Therapeutic Nanosystem Consisting of Singlet-Oxygen-Responsive Prodrug and Photosensitizer Excited by Two-Photon Light

Article abstract of DOI:10.1021/acsmedchemlett.7b00394

Using light as the sole stimulus and employing the generated singlet oxygen as a therapeutic agent and the trigger to activate chemo-drug release could serve as an elegant way to bring into full play the advantageous features of light and enhance therapeu

Full text of DOI:10.1021/acsmedchemlett.7b00394

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Letter
Therapeutic nanosystem consisting of singlet-oxygen-responsive
pro-drug and photosensitizer excited by two-photon light
Yi Lin, Xiao-Fang Jiang, Xiangyan Duan, Fang Zeng, Bo Wu, and Shuizhu Wu
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00394 • Publication Date (Web): 22 Dec 2017
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Therapeutic nanosystem consisting of singlet-oxygen-responsive pro-
drug and photosensitizer excited by two-photon light
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Yi Lin, Xiao-fang Jiang, Xiangyan Duan, Fang Zeng*, Bo Wu and Shuizhu Wu*
State Key Laboratory of Luminescent Materials and Devices, College of Materials Science and Engineering, South China
University of Technology (SCUT), Guangzhou, 510640, China
KEYWORDS: prodrug; liposomal nanosystem; drug delivery; photodynamic therapy; chemotherapy
ABSTRACT: Using light as the sole stimulus and employing the generated singlet oxygen as a therapeutic agent and the trigger to
activate chemo-drug release could serve as an elegant way to bring into full play the advantageous features of light and enhance
therapeutic efficacy through combination of chemotherapy and photodynamic therapy. Herein a liposomal drug system has been
developed by embedding a fluorescent photosensitizer and a prodrug into phospholipid vesicles. Upon one- or two-photon light
irradiation, the photosensitizer generates singlet oxygen, which removes the protecting group of the prodrug and subsequently caus-
1
es the release of the active drug chlorambucil. With the combined action of O2 and chlorambucil, highly controllable cytotoxicity
toward cancer cells was achieved. In addition, the fluorescent photosensitizer gives out fluorescent signal acting as the drug moni-
toring agent. This strategy may provide an efficient approach for cancer treatment and some useful insights for designing light-
stimulated
on-demand
therapeutic
systems.
As an important first-line treatment option for
cancers, chemotherapy does show its life-saving
potential; however, in many cases, it may as well
On the other hand, light has long been used to
1
10
induce the generation of singlet oxygen (O ) ,
2
11
12
heat , nitric oxide and etc., which served as the
active agents for disease treatment.
1-3
be notorious for its serious side-effects (e.g.
high toxic effects and non-specificity) resulted
from its damage to normal cells and tis-
sues/organs. Therefore, effective chemotherapy
should be able to distinguish between cancer cells
and normal cells, specifically killing cancer cells
while maintaining normal cells intact. In order to
achieve highly selective killing of cancer cells
and avoid side-effects, an ideal drug delivery sys-
tem is needed to control the release of anti-cancer
drugs only at the tumor site. To date, several ap-
proaches, including both the internal and external
To further enhance therapeutic efficacy, the
combinatorial approaches comprising chemother-
apy and photodynamic therapy were also em-
13
ployed . In these combination therapy systems,
usually a photosensitizer and a chemotherapy
drug were incorporated into the same drug deliv-
ery system; and singlet oxygen was generated by
the photosensitizer via light stimulation, while
the drug was usually released by the action of
another stimulant such as pH, enzymes or degra-
14-18
dation/disintegration of drug carrier
. While
4
-7
stimuli , have been used to trigger the drug re-
lease at the target locations. As an external stimu-
lus, light is particularly attractive due to the fact
that it can trigger various biological events in a
space- and time-controlled manner. In some pro-
drugs, the active drug molecules were coupled
with a light-labile protecting group which tempo-
rarily deactivated the active drug; upon light
stimulation, the active drug was released and
using light as the sole stimulus and employing
the generated singlet oxygen as both therapeutic
agent and the trigger to activate drug release
could serve as an elegant way to bring into full
play the advantageous features of light and en-
hance therapeutic efficacy through combination
of chemotherapy (the released drug) and photo-
dynamic therapy (singlet oxygen). And several
drug delivery systems based on this approach
have been developed and proved to be feasible
8
,9
subsequently unleashed its therapeutic action .
