An APN-activated NIR photosensitizer for cancer photodynamic therapy and fluorescence imaging

Article abstract of DOI:10.1016/j.biomaterials.2020.120089

Photodynamic therapy has been developed as a prospective cancer treatment in recent years. Nevertheless, conventional photosensitizers suffer from lacking recognition and specificity to tumors, which causing severe side effects to normal tissues, while th

Full text of DOI:10.1016/j.biomaterials.2020.120089

Journal Pre-proof
An APN-activated NIR photosensitizer for cancer photodynamic therapy and
fluorescence imaging
Xiao Zhou, Haidong Li, Chao Shi, Feng Xu, Zhen Zhang, Qichao Yao, He Ma, Wen
Sun, Kun Shao, Jianjun Du, Saran Long, Jiangli Fan, Jingyun Wang, Xiaojun Peng
PII:
S0142-9612(20)30335-5
DOI:
https://doi.org/10.1016/j.biomaterials.2020.120089
JBMT 120089
Reference:
To appear in: Biomaterials
Received Date: 3 February 2020
Revised Date: 18 April 2020
Accepted Date: 1 May 2020
Please cite this article as: Zhou X, Li H, Shi C, Xu F, Zhang Z, Yao Q, Ma H, Sun W, Shao K, Du J, Long
S, Fan J, Wang J, Peng X, An APN-activated NIR photosensitizer for cancer photodynamic therapy and
fluorescence imaging, Biomaterials (2020), doi: https://doi.org/10.1016/j.biomaterials.2020.120089.
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Credit author statement
Xiao Zhou: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original
Draft, Visualization
Haidong Li: Conceptualization, Methodology
Chao Shi: Formal analysis, Investigation
Feng Xu: Methodology, Formal analysis
Zhen Zhang: Formal analysis, Investigation
Qichao Yao: Conceptualization, Methodology
He Ma: Methodology, Formal analysis
Wen Sun: Formal analysis, Investigation, Supervision, Resources
Kun Shao: Resources, Supervision, Funding acquisition
Jianjun Du: Resources, Supervision, Funding acquisition
Saran Long: Resources, Supervision, Project administration, Writing - Review & Editing
Jiangli Fan: Resources, Supervision, Funding acquisition, Project administration
Jingyun Wang: Resources, Supervision, Funding acquisition
Xiaojun Peng: Resources, Supervision, Project administration, Funding acquisition, Writing -
Review & Editing, Data Curation, Conceptualization

An APN-activated NIR photosensitizer for cancer
photodynamic therapy and fluorescence imaging
Xiao Zhou,a Haidong Li,a Chao Shi,a Feng Xu,a Zhen Zhang,a Qichao Yao, a He Ma,a Wen Sun,ac
Kun Shao ac, Jianjun Du,ac Saran Long,*ac Jiangli Fan,ac Jingyun Wangb and Xiaojun Peng*ac
a.State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian 116024, P. R. China
b.School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian 116024, P. R. China
c.Shenzhen Research Institute, Dalian University of Technology, Nanshan District, Shenzhen 518057, P. R. China
E-mail: pengxj@dlut.edu.cn. Tel/Fax: +86-0411-84986306.
Abstract:
Photodynamic therapy has been developed as a prospective cancer treatment in recent
years. Nevertheless, conventional photosensitizers suffer from lacking recognition and
specificity to tumors, which causing severe side effects to normal tissues, while the
enzyme-activated photosensitizers are capable of solving these conundrums due to
high selectivity towards tumors. APN (Aminopeptidase N, APN/CD13), a tumor
marker, has become a crucial targeting substance owing to its highly expressed on the
cell membrane surface in various tumors, which has become a key point in the
research of anti-tumor drug and fluorescence probe. Based on it, herein an
APN-activated near-infrared (NIR) photosensitizer (APN-CyI) for tumor imaging and
photodynamic therapy has been firstly developed and successfully applied in vitro and
in vivo. Studies showed that APN-CyI could be activated by APN in tumor cells,
hydrolyzed to fluorescent CyI-OH, which specifically located in mitochondria in
cancer cells and exhibited a high singlet oxygen yield under NIR irradiation, and
efficiently induced cancer cell apoptosis. Dramatically, the in vivo assays on Balb/c
mice showed that APN-CyI could achieve NIR fluorescence imaging (λem = 717 nm)
for endogenous APN in tumors and possessed an efficient tumor suppression effect
under NIR irradiation.
Key words: Aminopeptidase N, enzyme-activated photosensitizer, PDT, NIR
fluorescence imaging
1. Introduction:
In recent years, photodynamic therapy (PDT) has been extensively developed as
a prospective cancer therapeutics.[1-5] The process of photodynamic therapy involves
delivering photosensitizer (PS) to the tumor site and irradiating with particular
wavelength in tumor region, which can activate the photosensitizer to convert oxygen

