Expanding Anti-Stokes Shifting in Triplet–Triplet Annihilation Upconversion for In Vivo Anticancer Prodrug Activation

Article abstract of DOI:10.1002/anie.201704430

A strategy to expand anti-Stokes shifting from the far-red to deep-blue region in metal-free triplet–triplet annihilation upconversion (TTA-UC) is presented. The method is demonstrated by in vivo titration of the photorelease of an anticancer prodrug. This new TTA system has robust brightness and the longest anti-Stokes shift of any reported TTA system. TTA core–shell-structured prodrug delivery capsules that benefit from these properties were developed; they can operate with low-power density far-red light-emitting diode light. These capsules contain mesoporous silica nanoparticles preloaded with TTA molecules as the core, and amphiphilic polymers encapsulating anticancer prodrug molecules as the shell. When stimulated by far-red light, the intense TTA upconversion blue emission in the system activates the anticancer prodrug molecules and shows effective tumor growth inhibition in vivo. This work paves the way to new organic TTA upconversion techniques that are applicable to in vivo photocontrollable drug release and other biophotonic applications.

Full text of DOI:10.1002/anie.201704430

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Accepted Article
Title: Expanding Anti-Stokes Shifting in Triplet-triplet Annihilation
Upconversion for In Vivo Anticancer Prodrug Activation
Authors: huang ling, yang zhao, he zhang, kai huang, jingyi yang, and
Gang Han
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201704430
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10.1002/anie.201704430
Angewandte Chemie International Edition
COMMUNICATION
Expanding Anti-Stokes Shifting in Triplet–Triplet Annihilation
Upconversion for In Vivo Anticancer-Prodrug Activation
Ling Huang[a] ‡, Yang Zhao[a] ‡, He Zhang[a], Kai Huang[a], Jingyi Yang[a], and Gang Han[a]*
Abstract: A strategy to expand anti-Stokes shifting from the far-red
to deep-blue region in metal-free triplet–triplet annihilation
upconversion (TTA-UC) is presented and its utility in the in vivo
titration of the photorelease of an anticancer prodrug is
demonstrated. This new TTA system has robust brightness and the
longest anti-Stokes shift of any reported TTA system. TTA core-
shell-structured prodrug delivery capsules that benefit from these
properties are developed; they can operate with low-power-density
far-red light-emitting diode (LED) light. These capsules contain
mesoporous silica nanoparticles preloaded with TTA molecules as
the core and amphiphilic polymers encapsulating anticancer prodrug
molecules as the shell. When stimulated by far-red light, the intense
TTA upconversion blue emssison in the system activates the
anticancer prodrug molecules and shows effective tumor growth
inhibition in vivo. This work paves the way for the design of new
organic TTA upconversion with regard to in vivo photocontrollable
drug release and other biophotonic applications.
remain in regard to inorganic UCNPs. For instance, due to the
intrinsic low absorption and emission cross-sections of the
contained lanthanide ions, such UCNPs have quite low quantum
yields that typically require relatively high-power-density laser
excitation. The long-term in vivo toxicity and systematic
clearance of inorganic lanthanide ions inside UCNPs are also
unclear.[3d, 6] These key limitations have led to the exploitation of
a more biocompatible upconversion strategy, particularly with
respect to the emerging organic-chromophore-based triplet–
triplet annihilation upconversion (TTA-UC). In regard to TTA-UC,
low-energy photons can be absorbed by
a
sensitizer
chromophore and then transferred to an acceptor chromophore
through a unique triplet−triplet energy-transfer process. Two
excited acceptor molecules subsequently undergo
a TTA
annihilation process, to generate one high-energy short-
wavelength photon (Scheme 1a). Compared to inorganic
UCNPs, TTA-UC offers some advantages due to its intense
absorption coefficient of sensitizers, high quantum yield and
brightness, as well as the concomitant low-power-density
excitation resource.[7] Therefore, TTA-UC-based materials are
potentially suitable for applications as photocontrollable drug-
