Rational Design of Organic Probes for Turn-On Two-Photon Excited Fluorescence Imaging and Photodynamic Therapy

Article abstract of DOI:10.1016/j.chempr.2018.12.001

The rational design of two-photon absorption (2PA) organic dyes with 2PA cross-section (δ) values tunable by mild stimuli under physiological conditions remains a challenge. Using a simulation-assisted structure screening method, we have designed an aceta

Full text of DOI:10.1016/j.chempr.2018.12.001

Article
Rational Design of Organic Probes for Turn-On
Two-Photon Excited Fluorescence Imaging
and Photodynamic Therapy
Chun-Lin Sun, Jun Li, Xiao-Zhen
Wang, ..., Hong-Bing Fu,
Jian-Nian Yao, Hao-Li Zhang
hbfu@cnu.edu.cn (H.-B.F.)
jnyao@iccas.ac.cn (J.-N.Y.)
haoli.zhang@lzu.edu.cn (H.-L.Z.)
HIGHLIGHTS
A simulation-assisted strategy is
used to design turn-on-type 2PA
dyes
The 2PA dyes are suitable for
imaging and photodynamic
therapy of tumor
Phototoxicity to normal tissue can
be reduced by turn-on in the
tumor area only
This design strategy is useful for
fast screening of new theranostic
reagents
We report a cost-effective theory-assisted molecular screening method for the
rational design of turn-on-type two-photon absorption (2PA) dyes. The obtained
dyes exhibit high efficiency in two-photon excited fluorescence and singlet oxygen
generation. The turn-on process can occur in mild and nontoxic conditions, even in
the intracellular environment. With such a turn-on feature, two-photon laser
confocal scanning microscopic imaging and two-photon excited photodynamic
therapy can be performed in a well-controlled manner for MCF-7 cells and
melanoma tumor.
Sun et al., Chem 5, 1–17
March 14, 2019 ª 2018 Elsevier Inc.
https://doi.org/10.1016/j.chempr.2018.12.001

Please cite this article in press as: Sun et al., Rational Design of Organic Probes for Turn-On Two-Photon Excited Fluorescence Imaging and
Photodynamic Therapy, Chem (2018), https://doi.org/10.1016/j.chempr.2018.12.001
Article
Rational Design of Organic Probes for
Turn-On Two-Photon Excited Fluorescence
Imaging and Photodynamic Therapy
Chun-Lin Sun,1 Jun Li,1,6 Xiao-Zhen Wang,1 Rong Shen,4 Sha Liu,4 Jian-Qiao Jiang,1 Ting Li,5
2,
1,7,
Qi-Wei Song,1 Qing Liao,3 Hong-Bing Fu,3, Jian-Nian Yao, and Hao-Li Zhang
*
*
*
SUMMARY
The Bigger Picture
Photodynamic therapy (PDT) is a
clinically accepted treatment for
tumors, which relies on the
photogeneration of singlet
oxygen with specific
The rational design of two-photon absorption (2PA) organic dyes with 2PA
cross-section (d) values tunable by mild stimuli under physiological conditions
remains a challenge. Using a simulation-assisted structure screening method,
we have designed an acetal terminated distyrylbenzene derivative (Ace-DSB),
which can be readily converted into aldehyde terminated molecules (Ald-DSB)
to exhibit a significant increase in d values at 760 nm. The conversion reaction,
which dramatically turns on the 2PA absorption capability of the molecules,
can occur in an intracellular environment. Furthermore, Ald-DSB exhibits
40 times higher efficiency of two-photon generation of singlet oxygen than
Ace-DSB. These turn-on features allow enhanced two-photon laser confocal
scanning microscopic imaging and two-photon excited photodynamic therapy
(2PE-PDT) for MCF-7 cancer cells and melanoma tumors. This work opens a
new avenue toward the design of turn-on-type two-photon absorption mole-
cules for both in vitro and in vivo tumor imaging and 2PA-PDT.
photosensitizers. Organic dyes
with two-photon absorption (2PA)
features allow PDT to be carried
out in deeper tissue than
conventional one-photon
absorption dyes. We report herein
the rational design of turn-on-type
two-photon absorption (2PA)
dyes for fluorescent imaging and
singlet oxygen generation by a
simulation-assisted structure
screening method. With such 2PA
dyes, microscopic imaging and
photodynamic therapy can be
performed in a well-controlled
manner according to the
INTRODUCTION
Organic molecules exhibiting two-photon absorption (2PA)1,2 activity are of interest
for many practical applications, including nonlinear optics,3,4 two-photon micro-
scopy,5,6 and photodynamic therapy.7,8 The two-photon excitation using near-
infrared (NIR) light gives a better definition of the focal spot and resolution, lower
phototoxicity, and deeper transmission in biological tissues compared with common
one-photon excitation using UV and visible light.9 To facilitate these applications,
the design and discovery of 2PA molecules with large 2PA cross-section (d) are
essential. However, two-photon absorption and excitation are intrinsically much
less efficient than the one-photon processes, and the occurrence probability of
2PA, reflected by the parameter d, is generally small for most organic molecules.
Therefore, developing an efficient method to design highly efficient 2PA dyes
with ideal functions remains a challenge.
requirements. The partial turn-on
state allows long-time imaging
with reduced light damage to
normal tissue, whereas the full
turn-on state allows efficient
photodynamic therapy to the
selected tumor tissue. This
simulation-assisted design
strategy paves a new way for the
rational design of 2PA
So-called ‘‘turn-on’’ fluorescence dyes are strongly desired in a variety of applica-
tions, such as bioimaging, biosensors, and photodynamic therapy.9–11 An ideal
turn-on dye is dark at the initial state but can be switched to brighter state after
being triggered by certain stimuli. To date, most of the turn-on dyes belong to
conventional one-photon absorption materials. Given the many advantages of
2PA, turn-on-type 2PA dyes are highly desirable for many biological applications.