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9-24
and effective
alkylthio)alkene
saturated phospholipid and 2-nitroimidazole
; these systems employed bis-
fluorescent property. Since liposomes can be de-
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19,20
21-23
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(
, aminoacrylates
, un-
graded via the lipid peroxidation process , in this
study, the molecular prodrug (diphenoxyethene
derivative) and the photosensitizer were encapsu-
lated into liposome, the resultant liposomal pro-
drug system can be stimulated by visible light or
infrared two-photon laser to generate singlet ox-
ygen, and then the prodrug can be triggered rap-
idly by singlet oxygen to release active drug mol-
ecules from liposomes (Scheme 1).
2
4
(
bioreducing agent activated via hypoxia or the
singlet-oxygen generation process)
2
5-26
as the
singlet-oxygen-sensitive moiety; despite of the
progress in this regard, new singlet-oxygen re-
sponsive moieties are still highly desired in terms
of high drug loading and overall performance as
well as the remarkable two-phonton properties.
Toward this aim, in this study, we employed di-
phenoxyethene as the singlet-oxygen-responsive
moiety which has symmetrical structure thus en-
suring two drug molecules can be incorporated
simultaneously, and developed a liposomal thera-
peutic system which consists of a singlet-oxygen-
responsive prodrug and a fluorescent photosensi-
tizer excitable by one- or two-photon light. In this
system, two sequential activations were involved:
singlet oxygen was generated by the photosensi-
tizer as a result of light irradiation, followed by
the removal of the protecting group and the sub-
sequent release of active drug. In addition, the
fluorescent photosensitizer also gives out fluores-
cent signal acting as the drug monitoring agent
and can be employed for imaging.
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The synthesis route for the prodrug (compound
6
) and the photosensitizer (compound 7) are
shown in Schemes S1 and S2. Compounds 1-5
were synthesized according to previously report-
2
9
ed methods. Compound 6 was obtained through
an esterification reaction between CHB and com-
pound 5. And compound 7 was synthesized from
cyclobutanone
and
N-methyl-N-(2-
hydroxyethyl)-4-aminobenzaldehyde. The struc-
As shown in Scheme 1, the prodrug molecule
designed herein contains two anticancer drug
Scheme 1. Schematic overview of the prodrug stimu-
lated by light to generate singlet oxygen which trig-
gers the drug release and the related anti-cancer ac-
tion.
Figure 1. (a) Absorption and Fluorescence spectra of the
PS (5 mM). (b) Photobleaching of ABDA (10 mM) by sin-
glet oxygen generated by PS (5 mM) solution over dif-
ferent periods of time under light irradiation (500 nm,
-2
30mW⋅cm ). (c) Photobleaching of ABDA (10 mM) by
singlet oxygen generated by PS (5 mM) solution over
different periods of time under light irradiation (840
-2
nm 55 mW⋅cm femtosecond laser). (d) Two-photon
absorption cross section of the PS using Rho-6G as a
reference.
27
chlorambucil (CHB) moieties which can be ac-
tures of the intermediates and the final product
were confirmed by proton nuclear Magnetic Res-
tivated and released by the reaction between the
prodrug with singlet oxygen. While for the pho-
tosensitizer (PS), a bis(arylidene)-cyclobutanone
derivative was designed to afford effective two-
photon excited photodynamic therapy and highly
1
onance spectroscopy ( H NMR) and mass spec-
trometry (MS) (Figures S1 - S9). And the lipo-
somal prodrug system (hereinafter referred to as
LIP-Pro-Stm) was obtained by loading the pro-
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drug and the PS into the liposomes with the parti-
cle size of around 90 nm (Figure S10).
First, we performed high resolution mass spec-
trometry (HR-MS) measurement for the mixture
solution containing the PS and the prodrug upon
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To demonstrate the photophysical property and
the singlet-oxygen generating capability of the
fluorescent photosensitizer (cyclobutanone deri-
vant), the absorption spectra, fluorescence spec-
500 nm light irradiation for 60 minutes. As
shown in Figure S17, the molecular weights of
CHB, the PS and the prodrug can be clearly
found. Furthermore, we carried out high perfor-
mance liquid chromatography (HPLC) tests (Fig-
ure 2a) so as to further confirm that the active
tra, two-photon excited fluorescence and the gen-
1
eration of O by the photosensitizer solutions
2
were determined under physiological conditions
(Figure 1, Figures S11 - S14). The absorption and
fluorescence spectra are shown in Figure 1a (and
Figures S11 and S12). It is clear that, the photo-
sensitizer has a strong absorption band at around
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10 nm and emission band at about 630 - 670
Figure 2. (a) Typical HPLC chromatogram of the pro-
drug, the PS, and the mixture of the prodrug (6 μM) and
the PS (4 μM) with one-photon light irradiation (500 nm
-2
and 30mW⋅cm ) for 60 minutes, and CHB. The mobile
phase was 80/20 methanol/water and the flow rate was
1.0 mL / min. (b) Percentage of CHB (as determined by
HPLC) released from the prodrug (6 μM) as a function
of time in the presence or absence of the PS (4 μM) up-
-2
on light irradiation (500 nm, 30mW⋅cm ).