delivery systems. Quite recently, green-to-blue TTA-UC
nanomicelles were fabricated to trigger the uncaging of blue-
light-sensitive coumarin-group-modified peptides, thus enabling
better subsequent cell targeting.[8] However, in vivo drug
photorelease and concomitant cancer treatment are formidable
challenges, as the green excitation source lacks deep-tissue
penetration depth and yields low quantum efficiency. Moreover,
such TTA-UC remains insufficient to activate a large number of
prodrug molecules for cancer treatment.[9] To address this
problem, some deep-tissue-penetrable TTA systems that are
excitable with longer wavelength light were proposed. For
In recent years, the development of stimulus-responsive
prodrug release and delivery systems has been of great interest
to the fields of chemistry and biology. These systems effectively
improve the therapeutic efficacy of drug release in malignant
sites with minimal side-effects.[1] Compared to other drug-
release strategies, light-induced prodrug activation is unique,
with noninvasive operation and high spatiotemporal
controllability.[2] In such a strategy, drug molecules are typically
modified and protected with light-sensitive chromophores, such
as coumarin, 2nitrobenzyl, and 7nitroindoline. However, these
chromophores typically require short-wavelength deep-blue light
(ca. 435 nm) or phototoxic ultraviolet (UV) light (ca. 365 nm) as
the excitation light sources; these methods also suffer rather
poor tissue penetration depth in their in vivo applications.[3]
To address this problem, long-wavelength light was recently
utilized in the therapeutic window (600900 nm) due to its
minimal absorption by tissue and its deep-tissue penetration.[4]
For example, lanthanide ion-doped inorganic upconversion
nanoparticles (UCNPs) can convert tissue-penetrable long-
wavelength light into high-energy short-wavelength photons to
trigger small-molecule drug release.[3c,5] However, challenges
example,
a
TTA system containing meso-tetraphenyl-
tetrabenzoporphine palladium PdTPBP (sensitizer) and perylene
(emitter) can upconvert 635 nm laser light to 475 nm photons
and was used for the photodissociation of ruthenium polypyridyl
complexes from PEGylated liposomes in water.[9c, 10a] However,
the existing system has limitations to its in vivo applications due
to its suboptimal efficiency and relatively high excitation power
density (2.3 W cm–2), which is beyond the biosafety threshold.[10a]
In addition, the anti-Stokes-shifted emission wavelength of 475
nm is not compatible with the typical deep blue/UV operation
wavelengths for biologically used caging groups.[3] To this end,
the development of a new TTA system with dramatically
improved anti-Stokes shifting from far red to deep blue and
robust brightness properties is highly desirable.
[a]
L. Huang, Dr. Y. Zhao, H. Zhang, Dr. K. Huang, J. Yang, Prof. Dr. G
Han
Department of Biochemistry and Molecular Pharmacology
University of Massachusetts Medical School
Worcester, MA 01605, United States.
E-mail: Gang.Han@umassmed.edu
‡ equal contribution
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10.1002/anie.201704430
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our new TTA system. In particular, we designed a TTA
upconversion core/shell structured nanocapsule (TTA-CS) in two
steps (Scheme S3). In the first step, TTA-MSNs are formed by
infusing a TTA-UC dye pair (20 M of BDP-F and 200 M of PEA)
in methyl oleate oil into mesoporous channels of silica
nanoparticles. In the second step, TTA-CS is further developed
by adding a layer of the prodrug coumarin–chlorambucil (Cou-C,
see Figure 2)[12] onto the TTA-MSNs that were made during the
first step. After uncaging, the prodrug (Cou-C) is able to convert
into hydrophilic chlorambucil, which can be released from the
TTA-CS and then kill the tumor cells.
Scheme 1. a) A Jablonski diagram of the photophysical processes of the
triplet photosensitizers and the TTA upconversion exemplified with BDP-
F as the triplet photosensitizer and PEA as the emitter; b) molecular
structure of BDP-F and PEA.
In this study, to achieve far red to deep blue TTA-UC, we
designed a metal-free iodized BODIPY dimer (BDP-F) molecule
to be used as a highly far-red-sensitive photosensitizer and 9-
phenylacetylene anthracene (PEA) as a deep blue emitter
(Scheme
1b).