The turn-on feature of the two-photon excited fluorescence dyes can reduce the
autofluorescence background and enhance imaging resolution.12,13 Moreover,
photosensitizers for future clinical
tumor therapy.
Chem 5, 1–17, March 14, 2019 ª 2018 Elsevier Inc.
1

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turn-on-type 2PA photodynamic therapy provides good control to the treatment
process and reduces phototoxicity.13,14 However, the development of 2PA dyes
with tunable d have met with limited success.11,15 The main difficulty in the design
of switchable 2PA emission dyes comes from the fact that the photophysical pro-
cesses associated with two-photon excitation and subsequent light emission are
much more complex than the conventional one-photon excitation process. Struc-
tural features that could dominate the d values are not well understood, so finding
an efficient mechanism to switch the 2PA efficiency on and off appears to be very
difficult.
There are two key problems that need be solved for the design of 2PA dyes with
tunable d value. First, we need to identify a relatively small structure change that
could significantly increase the d in a given 2PA framework. Second, an easily oper-
ated chemical reaction needs to be identified, which can trigger the aforemen-
tioned structural change so that it gives rise to a significant change in d value.
Given that the structure-property correlation of 2PA dyes is not well understood,
the first problem, i.e., identifying an appropriate structure change, is the more
challenging to solve.
Obviously, experimental screening of all the structural modification possibilities
among a large number of known 2PA frameworks is costly and impractical. There-
fore, a simulation-assisted structure screening method must be used to assist the
design of 2PA dyes. Unfortunately, 2PA process is a third-order nonlinear phenom-
enon and d is related to the imaginary part of the third-order polarizability so that
traditional theoretical calculations of d are too time consuming for a practical
screening of organic structures.
In this work we demonstrate, for the first time, the design of a turn-on 2PA probe by us-
ing a simple theoretical simulation method. We have chosen the distyrylbenzene (DSB)
structure as the model 2PA framework. The theory-assisted screening of different DSB
1State Key Laboratory of Applied Organic
Chemistry, Key Laboratory of Special Function
derivatives is based on a facile theoretical method analysis of the excited states.16 Aided
by the simulation, we found that through a simple conversion from ethylene glycol
acetal terminal to aldehyde terminal DSB structure, a 26-fold enhancement of the
d values is achieved. The rationally designed DSB molecules were successfully applied
in both turn-on in vitro and in vivo two-photon excited fluorescence (2PEF) imaging and
turn-on two-photon excited photodynamic therapy (2PE-PDT).
Materials and Structure Design, College of
Chemistry and Chemical Engineering, Lanzhou
University, Lanzhou 730000, P.R. China
2Beijing National Laboratory for Molecules
Science, State Key Laboratory for Structural
Chemistry of Unstable and Stable Species, and
Key Laboratory of Photochemistry, Institute of
Chemistry, Chinese Academy of Sciences, Beijing
100190, P.R. China
RESULTS AND DISCUSSION
3Beijing Key Laboratory for Optical Materials and
Photonic Devices, Department of Chemistry,
Capital Normal University, Beijing 100048, P.R.
China
Simulation-Assisted Molecular Design
The DSB structure is selected as the model framework because many DSB deriva-
tives are highly fluorescent and are widely used in various optoelectronic applica-
tions.17–23 In our previous work, we have discussed how small modification of DSB
frameworks could significantly affect their spectral properties.24–27 Our basic idea
here is to design carbonyl group modified DSB molecule as a turn-on-type 2PA im-
aging reagent, which is based on two considerations. First, an sp2 hybrid carbon in
carbonyl group connected directly to the DSB framework could extend the conjuga-
tion system. Previous studies have suggested that 2PA dyes extended p-conjuga-
tion with electron push-pull motifs is beneficial for high d values.10,28 Second, it is
very important that most carbonyl groups are relatively reactive, which helps to
find an appropriate trigger reaction for d value tuning. Several DSB derivatives
have been designed for initial screening, which is based on terminated substituted
1,4-dimethoxy-2,5-di((E)-styryl)benzene. The terminal substituent groups include
4School of Basic Medical Sciences, Lanzhou
University, Lanzhou 730000, P.R. China
5School of Life Sciences, Lanzhou University,
Lanzhou 73000, P.R. China
6MOE Key Laboratory of Space Applied Physics
and Chemistry, Joint Lab of Nanofluidics and
Interfaces, School of Natural and Applied
Sciences, Northwestern Polytechnical University,
Xi’an 710072, P.R. China
7Lead Contact
*Correspondence: hbfu@cnu.edu.cn (H.-B.F.),
jnyao@iccas.ac.cn (J.-N.Y.),
haoli.zhang@lzu.edu.cn (H.-L.Z.)
https://doi.org/10.1016/j.chempr.2018.12.001
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Figure 1. Structures of the DSB Compounds Studied in This Work
(A) The DSB derivatives designed for initial screening.
(B) ‘‘Turn-on’’ 2PA cross-section from Ace-DSB to Ald-DSB by mild and nontoxic reaction condition.
acyl chloride, amide, carboxylic acid, ester, methanimine, oxiane, acetal, and alde-
hyde with the molecular structures shown in Figure 1A. The possible transformation
among DSB carbonyl derivatives is shown in Figure S8. The bottom-right panel of
Figure 1A illustrates the two representative DSB molecules, including the alde-
hyde-containing molecule (Ald-DSB) and its ethylene glycol acetal (Ace-DSB).