molecules (3O2) into singlet oxygen (1O2) or ROS with extremely high reactive
activity.[6,7] These reactive oxygen species can damage cell structure through
oxidative stress and induce cell death.[8] Compared with traditional chemotherapy,
radiotherapy and surgery, photodynamic therapy possesses the advantages of
minimally invasive, negligible drug resistance and controllable treatment region
selection,[9,10] especially photosensitizers with near infrared (NIR) absorption and
emission own the advantages of deep tissue penetration and excellent anti-interference
capability, which are proper for application in vivo.[11-16] To date, plentiful NIR
photosensitizers based on different fluorophores have been developed.[17-20]
However, most photosensitizers suffered from lacking tumor specificity, and the
normal cells could also be damaged when exposed to irradiation ascribed to the “on”
state of conventional photosensitizers, which could lead to many serious
consequences.[21] To overcome this problem, activatable photosensitizers have been
proposed and applied in tumor imaging and therapy due to high selectivity to cancer
cells and relatively low toxicity to normal cells.[22,23] Meanwhile, because of the
activation, it is possible to distinguish cancer cells from normal cells. Up to now, a
number of activatable photosensitizers have been developed on the basis of some
overexpressed species in tumor microenvironment (TME).[24-31] Therein,
enzyme-activated photosensitizers are most promising because of superb selectivity
and admirable biological responsiveness,[32-34] which will be a key point in PSs’
studies and clinical value of cancer therapy in future.
Aminopeptidase
N(APN/CD13),
known
as
membrane
binding
zinc-ion-dependent type II metalloproteinase, can degrade neutral or basic amino
acids from the N-terminal of the protein polypeptide chain.[35,36] APN participates
in a number of important physiological and pathological processes in the body
accordingly, such as signal transduction, immune regulation, hydrolysis of bioactive
peptides and taking part in the coronavirus invasion process.[37] What's more,
numerous studies have shown that APN is highly expressed on the surface of most
tumor cells compared with normal cells because APN plays a crucial role in the
growth, invasion, metastasis and angiogenesis of malignant tumors.[38-41] Thus,
APN owns the potential to be a biomarker to recognize tumor cells and then has
become a noteworthy target in the research of fluorescence probes and anti-tumor
drugs.[42] However, most well-performing APN-activated fluorescence molecules
only placed emphasis on the ability of tumor imaging, which limited their further
applications.[43-46] Whereas photosensitizers activated by APN achieving the
integration of tumor imaging and photodynamic therapy are infrequent and
meritorious, which become a potent implement in the field of cancer therapeutics.
In this work, a NIR photosensitizer (APN-CyI) which could be activated by
APN not only for tumor imaging but also for photodynamic therapy was firstly
developed. As depicted in scheme 1, after being recognized, APN-CyI would be
hydrolyzed, thereby forming CyI-OH, which was a hemicyanine fluorophore with
NIR feature, good light stability, solubility and low biological toxicity,[47] especially,
with photodynamic effect due to the heavy atom effect of halogen elements. By
introducing iodine into the indole ring, the intersystem crossing (ISC) process was

