Inner salt-shaped small molecular photosensitizer with extremely enhanced two-photon absorption for mitochondrial-targeted photodynamic therapy

Article abstract of DOI:10.1039/c6cc09473b

Herein, we experimentally and theoretically demonstrate an unprecedentedly enhanced two-photon absorption in a small organic molecule by a simple introduction of an inner salt-shaped structure. Moreover, such an inner salt-shaped small molecule also exhibits superior singlet oxygen quantum yield and fascinating structure-inherent mitochondrial-targeting ability for highly efficient two-photon photodynamic therapy via a mitochondrial apoptosis pathway.

Full text of DOI:10.1039/c6cc09473b

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
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DOI: 10.1039/C6CC09473B
Laboratory for Organic Electronics and Information Displays & Institute of
Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center
for Advanced Materials (SICAM); Nanjing Tech University, Key Laboratory
of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM),
Jiangsu National Synergetic Innovation Center for Advanced Materials
(SICAM)
Fan, Quli; Nanjing University of Posts and Telecommunications (NUPT), Key
Laboratory for Organic Electronics and Information Displays & Institute of
Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center
for Advanced Materials (SICAM)

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Inner Salt-Shaped Small Molecular Photosensitizer with Extremely
Enhanced Two-Photon Absorption for Mitochondrial-Targeting
Photodynamic Therapy
Received 00th January 20xx,
Accepted 00th January 20xx
Wenbo Hu,ab Tingchao He,c Rongcui Jiang,a Jun Yin,d Lin Li,b Xiaomei Lu,b Hui Zhao,a Lei Zhang,a
Ling Huang,b Handong Sun,d Wei Huang*b and Quli Fan*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Here, we experimentally and theoretically demonstrate an drawbacks including stringent spectral overlap for energy-
unprecedentedly enhanced two-photon absorption in organic transfer, aggregation-enhanced non-radiative transitions, large
small molecule by a simple introduction of inner salt-shaped size-induced transport barrier, and complex fabrication or
structure. Moreover, such inner-shaped small molecule also synthesis. In contrast, directly improving the intrinsic δ of small
exhibits superior singlet oxygen quantum yield and fascinating molecular TP-PS, which has fascinating merits of tiny size, good
structure-inherent mitochondrial-targeting ability for highly- cell permeability, minimal perturbation to living systems, and
efficient two-photon photodynamic therapy via mitochondrial- easy synthesis,5 is more beneficial for the practical application
apoptosis pathway.
of TP-PDT. Unfortunately, traditional approaches that rely on
the expanded π-conjugated systems to improve δ often
become limited effectiveness or even invalid in strong polar
solvent,6 and also cause intrinsic limitations of increasing
molecular weight, poor water-solubility and synthetic
complexity. Worse still, these approaches cannot ensure an
effective transfer of singlet-to-triplet excited states for high
triplet-exciton yield, which is directly responsible for the 1O2
generation.7
Photodynamic therapy (PDT), relying on the cytotoxic singlet
oxygen (1O2) generated by photosensitizers (PSs) upon light
irradiation to kill diseased cells, is a very appealing noninvasive
cancer-therapeutic modality.1 Compared with one-photon (OP)
excited PDT (OP-PDT), two-photon (TP) excited PDT (TP-PDT)
exhibits more attractive potential in precise cancer therapy
owing to its increased penetration depth, intrinsic 3-
dimensional spatial-temporal selectivity and reduced off-target
photodamage.2 However, the ultralow two-photon absorption
cross-section (δ) of available TP-excited PSs (TP-PSs) with value
of tens GM (1 GM = 10-50 cm4 s photon-1) in strong polar
Besides, subcellular-targeting is an attractive strategy to
maximize the efficacy of TP-PDT.8 Among numerous
subcellular-targeting, mitochondrial-targeting is an optimal
subcellular site of action to magnify the destructiveness of 1O2
owing to its vital contribution in keeping the normal
solvent (necessary for biological samples) severely limits their
practical applications.2a,
3
To circumvent this problem,
continuous efforts have been made to construct energy-
transferring combination in which existing PS (energy
acceptor) is indirectly excited by the dye unit with large δ
(energy donor).4 Yet, this approach encounter some
Scheme 1. Schematically illustrating the design of small
molecular TP-PS for mitochondrial apoptosis-mediated TP-
PDT.
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physiological activity.9 Thereby, coupling mitochondrial-
targeting ability into small molecular TP-PS with is highly
desirable to maximize the efficacy of TP-PDT.10 But, to our
knowledge, no such small molecular TP-PS has heretofore
been proved viable for mitochondrial-targeting TP-PDT.