Ace-DSB hydrolysis yields Ald-DSB stoichiometrically under various mild conditions,
i.e., b-CD in water solution and room temperature (Figure 1B).29
The next step is to assess the potential 2PA performance of the molecules and select
a suitable structure for practical experimental measurement. Several theoretical
methods have been developed for the calculation of d values, the most well known
of which is called the summary over states (SOS).30 However, a full SOS calculation
for DSBs, which contains 210 or more electrons, would be time consuming. In fact, a
full ab initio calculation is unnecessary for initial structural screening. Instead we have
used a simpler method for screening purposes.
In our recent work, we reported a facile theoretical method for the fast screening of
small molecules as potential 2PA dyes. This method is based on analysis of the nat-
ural transition orbitals (NTOs) directly associated with the final state of 2PA transi-
tion.16,31 As a highly delocalized final excited state is likely to give a large d than a
localized one, visualization of the NTOs not only can help to understand the excited
state associated with the 2PA process but can also help to quickly identify candidate
molecules with high d. For centrosymmetric chromophores such as DSB derivatives
studied in this work, the 2PA transition is from the ground state (S0, 1Ag) to the
lowest excited state with Ag symmetry (S2 or/and S3).10,28 Therefore, we paid partic-
ular attention to the S2 and S3 states (for more details, see Figure S4).
We calculated the NTOs of the excited states (S2 and S3) of the DSB molecules shown
in Figure 2A. To investigate the excited states, we conducted time-dependent den-
sity functional theory (TD-DFT) calculations using the Gaussian 09 package.32 NTOs
are used to study the related ground and excited states. More details of NTO
calculation results and screening processes are provided in Figure S9. We have
considered possible reactions including esterification, amidation, hydrolysis,
chlorination, epoxidation, and deacetylation (conversion from acetal terminal to
the aldehyde). NTO analysis suggests that most reactions on the carbonyl groups
do not bring about obvious change to the S2 states, and therefore are unlikely to
enhance d very much. However, the conversion from the acetal terminated structure
(Ace-DSB) to the aldehyde terminated structure (Ald-DSB) is associated with a large
change of the NTO orbitals involved.
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Figure 2. NTOs and Energy Levels for Ace-DSB and Ald-DSB
(A) NTOs of S2 and S3 for Ald-DSB and Ace-DSB related to the 2PA process (detailed NTOs and data
shown in Figure S4), the H represents the hole and P represents the particles.
(B) Energy-level diagram and symmetry for the singlet state of Ald-DSB and Ace-DSB, where H
represents the highest occupied molecular orbital and L represents the lowest unoccupied
molecular orbital.
Figure 2B illustrates the calculated orbital energy level and symmetry for the singlet
states (to S4) of the Ald-DSB and Ace-DSB. The symmetry of ground state is Ag for
both molecules. The S2 state of the Ace-DSB has Au symmetry, suggesting that
the 2PA transition from S0 to S2 is forbidden. For the Ald-DSB symmetry of S2 state
is Ag, which allows 2PA transition to occur. It is noted that this symmetry analysis to
the different excited states does not consider possible vibronic coupling. Mean-
while, it is noted that the S3 state of Ace-DSB has Ag symmetry and lies very close
to its S2 state. Therefore, it is likely that the 2PA process of Ace-DSB may involve tran-
sition from S0 to S3. Nevertheless, the information from the orbital symmetry analysis
further verifies that the Ald-DSB is likely to have much a larger d than Ace-DSB.
As illustrated above, the TD-DFT based NTOs and symmetry analysis to the excited
states help us to quickly identify the Ace-DSB molecules as the promising candidate
for turn-on 2PA probe. It is noted that although NTO analysis provides only qualita-
tive information, it helps us to quickly screen out most of the candidates and focus on
only Ace-DSB and Ald-DSB molecules. More quantitative information can be ob-
tained from a significantly more expensive calculation method. Here we have veri-
fied the above calculation results regarding 2PA properties of the two molecules us-
ing the Dalton program, which is based on coupled cluster response theory.33,34
Details of such calculations are shown in Figure S5. In the 690- to 850-nm range,
the calculation predicts that the maximum d values for the Ace-DSB and Ald-DSB
are 10 and 3,500 GM, respectively, which confirms that the prediction based on
our NTO analysis is correct. The result of simulation of 2PA cross-section based on
a simplified sum-over-states model is consistent with Dalton calculations (details
shown in Figure S6). It is worth mentioning that the 2PA spectra calculation using
the Dalton program cost about 1,800 CPU hr, whereas the NTO calculation cost
only about 20 CPU hr. Given the simplicity of the NTO analysis, it is much easier
to be used in practice for fast molecule screening.
Spectroscopic Characterization
We then verified the above theoretical prediction by experimental measurements.
The synthesis and structural characterization data of the Ald-DSB and Ace-DSB are
provided in the Supplemental Information. The experimental one-photon absorp-
tion and emission spectra of the two molecules in diluted tetrahydrofuran (THF)
4
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Figure 3. Steady-State One-Photon Absorption and Emission Spectra
(A) Steady-state one-photon absorption and fluorescence of Ald-DSB (solid line) and Ace-DSB
(dashed line) in THF.
(B) Simulated absorption and fluorescence spectra. Inset shows the linear fitting to the calculated
and experimental energies including the two absorption peaks and the emission peak energy.
solution are shown in Figure 3A, with the key parameters summarized in Table 1. The
absorption spectra of the Ace-DSB show two prominent absorptions in the range of
300–500 nm. The lowest electronic transition (S0-S1) gives the maximum absorption
band around 395 nm, and the (S0-S2) transition gives the slightly weaker band at
around 327 nm. The emission wavelength is in the range of 400–600 nm with a
peak at 445 nm. The absorption and fluorescent emission bands of Ald-DSB show
bathochromic shifts compared with Ace-DSB. The first and second absorption peaks
are around 423 and 338 nm, respectively. The emission peak of Ald-DSB also
shows a bathochromic shift of about 40 nm compared with the Ace-DSB. The inset
of Figure 3A shows that the emission of Ace-DSB is blue and Ald-DSB is green.