nm. We then determined the fluorescence quan-
tum yield of the fluorescent photosensitizer using
rhodamine 6G (Rho-6G) as a reference as well as
its two-photon absorption cross section (Figure 1,
Figures S15 and S16). As shown in Figure 1d, the
PS has the strongest absorption cross section of
about 500 GM at 840 nm. In vitro reactive oxy-
gen species (ROS) generation by the PS was
Figure 3. Fluorescent microscopic images for HeLa and
A549 cells incubated in culture media with solution (4
μM PS and 6 μM prodrug) or Lip-Pro-Stm (50 μg/mL)
with the incubation time of 2 hours, respectively. The
demonstrated
bis(methylene)dimalonic acid (ABDA) as the
fluorescence probe for O (Figure 1b, Figure 1c,
using
9,10-anthracenediyl-
30
rd
th
control groups are placed in the 3 and 6 row. Scale
bar: 25 μm.
1
2
Figures S13 and S14).
drug CHB can be released from the prodrug mol-
ecule in the presence of PS upon light irradiation.
1
As the generation of O2 was confirmed via
both one-photon and two-photon excitation, we
then used it as a trigger to test whether the active
drug CHB could be released from the prodrug.
The
retention
times
of
CHB,
4-
(hydroxymethyl)phenol, the prodrug and the PS
are identified at 1.38 min, 1.63 min, 1.83 min and
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.06 min in HPLC chromatogram. For the pro-
upon light stimulation which is the key factor
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drug (6 µM) and the PS (4 µM) with one-photon
leading to cell apoptosis. While as the prodrug
was added during cell incubation together with
the PS and under proper light irradiation, the via-
-2
light irradiation (500 nm and 30mW⋅cm ) for 60
minutes, the peak intensities at 1.83 min and 2.06
min (corresponding to the prodrug and the PS
respectively) decrease and a new strong peak
emerges at 1.38 min which corresponds to that
for the active drug CHB; also another peak corre-
sponding to 4-(hydroxymethyl) phenol (the by-
product) can be observed. And the percentage of
CHB (as determined by HPLC) released from the
prodrug as a function of time in the presence or
absence of the PS was shown in Figure 2b. All
these results indicate that the active drug CHB
can be released upon light irradiation and the flu-
orescence of the photosensitizer can be utilized as
the reporting signal.
bility of the cancer cells dropped remarkably; this
1
result indicates that, the generated O
together
2
with the released active drug triggered by singlet
oxygen can greatly enhance cell apoptosis. In
Figure 4b, the interstrand DNA cross-linking ex-
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periments were assessed by alkaline agarose gel
electrophoresis. As for the prodrug in the pres-
ence of the PS (LIP-Pro-Stm) with the proper
light irradiation, we can see the linear DNA
Subsequently, we investigated the cell uptake
of the prodrug and PS in cancer cells (in both
HeLa cell line and A549 cell line). And the re-
sults are shown in Figure 3. For each cell line, we
treated the cells with both the solution (contain-
ing the prodrug and photosensitizer) and the Lip-
Pro-Stm (encapsulated with the prodrug and pho-
tosensitizer). In Figure 3, compared to the control
(without being treated with the solution of pro-
drug or Lip-Pro-Stm), all the treated groups show
strong red fluorescence of the PS in the cells, the
results indicate that both the solution containing
the prodrug and the PS as well as the Lip-Pro-
Stm can be transported into the cancer cells effi-
ciently, and can be applied for imaging the cancer
cell clearly.