Herein, we demonstrate the simple introduction of inner salt-
shaped structure into a conjugated organic small molecule can
remarkably improve its δ in strong polar solvent as well as
realize high 1O2 quantum yield and mitochondrial-targeting
ability for highly efficient TP-PDT (Scheme 1). By experimental
and theoretical investigations, inner salt-shaped organic small
molecular (DBA) exhibits two orders of magnitude enhanced δ
of 3260 GM in methanol than clinical used TP-PS, which is the
first our known example of organic small molecules (MW<600
Da) to reach the magnitude of thousand GM in strong polar
methanol. Meanwhile, DBA possesses a high 1O2 quantum
yield of 72.8%, probably because N atoms within DBA facilitate
the transfer of singlet-to-triplet excited states for triplet
photosensitized 1O2. Moreover, DBA also exhibits structure-
inherent mitochondrial-targeting ability and thus performs a
highly efficient mitochondrial apoptosis-mediated TP-PDT.
To obtain inner salt-shaped DBA, the precursor molecule DBD
structured with side-chain tertiary amines and π-conjugated
bis(phenylethynyl)benzene backbone (frequently used
backbone in designing TP-absorbing molecule) was first
synthesized (Fig. S1, Scheme 1).11 Upon hydrolysis, tertiary
amine group deprotonates the carboxylic acid, easily yielding a
water-soluble inner salt-shaped DBA. Notably, non-inner salt-
shaped DBD is also used as a representative control to
highlight the power of inner salt-shaped structure in improving
δ in strong polar solvent. The inner salt-shaped molecular
Fig. 1 (a) Normalized absorption and emission spectra of
DBD in tetrahydrofuran (THF) and DBA in methanol. (b)
Open aperture Z-scan data of DBD and DBA for the TP-
absorption cross-section at 740 and 720 nm, respectively.
photofrin (10 GM in methanol).2a To our knowledge, this δ is
the highest among the conjugated organic small molecule
(MW <600 Da) in strong polar solvent (methanol).
For a better understanding of such much-enhanced δ of DBA in
strong polar solvent, we performed the time-dependent
density functional theory calculations (See detailed description
in Supporting Information). It is known that δ is directly
proportional the molecular second hyperpolarizability (γ),13
thus γ and corresponding δ of DBD and DBA in both relatively
weak polar THF and strong polar methanol were computed
using the sum over states (SOS) formula.14 As shown in Table 1,
the attenuated γ and δ of DBD from THF to methanol indicate
that using traditional approach to improve molecular δ indeed
become limited effectiveness in strong polar solvent, which
agrees closely with previous reports.15 Conversely, DBA
exhibits an exceptionally enhanced γ and δ from THF to
methanol. Moreover, the magnitude of calculated δ of DBA in
methanol is reasonably consistent with our experimental δ,
theoretically confirming the feasibility of our approach. In view
of the closely relationship between molecular polarizability
(including γ) and molecular electric field,16 we reasoned that
the strong polar solvent produces a negative influence on
molecular electric field, thus leading to a decreased γ and δ.
While the charged structure of DBA in strong polar solvent can
provide an additional electric field that creates a positive
influence on molecular electric field, which then energetically
increase its γ and δ.
1
structure of DBA was verified by H-NMR spectra and single-
crystal X-ray diffraction; detailed synthetic procedures and
characterization were given in Supporting Information (Fig.S2-
6, Table S1).
The absorption maxima of DBA is located at 309 and 356 nm
(Fig.1a), which is about 20 nm blue-shift compared with that of
DBD (320, 381 nm). The emission spectrum maxima of DBA
(420 nm) also shows a 20 nm blue-shift when compared with
that of DBD (440 nm). Such blue-shift can be attributed to the
increased torsional angle of the bis(phenylethynyl)benzene
backbone in strong polar solvent, which in turn shortens the π-
conjugation system.12 Other OP-related optical properties,
including detailed discussions, are presented in Fig.S7 and
Table S2. Fig. S8 demonstrated the TP-absorption process of
DBD and DBA based on the Supporting Equation. 1. Before the
investigation of δ, we excluded the influence of linear
absorption of samples in the biological tissue transparency
window of 650 - 800 nm (Fig.S9). By open-aperture Z-scans
technology, we found the δ of DBD in THF upon the
femtosecond 740 nm excitation was 741 GM (Fig.1b).