Compared
to
conventional
BODIPY
Figure 1. a) UV-vis absorption spectra of BDP-F and 2,6-diiodio-BODIPY
(10 M ) in toluene at room temperature. b) The upconversion emission
photosensitizers, such as 2,6-diiodio-BODIPY ( = 85 000 M−1
cm−1 at 525 nm, Scheme S2), due to its large  core, BDP-F
presented broader and more intense absorption in the far-red
region from 600–670 nm (peaking at 615 nm,  = 1.77×105 M–1
cm–1; Figure 1a). Meanwhile, BDP-F has an outstanding triplet-
state lifetime (T = 243.6 µs; Figure S1) that is essential for the
TTA photosensitizers. To increase the anti-Stokes-shifted deep-
blue emission, 9-phenylacetylene anthracene (PEA) was
synthesized as a new emitter (Scheme S1). PEA presents
excellent fluorescence quantum yield in the deep-blue region
from 410–500 nm, peaking at 432 nm (f = 87%; Figure S2),
which makes it particularly suitable as the emitter.
spectra of BDP-F (20 M) and PEA (0.2 mM) in degassed toluene, ex
=
650 nm (100 mW cm–2), inset shows such TTA-UC can be observed with
the naked eye.
Firstly, we found that methyl oleate oil can efficiently prevent
oxygen quenching of TTA-UC, as seen in Figure S6a. There
was only 6% upconversion intensity reduction in air compared to
in argon. In addition, the TTA-UC system of BDP-F and PEA in
methyl oleate was quite stable; even after 20 days, the
upconversion emission intensity was reduced by only 9.0% in air
(Figure S6b). Subsequently, TTA-UC (20 M of BDP-F and 200
M of PEA) in methyl oleate oil was infused into mesoporous
channels of silica nanoparticles to form TTA-MSNs. The TTA-
MSNs were characterized by using transmission electron
microscopy (TEM). As shown in Figure S7, the TEM image
indicates that the TTA-MSNs consist of uniform spherical
We then further optimized the concentration ratio of BDP-F
and PEA in the TTA-UC system. The best combination was 20
M of BDP-F and 200 M of PEA in degassed toluene solution
(Figure S3, S4). At this optimal ratio, intense deep-blue
emission in the range of 410–500 nm can be observed by the
naked eye upon irradiation with 650-nm light (Figure 1b). This
TTA-UC system showed a high relative upconversion quantum
nanoparticles with
a
diameter of 218

16 nm. The
hydrodynamic diameter is 259  12 nm as measured by dynamic
light scattering (DLS) in deionized (DI) water. As shown in
yield (UC = 3.1%) when methyl blue was used as a reference.[7a, Figure S8, upon excitation with 650-nm light, the TTA-MSNs
10b, 10c]
The TTA-UC also presented excellent upconversion
brightness ( =    UC, 1054 at 100 mW cm–2 of 650-nm light).
More importantly, to our knowledge, our TTA-UC system
presents the longest anti-Stokes shift (Δ = 0.96 eV) of any
reported TTA upconversion system (see Table S1). Further, the
TTA upconversion intensity threshold (Ith) was studied (Figure
S5). Quadratic dependence of the upconversion emission was
observed for low-energy incident power density excitation and a
linear region observed at higher incident power densities in
deaerated toluene. The transition threshold (Ith) between the
quadratic and the linear regime occurs near 19.6 mW cm-2,
which is comparable to the value of reported TTA-UC
generated a deep, bright-blue upconversion emission that peaks
at 430 nm. By using methyl blue as the reference, the
upconversion quantum yield ( UC) of TTA-MSNs in water was
measured to be 2.0% (100 mW cm−2) in air. The photostability of
the TTA-MSNs was also investigated in air. No significant
changes in the TTA-UC were observed when TTA-MSNs were
continuously irradiated by using a 650-nm laser (100 mW cm−2)
for 30 min (Figure S9). This result suggests that the TTA-MSNs
have excellent photostability.
Secondly, as shown in Scheme S3, a TTA-CS was prepared
by adding a layer of the amphiphilic polymer F-127 encapsulated
deep blue light sensitive hydrophobic prodrug coumarin–
chlorambucil[12] onto the TTA-MSNs. In TTA-CS, chlorambucil
was chosen because it is a potent and cost-effective small-
molecule tumor inhibitor[9b,12] Moreover, the coumarin-based
group possesses high photocleavage efficiency and a deep-blue
absorption wavelength, the latter of which overlaps with the
systems.[11a]
A
power-dependence experiment provided
evidence for TTA-UC in BDP-F and PEA system in deaerated
toluene.[11b]
Because of these excellent photophysical properties, we
sought to construct a TTA-UC drug-delivery system based on
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10.1002/anie.201704430
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emission spectrum of PEA (Figure S10).[4c] As seen in Figure
S11a, the deep-blue upconversion emission of the resulting
TTA-CS is less than that of the TTA-MSNs, which suggests that
absorbs the 430 nm light was successfully encapsulated within
the system. The core/shell nanocapsule was further
characterized by using DLS. The hydrodynamic diameter of the
TTA-CS (358 ± 27 nm) is larger than the diameter of the TTA-
MSNs (Figure S11b), which suggests that the amphiphilic
polymer F-127 encapsulated prodrug is the external shell in the
system. The prodrug entrapment efficiency was calculated by a
previously reported method to be 79% (see Supporting
Information). The rate of release of prodrug from the TTA-CSs in
the absence of 650-nm light is rather insignificant (ca. 4.4% after
24 h; Figure S12).