The fluorescence quantum yields (V) of Ace-DSB and Ald-DSB were measured as
0.72 and 0.57, respectively.
We simulated the one-photon absorption spectra and fluorescence emission spectra
by TD-DFT methods (Figure 3B). The linear fitting to the calculated and experimental
transition energy shows good linearity with a small systematic error of 0.3 eV, indi-
cating that the simulations were reliable (Figure 3B, inset).26 The experiment and
simulation results of the one-photon properties, i.e., bathochromic shifts for Ald-
DSB compared with Ace-DSB, offer preliminary evidence that the electron orbital
delocalization of Ald-DSB is indeed higher than that of Ace-DSB.
The 2PA spectra of the Ald-DSB and Ace-DSB were measured by the 2PEF method.
Figure 4 shows the 2PA spectra between 730 and 840 nm. Both molecules show the
maximum 2PA absorption around 760 nm, corresponding well with the two-photon
absorption for the S0-S2 and S0-S3 transition. Meanwhile, the Ace-DSB and Ald-DSB
showed no obvious degradation upon the femtosecond laser irradiation for 2 hr
(120 fs, 1,000 Hz, 80 mW, Figure S10), indicating high photostability.
As shown in Figure 4, Ald-DSB exhibits very high d in the measurement range, the
largest d being about 2,421 GM. In contrast, the 2PA for Ace-DSB is much smaller,
with maximum d of only about 93 GM, similar to that of unmodified distyrylben-
zene. These 2PA measurement results are in good agreement with our theoretical
prediction, confirming that our NTO analysis method is also applicable to the
styryl-benzene-like molecules. Moreover, these results indicate that if we are
able to convert Ace-DSB to Ald-DSB, the d will enhance 26-fold, which is ideal
for turn-on applications. Although the F for Ald-DSB is slightly lower than that
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Table 1. Spectroscopic Parameters of Ald-DSB and Ace-DSB
Abs
exp
em
exp
l
l
F
t
d
FD
dFD
kr
knr
Ace-DSB
Ald-DSB
395
423
445
484
0.72
0.57
1.58
1.77
93
2421
0.18
0.28
17 (14)
678 (600)
0.47
0.32
0.17
0.24
l
Abs (nm) is the vertical transition wavelength of the lowest excited states, lem (nm) is the energy of the peak of the fluorescence band, F is the fluorescence quan-
tum yields, t (ns) is the lifetime of fluorescence, d (GM) is the 2PA cross-section, FD is the singlet oxygen quantum yield, and dFD presents the 2PA singlet oxygen
generation capability. The data in parentheses show the test value with tetraphenylporphyrin as a reference. The last two columns provide the rate constant for
radiative deactivation S1 to S0, kr (nsÀ1) and the rate constant for nonradiative deactivation, knr (nsÀ1).
of Ace-DSB, 2PA measurement shows that the Ald-DSB is significantly brighter
than Ace-DSB under the two-photon excitation fluorescence condition (inset of
Figure 4A). Figure 4B shows that the 2PEF emission from the Ace-DSB solution
was enhanced obviously upon the addition of b-CD, indicating the transformation
from Ace-DSB to Ald-DSB.
In Vitro Application
To examine Ald-DSB’s capability as 2PEF probe, we imaged Ald-DSB with MCF-7
cells by using two-photon laser confocal scanning microscopy (TPLCSM). The fluo-
rescence emission excited by the 760-nm laser is strong enough for the high-resolu-
tion microscopic imaging, as shown in Figure 5A.
z-stack confocal images (depth interval 0.5 mm) were taken continuously from the
bottom to the top via TPLCSM. The image stacks were processed by ImageJ soft-
ware to obtain the 3D reconstruction movie of MCF-7 cell (Video S1). Intense intra-
cellular luminescence was observed with femtosecond laser pulses at 760 nm, under
which no background fluorescence from the MCF-7 cells would interfere. Benefiting
from the high d of the Ald-DSB molecule, high signal strength can be obtained. The
large d and high photostability of Ald-DSB make it a good candidate for in vivo
TPLCSM imaging.
The Ace-DSB was also incubated with the MCF-7 cell, and the TPLCSM image is
shown in Figure 5B. Compared with the cells stained by Ald-DSB, those stained
by Ace-DSB are much darker. Ace-DSB can be transformed to Ald-DSB by treating
with b-CD in solution.29 This reaction was established in cell-culture conditions
and monitored by thin-layer chromatography and mass spectra (shown in Figure S3).
The b-CD is a Food and Drug Administration-approved biological safe material. The
mild and nontoxic reaction condition is suitable for intracellular translation. We
treated the Ace-DSB-stained cells with the b-CD solution, and the TPLCSM shows
significantly enhanced intensity (Figures 5B and 5C). These results confirm that the
turn-on 2PA in vitro has been achieved.