Figure 4. (a) Viabilities of HeLa cells upon treatment
with different formulations of varied components and
concentrations, as evaluated with MTT assay; the cells
were incubated in culture media with --- black bar: PS 0-
The viabilities of HeLa and A549 cells upon
treatment with the prodrug system were assessed
using MTT assays (Figure 4a); the results show
that, there is little cytotoxicity when treated with
either the prodrug or the PS alone (without light
irradiation), or even with both as long as it is un-
der the dark environment (namely without light
irradiation). With light irradiation, there is still
little cytotoxicity when the prodrug alone was
added. These results indicate that the prodrug
alone is stable in both dark environment and un-
der light irradiation. However, under light irradia-
tion, when only the photosensitizer was incubated
with cells, obvious cytotoxicity can be observed,
indicating that the PS can generate singlet oxygen
14 μM, dark; red bar: prodrug 0-14 μM, dark; blue bar: PS
4
μM, prodrug 0-14 μM, dark; pink bar: 500 nm light ir-
radiation for 60 minutes; green bar: prodrug 0-14 μM
500 nm light irradiation for 60 minutes; indigo bar: PS
0-14 μM, 500 nm light irradiation for 60 minutes; purple
bar: PS 4 μM, prodrug 0-14 μM, 500 nm light irradiation
for 60 minutes. (b) Agarose gel electrophoresis assay for
linear DNA (0.5 μg pBR322) crosslinking formation upon
treatment with different formulations; the DNA were
incubated in culture media with --- lane 1: buffer, lane 2-
6: 50 μg/mL LIP-Pro-Stm (equivalent to 4.07 μM of the
PS and 12.7 μM of the CHB) for 2 h after various time (15
min, 20 min, 30 min, 45 min and 60 min) of 500 nm light
irradiation; lane 7: DNA ladder. (c) Annexin V-
FITC/Propidium Iodide (PI) dual staining of HeLa cells
for detecting the time related apoptosis by flow cytome-
try. The HeLa cells were incubated in culture media for
15-60 min with 4 μM PS in the first row, with 4 μM PS
and 6 μM prodrug in the second row after of 500 nm
light irradiation and then further incubated in dark for
2h.
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crosslinking caused by the drug CHB which was
cer treatment and some useful insights for design-
ing other light-stimulated on-demand drug re-
lease systems.
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released from the prodrug. As a result, the linear
DNA which was crosslinked had a lower migra-
tion rate as shown in lanes 3-6 (the quantification
of Figure 4b and the control group are shown in
Figures S18-19). Thereafter, we used the flow
cytometry (Figure 4c) to further demonstrate the
time-related (15 to 60 min) anti-cancer effect of
the solution containing both the prodrug and the
PS as well as the PS solution only. And the flow
cytometry of the control, CHB, liposome with
equivalent amount of CHB were also tested as
shown in Figure S20. Upon light irradiation, as
for the prodrug in the presence of the photosensi-
tizer, the apoptotic percentage increases as the
irradiation time is lengthened. However, for the
control (with light irradiation only), the percent-
age for apoptotic cells is only 3.1%. These results
clearly display that, the prodrug and the photo-
sensitizer together with light irradiation can in-
duce significant cell apoptosis and could realize
highly controllable toxicity toward cancer cells.
And the IC of the PS (Figure 4a) and the Lip-
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Materials and methods, and supplemental Tables and
Figures (PDF)
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AUTHOR INFORMATION
Corresponding Author
* Tel: 86-20-22236262; E-mail: mcfzeng@scut.edu.cn;
shzhwu@scut.edu.cn
Author Contributions
Y. Lin, F. Zeng and S. Wu conceived the project and designed the
research strategy and experimental approach; Y. Lin synthesized
the compounds and performed the experiments, with assistance
from X. Jiang, X. Duan and B. Wu. The data was analyzed by Y.
Lin. The manuscript was written by Y. Lin, F. Zeng and S. Wu.
All authors have given approval to the final version of the manu-
script. F. Zeng and S. Wu supervised the research.
Notes
5
0
The authors declare no competing financial interest.
Pro-Stm (Figure S21) were calculated to be
6.03µM and 35.8 µg/mL (equivalent to 2.91 µM
of the PS and 4.55 µM of the CHB) which is
ACKNOWLEDGMENT
We acknowledge the financial support by NSFC (21574044,
21474031, and 51673066), the Science and Technology Planning
Project of Guangzhou (Project No. 201607020015), the Science
and Technology Planning Project of Guangdong (Project No.
2014A010105009), the Natural Science Foundation of Guang-
dong Province (2016A030312002) and the Fundamental Research
Funds for the Central Universities (2015ZY013).
3
2
lower than that of CHB as reported . In addition
the cytotoxicity of the Lip-Pro-Stm with two-
photon light was tested in Figure S21, showing
that the two-photon light can trigger the prodrug
system as well.