To demonstrate the in vitro effectiveness of our system, cell
viability experiments were conducted. Cancer cells (HeLa and
4T1 cells) were incubated with TTA-CS for 12 h and then a low-
power far-red LED was used. As shown in Figure S16, TTA-
MSNs have insignificant toxicity with and without light. In
addition, TTA-CS (0375 g mL-1) had negligible cell toxicity in
the absence of LED light. However, upon irradiation with the far-
red LED, TTA-CS presented significant toxicity towards both
HeLa and 4T1 cells. This result suggests that Cou-C is
successfully photocleaved by TTA-UC and the hydrophilic
anticancer drug chlorambucil is indeed released from TTA-CS
into the cells, causing cancer cell death (Figure 3). The IC50
under irradiation (half-maximal concentration of TTA-CS and
Cou-C to cause cell death) was calculated to be 235  5.9 g
mL– and 2.74 g mL–1 with HeLa cells, and 320  6.3 g mL–1
1
and 3.64 g mL–1 with 4T1 cells, respectively.
Figure 3. MTT assay of HeLa, 4T1 cell viability with different
concentrations of TTA-CS with and without light. a) HeLa cells; b) 4T1
cells. ex = 650 nm, photon fluence (360 J cm-2).
Prodrug photorelease mediated by far-red light was also
evaluated by the calcein-AM/PI co-staining method.[13] In the
absence of a far-red LED, we only observed bright green
emission in the cancer cells, which suggests that TTA-CS itself
did not kill cancer cells. However, in the presence of irradiation
with a far-red LED, red fluorescence was observed in the cells,
which suggests cell death. We did not observe significant cell
death with prodrug-free core nanoparticles (TTA-MSNs) in the
presence of LED irradiation (Figure S17). These results further
demonstrated that far-red-light triggered TTA upconversion can
activate prodrug photorelease and lead to cancer-cell-growth
inhibition.
Since TTA-CS showed excellent cancer cell inhibition in vitro,
we continued testing TTA-UC-induced prodrug release in vivo.
First, we prepared 4T1 tumor-bearing mice and divided them into
four groups (Group 1: only PBS injection + irradiation; Group 2:
TTA-CS injection but no irradiation; Group 3: TTA-MSNs and
irradiation; Group 4: TTA-CS injection and irradiation). After 60
min of intratumor injection, tumor sites were then irradiated with
a far red LED (100 mW cm–2). The treatment outcome of TTA-
CS to tumor was assessed by monitoring relative tumor volumes
in mice, and tumor tissue ablation was also evaluated by H&E
staining on tissue sections. As shown in Figure 4, no tumor-
growth inhibition or tumor-tissue necrosis was observed in
Group 1. Group 2 showed no tumor-growth inhibition or tumor-
tissue necrosis, which indicates that TTA-CS itself cannot inhibit
tumor growth. Group 3 also demonstrated insignificant cancer
treatment efficiency, which suggests that the low-power red LED
we used has low photothermal or other effects for cancer cell
killing. In marked contrast, the tumor growth in Group 4 was
Figure 2. The TTA-upconversion regulated activation of Cou-C from TTA-
CS with 650-nm-LED irradiation. “ON” and “OFF” indicate the initiation
and termination of LED irradiation, respectively, and the working power
density was 100 mW cm–2. Top inset: the photoactivation reaction of Cou-
C; bottom inset: schematic illustration of TTA-UC-mediated prodrug
activation process in TTA-CS.