improved and the singlet oxygen yield was also increased as well.[48] As a strong
electron donor, the hydroxyl group in CyI-OH greatly enhanced the NIR fluorescence
signal at 717 nm based on intramolecular charge transfer (ICT) effect compared with
APN-CyI. After activation, CyI-OH was specifically located on mitochondria
ascribed to the positive charge in hemicyanine fluorophore. Furthermore, the activated
molecule CyI-OH possessed higher 1O2 yield than APN-CyI, resulting in
significantly killing effect on cancer cells (HepG-2&4T1) compared to normal cells
(LO2). APN-CyI imaging and PDT experiments on tumor transplanted Balb/c mice
demonstrated that it could be activated by endogenous tumor APN in vivo, and it had
obvious inhibition on tumor, which declared APN-CyI might be a worthwhile tool in
tumor therapeutics. Hence, the functions of tumor imaging and PDT were effectively
integrated.
Scheme 1 The preparation scheme of APN-CyI for APN imaging and cancer therapy.
2. Experimental section:
2.1. General information and materials
Unless otherwise specified, all reagents were purchased from commercial
1
suppliers. The ultrapure water used in all experiments came from Milli-Q system. H
NMR and 13C NMR spectra were recorded on Bruker Avance II 400 MHz or 500
MHz spectrometer (TMS as the internal standard). Mass spectra were performed on
the Agilent Technologies HP1100LC/MSD MS and a Thermo Fisher LC/Q-TOF-MS
instruments. Absorption spectra were achieved on the Agilent Technologies CARY 60
UV-Vis spectrophotometer and fluorescence spectra were measured on a VAEIAN
CARY Eclipse fluorescence spectrophotometer. All the Spectrum experiments were
carried out in a quartz cell (10×10 mm). All the cell imaging experiments were
performed under the Olympus FV 1000 and FV1000-IX81 confocal microscopy

(Olympus, Japan). Cytotoxicity assays were analyzed by Thermo VarioskanTM LUX
multifunctional microplate reader. All the BALB/c mice were purchased from SPF
laboratory center of Dalian Medical University. And the mice imaging experiments
were carried out on NightOWL II LB983 small animal in vivo imaging system
(German).
2.2. Synthesis of photosensitizer APN-CyI
The synthetic route of APN-CyI is shown in Scheme S1.
2.2.1. Synthesis of compound 1
4-Aminobenzyl alcohol (400 mg, 3.25 mM), (tert-butoxycarbonyl)-L-alanine
(567 mg, 3 mM) , HATU(1.14 g, 3 mM) and DIPEA(776 mg, 3 mM) were dissolved
in 15 mL anhydrous tetrahydrofuran. The mixture was stirred at 0 ºC under nitrogen
protection for 10 minutes, then at room temperature for overnight. After the solvent
was removed under vacuum, the dark crude product was purified by silica gel column
chromatography with PE/EA = 2/1 to obtain compound 1 as a yellow white solid
1
(Yield: 82%). ESI-HRMS: m/z calcd for [M+Na]+: 317.1477, found: 317.1478. H
NMR (400 MHz, CDCl3) δ 8.92 (s, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 7.5 Hz,
2H), 5.43 (d, J = 5.2 Hz, 1H), 2.85 (s, 1H), 1.47 (s, 9H), 1.45 (d, J = 7.5 Hz, 3H).
2.2.2. Synthesis of compound 2
Compound 1 (200 mg, 0.67 mM), 4-toluene sulfonyl chloride (152.5 mg, 0.8
mM) were dissolved in 15 mL anhydrous dichloromethane, Triethylamine (0.5 mL)
was added dropwise . Then the mixture was stirred at 0 ºC under nitrogen protection
for 3 hours. The solvent was removed at vacuum and the crude product was further
purified through silica gel column chromatography with DCM to obtain compound 2
as a white solid (Yield: 22%). Compound 3 was unstable and directly used in next
step.
2.2.3. Synthesis of compound 3
Compound 3 was synthesized according to previous literature.[33] ESI-HRMS:
m/z calcd for [M-I-]+: 314.0400, found 314.0407.
2.2.4. Synthesis of compound 4
Anhydrous N, N-Dimethylformamide (5.6 mL, 72.5 mM), trichloromethane (25
mL) were stirred at 0 ºC. Then PBr3 (6.2 mL, 65.3 mM) was added dropwise through
a constant pressure drip funnel. Then the reaction was stirred for 1 hour. A mass of
yellowish solids were formed in reaction. Cyclohexanone (2.5 mL, 24.2 mM) was
added to the mixture. The reaction was stirred at 25 ℃ for overnight. Pour the red
solution on 100 g ice and add NaHCO3 until the solution is neutral. After separation,
the aqueous layer was extracted with DCM (25 mL), the organic layer was dried with
MgSO4. Then the solvent was removed at vacuum to obtain compound 4 as a red oily
liquid (3.42 g) and no further purification is required.

