (Figure S5). The
excellent photobleaching, photothermal and RONS (such as
H2O2 and ONOO−) resistance of C-4 NPs was an important
inherent property of phototherapeutic agents for tumor imaging
and therapy,[27] which has been first confirmed (Figure S6). C-4
NPs also showed concentration- and laser-power-dependent
temperature changes, and continuous generation of singlet
oxygen with irradiation time (Figure S7), indicating their
promising synergistic PDT/PTT performance. They also
displayed high photothermal conversion efficiency as 35.9%
(Figure S8), which was higher than common photothermal
agents.[28]
cytotoxicity of C-4 NPs. The generation of singlet oxygen in vitro
was validated by cell imaging (Figure 3b). The laser-power-
dependent Hela cells viability was investigated. Hela cells were
treated with C-4 NPs (15 μM), C-4 NPs under hypoxia (oxygen,
5%), C-4 NPs plus vitamin C (VC) or saline, respectively. The
cell viability showed negative correlations with laser power
(Figure 3c). Under the same irradiated condition, the cells
incubated with C-4 NPs plus hypoxia exhibited higher cells
viability than those treated by C-4 NPs, but lower than those
treated by C-4 NPs plus VC (Figure 3c). The results manifested
that photodynamic performance of C-4 NPs existed in
phototherapy, which acted as an important part even under
hypoxia. The fluorescence live/dead cell imaging and flow
cytometry experiments also verified the same results (Figure 3d,
S9 and S10).
Inspired by the concentration-dependent photoacoustic,
fluorescence and photothermal properties of C-4 NPs in vitro,
the EPR effects of C-4 NPs were investigated by FI and
photoacoustic imaging (PAI). Tumors exhibited the brightest
fluorescence signal with high signal-to-noise ratio (SNR) as
about 3.0 at 6 h post intravenous injection of C-4 NPs (150 μM,
200 μL) (Figure 4a and 4c), suggesting the effective
accumulation of C-4 NPs in tumors. Meanwhile, the tumors also
presented obviously enhanced photoacoustic signal (SNR was
about 2.5) compared to those before injection (Figure 4b and
4d). The C-4 NPs were mainly
To explore the excellent phototherapeutic performance of C-
4 NPs in vivo, the in vitro concentration-dependent Hela cells
and 3T3 cells viability for C-4 NPs was first investigated to
evaluate their dark cytotoxicity. The cells still retained a high
viability (>83%) even treated with various concentrations of C-4
NPs (up to 150 μM) (Figure 3a), indicating almost no dark
cytotoxicity of C-4 NPs. The generation of singlet oxygen in vitro
Figure 3. (a) The viability of cells treated with different concentrations of C-4
NPs, respectively. (b) Cell imaging for C-4 NPs (15 μM)-mediated ROS
generation. The scale bar of all images was 20 μm. (c) The viability of cells
incubated with C-4 NPs (15 μM) or not, and other treatment under various
laser irradiation (730 nm, 5 min). (d) The imaging of Hela cells treated with C-4
NPs only, C-4 NPs + VC + laser, C-4 NPs + laser, C-4 NPs plus hypoxia, PBS
and laser, respectively (15 μM, 730 nm, 0.4 W cm-2, 5 min). All the images
share the same scale bar of 20 μm.
Figure 4. (a) Fluorescence and (b) photoacoustic imaging of mouse before
and after injection of C-4 NPs (150 µL, 200 µM). (c) and (d) were the average
signal intensity of (a) and (b), respectively. (e) C-4 NPs distribution in different
organs 6 h after its injection. (f) The mice tumor sections imaging after C-4
NPs (150 µL, 200 µM) injection or not for 6.5 h. VC acted as singlet oxygen
quencher, which was intratumorally injected after C-4 NPs (150 µL, 200 µM)
injection for 6 h. 0.5 h later, the tumor part was irradiated (730 nm, 0.4 W cm-2,
5 min). The scale bar of all images was 20 μm.
3
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distributed in tumors and livers at 6 h post intravenous injection
of them (Figure 4e), which could confirm the same results.
Finally, the weaker or even negligible FI signal was observed
after 20 h (Figure 4a), suggesting that C-4 NPs in tumor parts
could be completely metabolized. Besides, hypoxia in tumors
could weaken PDT effect, and singlet oxygen generation in
tumor parts was also validated by confocal microscopy images
of tumor section mice injected with C-4 NPs, suggesting
sufficient singlet oxygen generation for PDT in tumor sites during
phototherapy (Figure 4f).