Next, we tested the feasibility of prodrug activation using our
TTA-CS system (Figure 2). The activation of the prodrug
process was quantified by using the fluorescence measurement
because the fluorescence at 498 nm of the coumarin moiety
decreases when it is removed from the chlorambucil molecules
(Figure S13). As shown in Figure S14, when we irradiated
TTA-CS with a 650-nm light-emitting diode (LED) (100 mW cm–
2), the prodrug was uncaged, resulting in > 48% activation of the
prodrug within 30 min, and a maximum photorelease of around
82% of the prodrug after 60 min. These results confirm that the
prodrug can be activated by the TTA-upconversion process
using far-red light. A similar nanocapsule consisting of the
photosensitizers without the PEA emitter (termed BDP-F-CS)
was designed as
a non-emissive control. Excluding the
possibility of the direct uncaging of chlorambucil by 650 nm light
(Figure S15), upon 650 nm irradiation of BDP-F-CS for 60 min,
no observable uncaged chlorambucil was detected. Moreover,
the photorelease of chlorambucil was clearly dependent on the
ON–OFF pattern of the LED excitation source (Figure 2). This
result indicates that the release dose and duration can be
precisely titrated by using far-red light under a low power density
of 100 mW cm−2.
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remarkably suppressed, and the tumor tissue showed obvious
necrosis. These results indicate that the deep-blue
upconversion-induced chlorambucil release from the prodrug
(Cou-C) does lead to tumor-tissue ablation. To our knowledge,
this is the first time that TTA-UC-induced photocleavage-based
prodrug photorelease has been realized in vivo upon low-power
irradiation with a far-red LED.
This research was supported by the National Institutes of Health
R01MH103133 the Human Frontier Science RGY-0090/2014,
and UMass OTCV award.
Keywords: Triplet–triplet annihilation upconversion
Stokes shifts • BODIPY •Prodrug activation • Anticancer
• Anti-
To determine the potential toxicity and side effects of TTA-CS,
we measured the body-weight loss of the mice. As shown in
Figure S18, mice treated with TTA-CS did not show apparent
weight loss. Nine days after an intravenous injection of TTA-CS,
the treated mice and untreated age-matched healthy mice were
sacrificed, and the major organs including heart, liver, spleen,
lung, and kidney were collected for H&E staining to evaluate the
toxicity effects. No noticeable sign of organ damage was
observed in the H&E-stained organ slices, which suggests that
TTA-CS is safe for in vivo cancer treatment applications (Figure
S19). A serum analysis was performed, as shown in Table S2;
we observed no abnormal results from this analysis, which
suggests that no observable inflammation was induced.
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Figure 4. a) Illustration of the photocleavage drug release via TTA-UC. b)
Tumor-growth inhibition by TTA-CS-mediated drug release in 4T1 tumors;
Values are means ± s.e.m. (n = 5 mice per group). c) H&E staining of
tumor tissue sections from different treatment groups
9 days after
treatment, scale bar represents 50 µm. d) Representative digital photos of
tumors for the four groups of mice. Photon flux (180 J cm-2).
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In conclusion, we designed the longest known anti-Stokes
shift TTA-UC system. This new TTA system has robust
brightness from the far-red to deep-blue regions. Thanks to
these properties, we were able to develop a TTA core-shell-
structured drug-delivery capsule that can effectively activate
[7]
anticancer prodrug release in vitro and in vivo with
a
biocompatible far-red LED light. This new organic TTA
upconversion system not only offers a new nanoplatform for
spatiotemporally controlled cancer therapy, but also has great
potential for numerous photonic and biophotonic applications.
Acknowledgements
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Entry for the Table of Contents (Please choose one layout)
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COMMUNICATION
Far Red light means go for organic
upconversion prodrug activation:
Ling Huang, Yang Zhao He Zhang, Kai
Huang, Jinyi Yang, and Gang Han
Cou-C
TTA-UC
A strategy is presented for a new
triplet-triplet annihilation upconversion
that has robust brightness and the
record longest anti-Stokes shift from
far red to deep blue. TTA core-shell-
Page No. – Page No.
Expanding Anti-Stokes Shifting in
Triplet–Triplet Annihilation
Upconversion for In Vivo Anticancer
Prodrug Activation
structured
prodrug
photorelease
nanocapsules are developed that
operate with a low-power density far-
red-LED light and show effective
prodrug activation control ability and
potent tumor-growth inhibition in vivo.
Longest anti-Stokes Shifting TTA-
upconversion for prodrug activation
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