2.2.5. Synthesis of compound 5
Compound 4 (2.27 g, 12 mM), 2-hydroxy-4-methoxybenzaldehyde (1.52 g, 10
mM) and Caesium carbonate (8.0 g) were stirred in 15 ml N,N-Dimethylformamide at
25 ºC for 16 hours. Then the dark mixture was diluted with 50 mL water, extracted
with 50 ml DCM. The organic layer was washed with (50 mL × 3) water, dried over
Na2SO4. The solvent was dried under reduced pressure and the crude product was
purified by silica gel column chromatography with PE/EA = 10/1 to obtain yellow
crystalline compound 5 (Yield: 37.7%). ESI-HRMS: m/z calcd for [M+H]+: 243.1021,
1
found 243.1018. H NMR (400 MHz, CDCl3) δ 10.32 (s, 1H), 7.08 (d, J = 8.7 Hz,
1H), 6.66 (m, J = 7.3 Hz, 3H), 3.85 (s, 3H), 2.62 – 2.52 (m, 2H), 2.45 (t, J = 5.9 Hz,
2H), 1.77 – 1.48 (m, 2H).
2.2.6. Synthesis of CyI-OMe
Compound 5 (363 mg, 1.5 mM), compound 3 (794 mg, 1.8 mM) and potassium
carbonate (415 mg, 3.0 mM) were dissolved in 15 ml acetic anhydride. The mixture
was stirred at 80 ºC for 12 hours. 50 mL water was added to dilute the solvent. Then
the dark blue mixture was extracted with 100 mL DCM. The organic layer was
washed by 50 mL saturated NaCl solution for three and dried over Na2SO4. The
solvent was removed under reduced pressure and the crude product was purified by
silica gel column chromatography with DCM/MeOH = 30/1 to obtain CyI-OMe as a
blue solid with metallic sheen (Yield: 91.1%). ESI-HRMS: m/z calcd for [M-I-]+:
538.1237, found 538.1235. 1H NMR (500 MHz, CD3OD) δ 8.77 (d, J = 14.7 Hz, 1H),
8.01 (d, J = 1.5 Hz, 1H), 7.86 (dd, J = 8.4, 1.5 Hz, 1H), 7.57 – 7.46 (m, 2H), 7.32 (d, J
= 8.4 Hz, 1H), 7.07 (d, J = 2.2 Hz, 1H), 7.02 (dd, J = 8.6, 2.4 Hz, 1H), 6.46 (d, J =
14.7 Hz, 1H), 4.34 (q, J = 7.3 Hz, 2H), 3.97 (d, J = 5.3 Hz, 3H), 2.87 – 2.78 (m, 2H),
2.74 (t, J = 6.1 Hz, 2H), 2.02 – 1.88 (m, 2H), 1.82 (s, 5H), 1.46 (t, J = 7.3 Hz, 3H).
2.2.7. Synthesis of CyI-OH
CyI-OMe (100 mg, 0.15 mM) was dissolved in 15 mL anhydrous
dichloromethane and stirred at 0 ºC. Then 0.5 mL BBr3 was dissolved in 1 mL
anhydrous dichloromethane and added dropwise to the mixture in 10 min by an
injection syringe. After addition, the solution gradually changeed from blue to yellow
and the mixture was react at 25 ºC for 12 hours. After the reaction was finished, 20
mL water was added into the mixture gradually at an ice bath. NaHCO3 was added
until the solution is neutral. The mixture was extracted with 100 mL DCM and the
organic layer was washed by 50 mL water. The solvent was dried over Na2SO4 and
removed by vacuum. The crude product was purified by silica gel column
chromatography with DCM/MeOH = 15/1 to obtain CyI-OH as a blue solid with
metallic sheen (Yield: 83%). ESI-HRMS: m/z calcd for [M]+: 524.1081, found:
524.1087. 1H NMR (500 MHz, CD3OD) δ 8.58 (d, J = 14.1 Hz, 1H), 7.90 (d, J = 1.5
Hz, 1H), 7.83 – 7.66 (m, 1H), 7.61 (s, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.14 (d, J = 8.4
Hz, 1H), 6.83 (dd, J = 8.7, 2.2 Hz, 1H), 6.74 (d, J = 1.9 Hz, 1H), 6.22 (d, J = 14.3 Hz,
1H), 4.18 (q, J = 7.2 Hz, 2H), 2.85 – 2.76 (m, 2H), 2.72 (t, J = 6.1 Hz, 2H), 2.04 –
1.87 (m, 2H), 1.76 (s, 6H), 1.40 (t, J = 7.2 Hz, 4H).