We measured the singlet oxygen quantum yields (VD) of the DSBs by the singlet
oxygen luminescent method, using rose bengal as a reference. The singlet oxygen
1Dg emission spectra are shown in Figure 6 and the determined VD is displayed in
Table 1. The VD of Ald-DSB is about 0.28, which is twice that of the Ace-DSB, i.e.,
0.14. However, the efficiency of singlet oxygen generated by the two-photon exci-
tation is defined not only by VD but also by d. In fact, the factor dVD is a useful figure
with which to estimate the two-photon excited photodynamic therapy (2PE-PDT) ef-
ficiency. The dVD values are 678 for Ald-DSB and 17 for Ace-DSB, indicating that
Ald-DSB is 40 times more efficient than Ace-DSB in singlet oxygen production. In
Figure 6, two-photon excited luminescence of singlet oxygen sensitized with Ace-
DSB or Ald-DSB was used to directly evaluate their dVD, which gave dFD values of
6
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Figure 4. 2PA Spectra of Ald-DSB and Ace-DSB in Solution
(A) 2PA cross-section of Ald-DSB and Ace-DSB in THF solutions. The inset shows the corresponding
2PA fluorescence photographs of the Ace-DSB (left) solution and the Ald-DSB (right) solution under
760-nm illumination.
(B) 2PEF emission changes upon the addition of b-CD.
14 and 600 for Ace-DSB and Ald-DSB, respectively. Figure S10 shows the turn-on
singlet oxygen process through transformation from Ace-DSB to Ald-DSB.
Next, the 2PE-PDT of the two compounds were studied. The MCF-7 cancer cells
were incubated with different concentrations of Ace-DSB and Ald-DSB for 24 hr
and then divided into two equal series. One series was illuminated with 760 nm
for 20 min. Another series was kept in the dark as the control group. After illu-
mination, all cells were incubated in darkness for 24 hr before the viability test.
The cell viability of control-group data exhibits low cytotoxicity for both Ald-DSB
and Ace-DSB in darkness. The cell viabilities were higher than 90% even when
the concentration was as high as 32 mg/mL (about 80 mM). After the irradiation
(control experiments confirmed that the laser does not damage the cells), the
viabilities were very different between the cell samples containing Ald-DSB and
Ace-DSB.
There is no obvious viability change in the cell containing Ace-DSB, indicating weak
2PE-PDT effect. In contrast, Ald-DSB resulted in a viability lower than 20%, suggest-
ing a good 2PE-PDT effect (Figure 7). The lower 2PE-PDT efficiency of Ace-DSB can
be attributed to its lower singlet oxygen production efficiency.
Finally, we tested the capability of Ace-DSB as 2PE-PDT turn-on reagent. The
cellular phototoxicity of Ace-DSB in the presence of b-CD was carried out on
MCF-7 cells under NIR illumination. Figure 8 shows the 2PE-PDT results during
the turn-on process. In this experiment, the MCF-7 cells incubated with Ace-DSB
(8 mg/mL, 20 mM) were divided into two batches. One batch was incubated in
the standard cell-culture medium and the other was incubated in the cell-culture
medium containing b-CD (16 mg/mL, 28 mM) for 24 hr. Half of each batch was
then illuminated with 760-nm light (10 mW cmÀ2, 120 fs, 1,000 Hz) for 20 min
(others remained in darkness). After the illumination, all cells were incubated in
the dark for another 24 hr. The cell viabilities were monitored by using microscopy,
as shown in Figure 8.
Figures 8A and 8B show that there is no obvious cell death to the Ace-DSB stained
cells after the illumination, indicating that Ace-DSB has low 2PE-PDT efficiency. In
contrast, in the cell batch containing b-CD distinct cell death was observed within
the irradiation area, as shown in Figure 8D. As discussed above, the Ald-DSB has
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Figure 5. In Vitro Bioimage Application
(A) TPLCSM image of MCF-7 incubated with Ald-DSB upon excitation at 760 nm; the inset shows the
high-resolution image.
(B and C) Before (B) and after (C) treatment with b-CD in same exposure time.
much higher dVD than its precursor Ace-DSB. Therefore, the Ald-DSB generated a
significant amount of singlet oxygen upon two-photon excitation, which resulted in
the death of almost all cancer cells.
The above results demonstrate that the low 2PE-PDT active precursor Ace-DSB can
be converted into Ald-DSB under cell-culture conditions when treated by a low con-
centration of b-CD. Due to the high 2PA fluorescence intensity of the Ald-DSB, the
treated area can be readily imaged by TPLCSM. Furthermore, the turn-on regions
can be selectively illuminated by NIR light to produce singlet oxygen, which is
8
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Figure 6. Singlet Oxygen Quantum Yields
(A) Photoexcited luminescence of singlet oxygen sensitized with Ace-DSB, Ald-DSB, and rose
bengal (RB) as a reference.
(B) Photoexcited luminescence of singlet oxygen sensitized with Ace-DSB, Ald-DSB, and
tetraphenylporphyrin (TPP) as a reference.
used in 2PE-PDT. Such a strategy may be applied in the treatment of various cancers
or disease, such as skin cancer and fundus lesions, with higher selectivity and fewer
side effects.
In Vivo Application
The mice tumor model (the building process of the model is described in the
Experimental Procedures) was used for evaluating the turn-on TPLCSM in vivo image
(Figure 9A). The mice were injected with Ace-DSB at the tumor location. The
TPLCSM in Figure 9B shows only very low signal and most of the emission can be
attributed to the background luminescence. After b-CD injection as the turn-on stim-
ulant, TPLCSM (Figure 9C) shows the details of in vivo tumor cells. Compared with
Figure 9B, the tumor cell signal is very easily distinguished from the background
luminescence.