Interestingly, despite possessing comparable π-conjugated
system, DBA in strong polar methanol still exhibits a nearly 5-
fold enhanced δ of 3260 GM upon the femtosecond 720 nm
excitation relative to that of DBD in THF and two orders of
magnitude enhancement when compared with commercial PS
Table 1. Calculated molecular second hyperpolarizability (γ)
and two-photon absorption cross-section (δ) of DBD and
DBA in different solvent.
γ
a)
δ
ωTriplet
[eV]
Molecule
DBD
Solvent
[×10-34
aus]
[GM]
THF
102.0
34.1
574.0
186.6
264.7
4857.4
2.53
2.52
2.60
2.58
Methanol
THF
40.4
DBA
Methanol
758.4
a) Calculated energy level of the lowest triplet
2 | J. Name., 2012, 00, 1-3
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experiment was performed with DBA and a commercial Mito-
tracker (MTR) after the exclusion of optical interferences exist
between two dyes (Fig.S13). The characteristic punctate
fluorescence of mitochondria and bright yellow signals in the
merge of DBA and MTR (Fig.2c3) mean that DBA can
selectively target mitochondria. Furthermore, mitochondrial
fractions were further separated from cytoplasm fractions. The
PL intensity of the mitochondrial fractions was higher than
that of the cytoplasm fractions (Fig.2d), again demonstrating
the mitochondrial-targeting ability of DBA. Such structure-
inherent mitochondrial-targeting ability could be attributed to
the weak positive-charged property of DBA (Fig. S14), which
may have a stronger affinity to the more negative-charged
mitochondrial membrane than to the plasma membrane.20
Given the key role of reactive oxygen species (ROS, e.g., 1O2) in
PDT, we first verified the ROS generation of DBA in
intracellular environment under TPE (Fig. S15). Fig.3a showed
the time-dependent cell viability profile of Hela cells that were
exposed to 1 kHz femtosecond laser irradiation at 750 nm with
the power density of ca 1.0 W cm−2 to quantitatively evaluate
the TP-PDT efficiency. The viability of DBA stained cells upon
only 5 min TPE dramatically decreased to 55.8%, while no
obvious decrease in cellular viability for classic PS TMPyP4 or
TPE alone, revealing a tremendous potential of DBA in highly
efficient TP-PDT. Further, TP-PDT activity of DBA against cancer
Figure 2. a) Normalized emission of DBA in methanol solution (solid
line, dash line and dot line represent without, with saturation of
nitrogen at room temperature and oxygen-free, respectively, under
pulsed 375 nm excitation). b) Phosphorescence luminescence (PL)
spectra of 1O2 generated by DBA under 365 nm irradiation.
5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin
tetra(p-
toluenesulfonate) (TMPyP4) in D2O at 410 nm irradiation was used
as the standard. c) TP-fluorescence confocal image of Hela cells
treated with DBA or Mito-tracker MTR, and merged image. Scale
bar = 10 µm. d) Fluorescence spectra of mitochondrial and
cytoplasmic fractions of DBA (10 μM) treated Hela cells.
cells was investigated by using
a fluorescence probe
(propidium iodide, PI) to distinguish viable cells from dead
cells. Compared with Hela cells stained by TMPyP4, Hela cells
Typically, a TP-PS firstly creates an excited singlet state upon
TP-excitation (TPE) and then undergoes singlet-to-triplet
intersystem crossing (ISC) process for triplet photosensitized
1O2. To verify the existence of suitable triplet state in DBA, the
OP-excited emission spectrum of DBA-dissolved methanol
solution without (control) or with saturation of nitrogen were
investigated, in which nitrogen-saturated methanol solution
was used to eliminate the quenching of triplet state emission
caused by molecular oxygen (3O2).17 The occurrence of obvious
bump at 480 nm in Fig.2a indicates the existence of triplet
state. Further, a triplet state emission of DBA in methanol
solution under nearly vacuum state (oxygen-free) was
observed at 480 nm, which is closely consistent with our
calculated energy level of DBA at the lowest triplet excited
states of ωTriplet =2.58 ev, 480 nm in Table 1. Furthermore, two
triplet state lifetime were found to be 10.85 μs (70%) and 1.32
μs (30%) from the fitting of the emission decay (Fig.S10), which
is long enough to interact with 3O2 to yield 1O2. The shorter
lifetime (1.32 μs) could attributed to the intermolecular
stained by DBA exhibit
a significant cell morphological
disruption and strong apoptotic fluorescence (Fig.3b), thus
again demonstrating the higher TP-PDT efficiency of DBA.