2.2.8. Synthesis of Boc-APN-CyI
Compound 2 (60 mg, 0.13 mM), CyI-OH (87 mg 0.13 mM), potassium
carbonate (90 mg, 0.65 mM) and sodium iodide (15 mg, 0.1 mM) were dissolved in
20 mL acetonitrile. The mixture was stirred at 80 ºC for 5 hours. The solvent was
removed by vacuum, and the crude product was purified by silica gel column
chromatography with DCM/MeOH = 20/1 to obtain Boc-APN-CyI as a blue solid
(Yield: 77%). Boc-APN-CyI was no need to characterize the structure and directly
used in next step.
2.2.9. Synthesis of APN-CyI
Boc-APN-CyI (60 mg, 0.065 mM) was dissolved in 10 mL anhydrous
dichloromethane. Then 1 mL trifluoroacetic acid was dissolved in 1 mL dry DCM and
added dropwise to the mixture above at 0 ºC. After added, the mixture was stirred at
room temperature for another 1 h. The solvent was removed under reduce pressure
and the crude product was purified by silica gel column chromatography with
DCM/MeOH = 10/1 to obtain APN-CyI as a blue solid with metallic sheen (Yield:
1
68%). ESI-HRMS: m/z calcd for [M-I-]: 700.2031, found 700.2041. H NMR (400
MHz, CD3OD) δ 8.79 (d, J = 14.7 Hz, 1H), 8.04 (s, 1H), 7.89 (d, J = 8.5 Hz, 1H),
7.70 (d, J = 8.5 Hz, 2H), 7.53 (t, J = 8.6 Hz, 4H), 7.36 (s, 1H), 7.16 (d, J = 2.0 Hz,
1H), 7.10 (dd, J = 8.7, 2.3 Hz, 1H), 6.48 (d, J = 14.7 Hz, 1H), 5.27 (s, 2H), 4.35 (q, J
= 7.2 Hz, 2H), 4.10 (q, J = 7.0 Hz, 1H), 2.88 – 2.78 (m, 2H), 2.75 (t, J = 5.9 Hz, 2H),
2.03 – 1.90 (m, 3H), 1.84 (s, 6H), 1.62 (d, J = 7.0 Hz, 4H), 1.47 (t, J = 7.2 Hz, 4H),
1.31 (s, 2H). 13C NMR (101 MHz, CD3OD) δ 177.69, 169.36, 164.01, 156.04, 147.30,
145.70, 142.55, 139.34, 136.31, 133.97, 133.15, 130.32, 129.72, 128.81, 121.27,
117.58, 116.39, 115.70, 115.29, 103.92, 102.73, 91.84, 71.53, 51.79, 50.96, 41.36,
30.06, 28.26, 25.09, 21.67, 17.66, 12.71.
2.3.Determination of the detection limit
The imaging detection limit (DL) of APN-CyI was determined by fluorescence
titration under different concentrations of APN (0-20 ng/mL). The fluorescence
intensity of APN-CyI blank samples was measured several times to obtain the
standard deviation. The detection limit is calculated by the following equation:
Detection limit = 3σ/k
Where σ is the standard deviation of blank samples, and k is the slope of
fluorescence intensity (F717 nm) to APN concentrations.
2.4.Singlet oxygen detection in vitro
The singlet oxygen was detected according to the following method, DPBF (1,
3-diphenlisobenzofuran) was used as singlet oxygen capture agent. In methanol, the
DPBF absorbance at 415 nm was adjusted to about 1.0, subsequently, APN-CyI and