The mice tumor model was then used for evaluating the turn-on 2PA-PDT therapeu-
tic effect, as shown in Figure 10A, and details are given in the Experimental Proced-
ures. Melanoma is the most dangerous form of skin and eye cancer and is the most
suitable cancer for PDT treatment.14 When light treatment of 80 mW/cm2 was com-
bined with Ace-DSB (turn-on by b-CD) for 15 days, the tumors in the treated group
were significantly smaller than in the control group and the Ace-DSB group
(Figure 10B). From histopathological analysis using H&E staining (Figures 10C,
10D, and S14), we observed that the tumor tissue from the control group displayed
compact tumor cells with an intact structure. No significant difference between Ace-
DSB-injected and control groups was detected, which suggests that the tumor tissue
is not affected by Ace-DSB/b-CD injection (Figure S14). In the Ace-DSB/b-CD and
irradiation groups (Figure 10D), the tumor tissue was no longer structurally inte-
grated, and there were many sections and numerous nuclear fragments. With the
help of TPLCSM imaging of the tumor tissue section, the details can be observed.
Before 2PA-PDT the morphology of tumor cells was normal, whereas cavity and
cell disintegration were observed on the tumor image after the 2PA-PDT treatment
(Figures 10E and 10F).
The trends of tumor size and percentage survival are reported in Figures 10G and
10H. The final relative tumor volume of the control group was about 6.5
(650 mm3). The Ace-DSB group showed very similar tumor-growing trend with the
control group, suggesting no inhibitory effect on the tumor. In the turn-on group,
the relative tumor volume was reduced obviously compared with the control group,
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Figure 7. Cytotoxicity of Ace-DSB and Ald-DSB
Viability of MCF-7 cells incubated with (A) Ace-DSB and (B) Ald-DSB for 24 hr and then illuminated
by a 760-nm laser (10 mW cmÀ2, 1,000 Hz, 120 fs) for 20 min (2PE-PDT series) or no light (dark series).
The error bars denote the SD of three replicates.
indicating significant inhabitation to the tumor tissue due to 2PE-PDT. The mice sur-
vival percentages after the 2PE-PDT are shown in Figure 10H. The survival percent-
age of the control and Ace-DSB-only group fell rapidly after 40 days, and all the mice
died after 60 days. In contrast, the survival percentage of the Ace-DSB/b-CD group
remained 40% after 70 days Figure 10H indicates that the 2PA-PDT using Ace-DSB/
b-CD significantly prolonged the survival of the tumor-bearing mice and inhibited
tumor growth. The above results demonstrate that we have successfully achieved
both turn-on 2PEF tumor cell imaging and turn-on 2PA-PDT on melanoma tumor
cells. It is noted that the current experiments were performed on melanoma cells,
which are exposed at the skin surface. Further molecular optimization and more so-
phisticated drug-delivery systems may be needed to take advantage of the unique
turn-on feature of the dyes and perform 2PA-PDT treatments for other tumors.
In summary, we have demonstrated that by using NTO analysis of the excited states, fast
screening of the potential 2PA dyes can be achieved. We have used this method to
quickly evaluate a series of carbonyl functionalized distyrylbenzene derivatives for their
potential as turn-on 2PA dyes without the need of actually synthesizing all the mole-
cules. Under consideration of both the 2PA activity and chemical reactivity, we have
identified the acetal terminated molecule Ace-DSB as a preferable structure for the
2PA probes. We then conducted more careful theoretical and spectroscopic character-
ization focusing only on this structure. The measurements confirmed that the conversion
of the Ace-DSB into Ald-DSB under mild conditions gave rise to a 26-fold increase of the
d values at 760 nm, along with a 40-fold increase in efficiency of two-photon generation
of singlet oxygen. The conversion reaction, which dramatically turns on the 2PA absorp-
tion capability of the molecules, can occur under mild and physiological conditions. This
turn-on feature of the Ace-DSB molecule allows enhanced imaging of cancer cells using
TPLCSM. Importantly, by controlled conversion of Ace-DSB into Ald-DSB, we have suc-
cessfully achieved turn-on two-photon excited photodynamic therapy for MCF-7 cancer
cells. These probes were also successfully applied in in vivo 2PA photodynamic therapy
on melanoma tumor using a mouse model. This work highlights the importance of the-
ory-assisted molecular screening methods for the cost-effective rational design of novel
2PA-based theranostic reagents.
EXPERIMENTAL PROCEDURES
Calculation
All the calculations were performed with the Gaussian 09 software package.32 The
ground states of these four molecules were optimized using DFT with the B3LYP
10
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Figure 8. Cellular Phototoxicity in Response to 2PE-PDT by Turn-On Process
(A and B) Viability of MCF-7 cells incubated with Ace-DSB and no b-CD (A) in darkness and (B)
illuminated by the 760-nm laser.
(C and D) Viability of MCF-7 cells incubated with Ace-DSB and added b-CD, when deprotection
happens, (C) in darkness and (D) illuminated by the 760-nm laser; the box shows the illuminated
area.
functional (DFT/B3LYP/6-31G++(d, p)), and the excitation energies were calcu-
lated using the TD-DFT at the B3LYP functional (DFT/B3LYP/6-311G++(2d,2p)).
Adiabatic TD-DFT in the Kohn-Sham form was used for calculating the excited-
state structures.35,36 The S1 excited states were then optimized using the restricted
configuration interaction (singlet) (DFT/6-31G(++d,p)) approach, and the S1-S0
electronic transitions from the relaxed excited states were obtained from TD-
DFT calculations using the optimized excited states as inputs with the B3LYP
functional (DFT/B3LYP/6-311G++(2d,2p)). Natural transition orbitals (NTO)37
calculations were performed after TD-DFT to describe the physical meanings of
the orbitals of the hole and electron on the excitation states. Inhomogeneous
line-broadening parameter for all absorption spectra calculations has been fixed
to G = 0.17 eV for all chromospheres based on a typical line width of respective
experimental spectra.38 The transition dipole moments between the excited states
were calculated by the Multiwfn wavefunction analyzer software with the Gaussian
fchk and out file as input.39
Synthesis
The synthesis and structural characterization of the DSB molecules are shown in
Figures S1 and S2.