C-4 NPs plus light and VC (PTT), only C-4 NPs, only light and
only saline, respectively. The tumors in PDT+PTT and PTT
groups could be effectively inhibited, and they were removed
before the seventh therapy, indicating the excellent
phototherapeutic effects. The PDT+PTT group presented slightly
better effects than PTT group (Figure 5b), due to the fact that
the aggravated hypoxic environment weakened the singlet
oxygen generation during phototherapy with time. Finally, the
removed tumors exhibited no recurrence after 20 d treatment
(Figure 5b). The tumors sizes in the rest groups presented
slightly better effects than PTT group (Figure 5b), due to the
fact that the aggravated hypoxic environment weakened the
singlet oxygen generation during phototherapy with time. The
tumors sizes in the rest groups presented obvious growth with
sizes about 7 - 10 times of the original value after 20 d treatment
(Figure 5b). The mice weight in all groups exhibited almost no
fluctuation (Figure 5c), and the histological hematoxylin and
eosin staining showed no obvious pathological discrepancy
among all groups in tumors and main organs (Figure 5d),
suggesting that the only C-4 NPs or only laser did almost no
harm to mice.
Rational temperature change was key for highly efficient
tumor therapy.[28] The tumor part of mice at 6 h post intravenous
injection of C-4 NPs in different concentrations was irradiated,
respectively,
which
showed
concentration-dependent
temperature increase (Figure 5a). The tumor temperature was
51.3 °C after 5 min irradiation (Figure 5a),[29] manifesting the
promising PTT in vivo with C-4 NPs at 150 μM.
Inspired by the excellent photothermal and photodynamic
performance, and good EPR effect of C-4 NPs in vivo,
phototherapy of tumor-bearing mice was conducted after
intravenous injection of samples for 6.5 h in vivo. The tumor-
bearing mice were treated with C-4 NPs plus light (PDT+PTT),
Figure 5. Photothermal therapy in vivo. (a) Photothermal imaging of mice intravenously injected with C-4 NPs in different concentrations. (b) The tumor size and
(c) body weight of Hela tumor-bearing mice with various treatments during treatments, respectively. (d) H&E staining of tumor and major organs sections obtained
from the mice with different treatments. All the images share the same scale bar of 300 μm. Images were taken at 25 oC.
4
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COMMUNICATION
In summary, highly efficient PTA C-4 NPs based on aza-
BODIPY with D-A structure has been rationally developed via
perturbation theory. The systematic investigation of theoretical
calculation, fluorescence quantum yield, photothemal conversion
efficiency and singlet oxygen generation ability have confirmed
that the rational optimization of donor–acceptor structure, heavy
atom number and their functionalization position can effectively
decrease energy gap between the singlet and triplet states and
increase spin–orbit coupling constant, which contributes to the
enhanced intersystem crossing rate for singlet oxygen
generation and nonradiative transition for photothermal
conversion efficiency simultaneously. Noteworthily, water
Program for Support of Top-Notch Young Professionals,
Scientific and Technological Innovation Teams of Colleges and
Universities in Jiangsu Province (TJ215006).
Conflict of interest
The authors declare no conflict of interest.
Keywords: phototherapeutic agent • aza-BODIPY • heavy atom
effect • perturbation theory • multimode imaging
soluble
C-4
NPs
showed
concentration-dependent
References:
photoacoustic and fluorescent signal, high photothermal
conversion efficiency (η = 35.9%) and abundant singlet oxygen
generation ability. FI, PAI and PTI of C-4 NPs in vivo manifest
that C-4 NPs can efficiently accumulate in tumor parts via EPR
effects. The C-4 NPs show efficient therapeutic performance
without recurrence when utilized in vivo with mild condition,
demonstrating their efficient phototherapeutic performance. This
study will offer more interesting exploration on the
multifunctional nanotheranostic agents for potential clinical
applications.
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Acknowledgements
The authors acknowledge financial support from the National
Funds for Distinguished Young Scientists (61825503), the
National Program for Support of Top-Notch Young Professionals,
the Priority Academic Program Development of Jiangsu Higher
Education Institutions (YX03001), National Natural Science
Foundation of China (51473078 and 21671108), National
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Entry for the Table of Contents
The aza-BODIPY (C-4) based phototherapeutic agent (PTA) with enhanced anticancer efficacy has been rationally designed via
perturbation theory. According to the theory, C-4 with improved singlet oxygen generation and photothermal conversion efficiency
simultaneously has been obtained through the rational optimization of structure. Finally, the PTA successfully cures tumors without
recurrence under multimode imaging guided phototherapy.
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