1
Furthermore, an effective O2 scavenger (NAC) was employed
interaction-induced fast decay.18 Finally, DBA displayed an
1
obvious O2 characteristic emission band at
∼
1270 nm (Fig.2b)
and its 1O2 quantum yield was calculated to be 72.8% using
TMPyP4 in D2O as standard.19 Although relevant mechanism
remains elusive, we speculate that the introduction of N atoms
improves the spin-orbit coupling probability and thus
enhances its singlet-to-triplet ISC process.
Fig. 3 (a) Time dependent cell viability of Hela cells treated
with DBA (2 μM) or TMPyP4 (2 μM) after TPE and TPE alone.
(b) Fluorescence images of PI staining Hela cells after TP-PDT
treatments at 750 nm laser; Scale bars: 25 μm. (c) Western
blotting results of TP-PDT activity of DBA (20 μM) on Hela cell
lysates against anti-caspase-3 and PARP antibodies. (d) The
proposed mitochondrial apoptosis pathway of Hela cells.
In view of bioapplications, the low dark-cytotoxicity, excellent
biocompatibility and chemical stability of DBA were
demonstrated in Fig.S11 and Fig.S12. To determine the
mitochondrial-targeting of DBA,
a standard co-staining
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1
to clarify the role of O2 in cell death. In the presence of NAC, Synergetic Innovation Center for Organic Electronics and
the unobservable apoptotic fluorescence reflected an efficient Information Displays, the Natural Science Foundation of
1
inhibition of cell death, indicating that the generated O2 from Jiangsu Province of China (No. BZ2010043, NY211003,
DBA with the assistance of TPE was responsible for the cell BM2012010), the Key Laboratory of Optoelectronic Devices
death. The above results verify that DBA is superior to TMPyP4 and Systems of Ministry of Education and Guangdong Province
as efficient TP-PS for TP-PDT on cancer cells.
(GD201603).
To elucidate the apoptosis pathways of DBA, western blotting
assay was utilized to monitor the caspase activity of the
apoptotic HeLa cell lysates. As known, deleterious caspase-3 is
mitochondria-mediated caspase and its activation is
Notes and references
1. (a) D. E. J. G. J. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev.
Cancer, 2003, 3, 380; (b) J. P. Celli, B. Q. Spring, I. Rizvi, C. L.
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considered to be
a
commitment to cell death.21 Once
activated, the caspase-3 is responsible for the cleavage of
downstream poly(ADP-ribose) polymerase (PARP) that
ultimately leads to cell death.9 As shown in Fig.3c, distinct
changes were detected in the expression of caspase-3, PARP,
and cleaved PARP of the cells with different treatment. A
dramatic activation of caspase-3 was observed upon treatment
with the DBA, whereas low/no caspase-3 activation was
observed with TMPyP4, indicating DBA benefiting from the
intrinsic mitochondrial-targeting is considerably superior to
TMPyP4 for activation of caspase-3. Then the clear appearance
of cleaved PARP bands in DBA stained Hela cell lysates further
demonstrated that the superior TP-PDT capability of DBA is
associated with caspase-3 pathway. Based on above results, a
schematic of mitochondrial apoptosis model through caspase-
3 pathway was presented in Fig.3d. Upon receiving an
apoptotic 1O2 stimulus generated in mitochondria, cytochrome
c is released from the intermembrane space of mitochondria
into the cytoplasm. Then cytchrome c in cytoplasm executes a
series of functions to recruit and to facilitate downstream
activation of caspase-3, which subsequently results in the
cleavage of PARP and final cell apoptosis. To the best of our
knowledge, this is the first report of mitochondrial apoptosis-
mediated TP-PDT using such purely organic small molecules.
In conclusion, our experimental and theoretical investigation
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on inner salt-shaped small molecule demonstrates
a
convenient and effective approach to achieve synthetic small
molecular TP-PS with remarkably enhanced δ, high 1O2
quantum yield and fascinating mitochondrial-targeting ability,
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versatile TP-absorbing small molecule for various applications,
especially in biomedical fields. Meanwhile, our results present
the first demonstration of highly efficient mitochondrial-
targeting TP-PDT via intrinsic apoptosis pathway, moving an
important step forward to the understanding of TP-PDT in
future cell biology and clinical theranostic. Importantly, the
amino and carboxyl group (that are ubiquitous in living
organisms) within DBA makes it a promising parent molecule
for further modification toward stimulus-cleavable segments
(e.g. pH, reactive oxygen species and enzyme) in the
development of novel TP-related smart diagnostic or
therapeutic applications.
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This work was financially supported by the National Basic
Research Program of China (No. 2012CB933301), the National
Natural Science Foundation of China (No. 21674048,
21574064, 61378081, 11404219, 61136003, and 61505076),
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