Absorption and Fluorescence Spectra
Steady-state absorption spectra were recorded using a T6 UV-vis spectrometer
(Purkinje General, China). Fluorescence measurements were performed on an
LS55 fluorescence spectrometer (PerkinElmer, USA). The solutions were bubbled
with N2 for 10 min before fluorescence measurement. Absolute fluorescence quan-
tum yield (V) of Ald-DSB (0.01 mM in THF) was measured by an FLS920 fluorescence
spectrometer using an integrating sphere (Edinburgh Instruments, UK). Relative
fluorescence quantum yield was measured in THF solution with Ace-DSB in THF as
a reference, using the relative methodology based on the following equation: Vx/
Vr = (Ar/Ax) (Dx/Dr) (nx/nr)2, where A is the absorbance at the excitation wavelength,
n the refractive index, and D the integrated luminescence intensity. r and x stand for
reference and sample.
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Figure 9. In Vivo Bioimaging Application
(A) Diagram of in vivo turn-on TPLCSM image of tumor stained by the Ace-DSB/b-CD.
(B and C) Before (B) and after (C) treatment with b-CD in same exposure time.
For measurements of the fluorescence time profiles, the time-correlated single-
photon counting method using a nanosecond pulsed LED sources (376 nm, full
width at half-maximum ca. 800 ps) with 40-MHz repetition rate was employed. A
photomultiplier tube and a counting board (PicoQuanta, PicoHarp 300, Germany)
were used for signal detection (Figure S7).
Singlet Oxygen Quantum Yields
The singlet oxygen quantum yield VD was estimated by the singlet oxygen lumines-
cent method. Singlet oxygen phosphorescence (near 1,270 nm) was recorded with a
liquid nitrogen-cooled, solid indium-gallium-arsenic detector (Edinburgh Instru-
ments), and VD values were deduced from an analogous methodology similar to
that for fluorescence quantum yields (see above) with rose bengal as a reference
(VD = 0.76 in methanol).40,41 We measured the two-photon excited 1O2 generation
ability (dFD) by using tetraphenylporphyrin as a reference (l2PA = 790 nm, d = 12 GM,
FD = 0.6, dFD = 7.2).42 The InGaAs detector (NIRvana-640, Princeton Instruments)
was used to detect 1O2 emission.
2PA Cross-Section
2PA cross-sections were determined via a comparative method that measured
the 2PEF by using rhodamine B as a reference. The fundamental of a mode-locked
Ti:sapphire laser (690–850 nm, Tsunami) was focused into a quartz cuvette with
an optical geometry and detected with a liquid-nitrogen-cooled charge-coupled
device (SPEC-10-400 B/LbN, Roper Scientific) attached to a polychromator (Spectro-
pro-550i, Acton).
12
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Photodynamic Therapy, Chem (2018), https://doi.org/10.1016/j.chempr.2018.12.001
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Photodynamic Therapy, Chem (2018), https://doi.org/10.1016/j.chempr.2018.12.001
Figure 10. In Vivo 2PA-PDT Application
(A) In vivo studies of Ace-DSB/b-CD as cancer turn-on 2PA-PDT agent, whereby an extending femtosecond laser pulse was used to irradiate the tumor.
(B) In vivo tumor inhibition assays of Ace-DSB/b-CD. Experimental conditions are described in the text; H&E-stained paraffin section image was used as
control.
(C–F) Before 2PA-PDT (C) and after 2PA-PDT (D); the TPLCSM image of histological sections of tumor stained with Ace-DSB/b-CD for (E) control and (F)
after 2PA-PDT.
(G) Relative tumor volume (compared with the same tumor on day 0, V0) with 2PE-PDT; the arrows show the 2PE-PDT treatments (t test, **p < 0.01).
(H) Percentage of survival after 2PE-PDT.
Cell Culture
MCF-7 human breast cancer cells were cultured in DMEM (Gibco) containing 10%
bovine calf serum and 1% penicillin and streptomycin at 37ꢀC in a humidified
atmosphere composed of 95% air and 5% CO2. Cells were removed with a
solution of 0.05% trypsin in 0.53 mM EDTA, resuspended in serum-free medium
(105 cells/mL) for cell seeding, and allowed to attach to the surface for 2 hr before
the addition of serum-containing media. For passage, cells were resuspended in
the same 10 mL of the medium that they were growing in, and 3 mL was transferred
to 7 mL of fresh medium in a new flask. Cells were seeded into a Petri dish, 12-well
plate, or confocal dish for different experiments.
Cell Staining and Two-Photon Fluorescence Microscopy Imaging
Cells were cultured on 12-well glass slides or confocal dish under normal culture con-
ditions. After a 48 hr incubation period, the medium was removed and cells were
incubated with 50 mM Ace-DSB or Ald-DSB in PBS (containing 0.5% DMSO) for
10 min, followed by two washes with PBS. The cell was visualized with 203, 403,
and 633 objectives. The cell images were captured sequentially on an Olympus
FluoView FV1000 confocal laser scanning microscope (Olympus) with 760-nm
excited light. Stacks of 18 optical sections (1,024 3 1,024 pixel arrays) were collected
at 0.55-mm intervals in the z dimension. The 3D microscopy construction of MCF-7
cell images is shown in Video S1.