ABBREVIATIONS
In summary, we have successfully developed a
prodrug system which can be sequentially trig-
1
O , singlet oxygen; CHB, chlorambucil; PS, photosensitizer;
2
1
LIP-Pro-Stm, the liposomal prodrug system; H NMR, proton
gered by light (to generate singlet oxygen) and
nuclear Magnetic Resonance spectroscopy; MS, mass spec-
trometry; Rho-6G, rhodamine 6G; ROS, reactive oxygen spe-
cies; ABDA, 9,10-anthracenediyl-bis(methylene)dimalonic
acid; HR-MS, high resolution mass spectrometry; HPLC, high
performance liquid chromatography.
1
O (to activate the release of active drug mole-
2
cules). The system can be activated by one-
photon or two-photon light irradiation to release
CHB active drug molecules; and the fluorescent
photosensitizer also serves as the tracking and1
imaging agent. With the combined action of O2
and CHB, the cancer cells undergo apoptosis rap-
idly. This new strategy is the first to employ light
as the stimulant and simultaneously employ the
fluorescent photosensitizer (singlet oxygen) as
the trigger for drug release (covalent bond cleav-
age) in cancer cells, therapeutic agent and imag-
ing agent in combination with anti-cancer drug;
and it may provide an efficient approach for can-
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A liposomal therapeutic nanosystem features one- or two-photon light-responsive drug release to activate both chemotherapy and
photodynamic therapy as well as self-imaging.
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Scheme 1. Schematic overview of the prodrug stimulated by light to generate singlet oxygen which triggers
the drug release and the related anti-cancer action.
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Figure 1. (a) Absorption and Fluorescence spectra of the PS (5 mM). (b) Photobleaching of ABDA (10 mM)
by singlet oxygen generated by PS (5 mM) solution over different peri-ods of time under light irradiation
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(500 nm, 30mW·cm ). (c) Photobleaching of ABDA (10 mM) by singlet oxygen generated by PS (5 mM)
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solution over different periods of time under light irradiation (840 nm 55 mW·cm femtosecond laser). (d)
Two-photon absorption cross section of the PS using Rho-6G as a reference.
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Figure 2. (a) Typical HPLC chromatogram of the prodrug, the PS, and the mixture of the prodrug (6 µM) and
-2
the PS (4 µM) with one-photon light irradiation (500 nm and 30mW·cm ) for 60 minutes, and CHB. (b)
Percentage of CHB (as determined by HPLC) released from the prodrug (6 µM) as a function of time in the
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presence or absence of the PS (4 µM) upon light irradiation (500 nm, 30mW·cm ).
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Figure 3. Fluorescent microscopic images for HeLa and A549 cells incubated in culture media with solution (4
µM PS and 6 µM prodrug) or Lip-Pro-Stm (50 µg/mL) with the incubation time of 2 hours, respectively. The
control groups are placed in the 3rd and 6th row. Scale bar: 25 µm.
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Figure 4. (a) Viabilities of HeLa cells upon treatment with different formulations of varied components and
concentrations, as evaluated with MTT assay; the cells were incubated in culture media with --- black bar:
PS 0-14 µM, dark; red bar: prodrug 0-14 µM, dark; blue bar: PS 4 µM, prodrug 0-14 µM, dark; pink bar:
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00 nm light irradiation for 60 minutes; green bar: prodrug 0-14 µM 500 nm light irradiation for 60
minutes; indigo bar: PS 0-14 µM, 500 nm light irradiation for 60 minutes; purple bar: PS 4 µM, prodrug 0-
1
4 µM, 500 nm light irradiation for 60 minutes. (b) Agarose gel electrophoresis assay for linear DNA (0.5 µg
pBR322) crosslinking formation upon treatment with different formu-lations; the DNA were incubated in
culture media with --- lane 1: buffer, lane 2-6: 50 µg/mL LIP-Pro-Stm (equivalent to 4.07 µM of the PS and
12.7 µM of the CHB) for 2 h after various time (15 min, 20 min, 30 min, 45 min and 60 min) of 500 nm light
irradiation; lane 7: DNA ladder. (c) Annexin V-FITC/Propidium Iodide (PI) dual staining of HeLa cells for
detecting the time related apoptosis by flow cytometry. The HeLa cells were incubated in culture media for
1
5-60 min with 4 µM PS in the first row, with 4 µM PS and 6 µM pro-drug in the second row after of 500 nm
light irradiation and then further incubated in dark for 2h.
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