In Vitro 2PE-PDT
MCF-7 human breast cancer cells were seeded into a confocal dish (100,000 cells/mL)
and cultured for 24 hr. The cells were then cultured for 4 hr with or without 8 mg/mL
(about 20 mM) Ald-DSB or Ace-DSB in fresh culture medium containing 0.5%
DMSO. After incubation with the DSB molecules, cells were washed twice, main-
tained in fresh culture medium, and submitted (or not) to laser irradiation. The
b-CD added to the culture medium for the turn-on stimuli was 16 mg/mL (about
28 mM). The entire area of the well was irradiated at 760 nm (10 mW/cm2 120 fs,
1,000 Hz) for 20 min.
Cytotoxicity
To assess the cytotoxicity of the Ald-DSB or Ace-DSB with (without) laser irradiation,
we observed the viability after staining by using a LIVE/DEAD kit. Cells were cultured
in a 24-well microplate (approximately 100,000 cells per well). After culture for 24 hr,
a calcein and ethidium homodimer-1 PBS solution was added and cultured for
20 min in normal culture environment. We determined the cell viability by using a
confocal microscope (Carl Zeiss LSM710) whereby we calculated viability by count-
ing live (green) and dead (red) cells, comparing those numbers with that of the con-
trol well, and expressing them as percentages.
In Vivo Imaging and 2PA-PDT
BALB/c nude mice (6 weeks old) were purchased from Beijing Vital River Laboratory
Animal Technology Co., Ltd., and all experimental procedures and protocols in the
14
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Photodynamic Therapy, Chem (2018), https://doi.org/10.1016/j.chempr.2018.12.001
study were approved by the Ethics Committee of Lanzhou University, China. Accord-
ing to the requirements for Animal Ethics, all experiments were made to minimize
the number of mice (20 mice per group) used and their suffering. Each 6-week-old
female BALB/c nude mouse was inoculated subcutaneously with B16-F10 melanoma
in mouse cells suspended in 0.2 mL of sterile saline solution. When the tumors grew
to about 100 mm3, the mice were distributed randomly into groups for the analysis of
tumor development with PBS and Ace-DSB/b-CD treatments. One milligram of Ace-
DSB was dissolved in 2 mL of 20% fat emulsion (Intralipid) by ultrasonication for
10 min (concentration: 1.25 mM); 4.5 mg b-CD was dissolved in 2 mL PBS to form
a solution whose concentration was 2 mM (Figure S13). We have measured the
d spectra in different solutions (Figure S12), which show negligible solvent effect.
The dose of 50 mL of PBS (control) or Ace-DSB was injected into the tumors per
mouse. The turn-on 2PEF was achieved by injection of 100 mL of b-CD solution.
The in vivo tumor images were captured sequentially on an Olympus FluoView
FV1000MPE confocal laser scanning microscope (Olympus). The 2PA PDT was per-
formed by the home-made instrument shown in Figure 10A. The average power den-
sity of laser (760 nm, 120 fs, 1,000 Hz) delivered to the tumor was measured with a
thermoelectric optical energy meter and was 80 mW/cm2. Each 2PE-PDT process
was performed for 10 min three times.
The initial tumor volumes were all pre-designed to be approximately 100 mm3
before 2PE-PDT. To reduce individual initial tumor size differences, we introduced
the relative tumor volume, which we calculated by using the following formula: rela-
tive tumor volume = Vx/V0, where Vx is the absolute tumor volume of the respective
tumor on day x, and V0 is the absolute tumor volume of same tumor on day 0, when
the treatment started.
In the in vivo theranostic process, the turn-on degree of the dyes can be controlled
according to the experimental requirements (Figure S15). Partial turn-on state allows
imaging with reduced light damage to normal tissue, and the full turn-on state allows
efficient photodynamic therapy to the selected tumor tissue. For example, the dyes
can be partially turned on by adding a 10% dose of b-CD solution, and enabled high-
quality TPLCSM imaging with a low level of light toxicity. The full dose of b-CD was
then imposed to fully turn on the photosensitizers for 2PE-PDT (Figure S15).
H&E Staining
Tumors and major organs were fixed with 10% formalin for at least 24 hr. Samples
were then embedded in paraffin, sectioned, and stained with H&E. Histopatholog-
ical changes were observed with a light microscope (DMI4000B; Leica, Germany).
The TPLCSM images of histological sections of tumor stained with Ace-DSB/b-CD
were captured on a Leica SP8 DIVE.
DATA AND SOFTWARE AVAILABILITY
X-ray crystallographic data can be obtained free of charge from the Cambridge
Crystallographic Data Center (www.ccdc.cam.ac.uk/data_request/cif) under acces-
sion number CCDC: 934402.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, 15 fig-
ures, 1 video, and 1 data file and can be found with this article online at https://doi.
org/10.1016/j.chempr.2018.12.001.
Chem 5, 1–17, March 14, 2019
15

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Photodynamic Therapy, Chem (2018), https://doi.org/10.1016/j.chempr.2018.12.001
ACKNOWLEDGMENTS
This work is supported by National Natural Science Foundation of China (21702085
21602176, 51525303, 21233001, 21190034, 21573251, and 21673144), the Funda-
mental Research Funds for the Central Universities (lzujbky-2017-11), the Beijing
Natural Science Foundation of China (2162011), and High-Level Teachers in Beijing
Municipal Universities in the Period of 13th Five-Year Plan (CIT&TCD20180331).
AUTHOR CONTRIBUTIONS
Conceptualization, C.-L.S. and J.L. equally; Investigation, C.-L.S., J.L., X.-Z.W.,
R.S., S.L., J.-Q.J., T.L., Q.-W.S., and Q.L.; Writing – Original Draft, C.-L.S. and J.L.;
Writing – Review & Editing, H.-L.Z.; Supervision, H.-B.F., J.-N.Y., and H.-L.Z.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: May 16, 2018
Revised: July 12, 2018
Accepted: November 30, 2018
Published: January 10, 2019
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