CyI-OH with the same concentration (5 μM) was added to quartz cells respectively.
Then the quartz cells were exposed at 690 nm irradiation (3.0 mW). And the
absorbtion spectra of the mixture were detected every 5 minutes.
2.5.Cell incubation
Hepatoma carcinoma cells (HepG-2 cells), normal liver cells (LO2 cells) and
breast cancer cells (4T1 cells) were purchased from Institute of Basic Medical
Sciences (IBMS) of the Chinese Academy of Medical Sciences. And all cells were
incubated with Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) with 10 %
fetal bovine serum (Invitrogen). The cells were treated at 37 ºC under a humidified
atmosphere containing 5% CO2.
2.6.Cytotoxicity assays
Cell viability was determined by the reduction of MTT (3-(4,
5)-dimethylthiahiazo (-2-yl)-3, 5-diphenytetrazoliumromide) to formazan by succinic
acid dehydrogenase in the mitochondria of living cells (MTT assay). HepG-2 and
LO2 cells were incubated in 96-well microplates (Nunc, Denmark). Every wells were
seeded a density of 1 × 105 cells/ml cells in 100 μL DMEM medium containing 10 %
FBS. After 24 h incubation, the dark groups were cultured in 100 μL DMEM medium
with different concentrations of APN-CyI (4, 3, 2.5, 2, 1.5, 1, 0.5, 0.25, 0 μM) for 12
h. And the irradiation groups were cultured in 100 μL DMEM medium with different
concentrations of APN-CyI (4, 3, 2.5, 2, 1.5, 1, 0.5, 0.25, 0 μM) for 100 min,
subsequently, the 96-well microplates with cells were exposed under 660 nm
irradiation (20 mW/cm2) for 20 min, then the cells were incubated for another 12 h.
After 12 h incubation, the medium was removed and 100 μL medium with MTT (0.5
mg/mL) was added to each well and the plates were incubated at 37 ºC for another 4
hours (MTT mother liquor of 5 mg/mL was diluted 10 times with medium). Then the
medium was removed, the formazan was dissolved in 100 μL DMSO for each well.
And the fluorescence intensity of the solutions in each well was measured by a
multifunctional microplate reader at 570 nm and 630 nm. The cell viability was
calculated by the following formula:
Cells viability (%) = (OD570 – OD630) dye / (OD570 – OD630) control × 100
2.7.Imaging endogenous APN activity of living cells
HepG-2, LO2 cells were plated to cell culture dishes with a density of 1 × 104
cells/mL and incubated for 24 h at 37 ºC under a humidified atmosphere containing 5%
CO2. Then the control groups were treated with PBS, another groups were treated
with 2 μM APN-CyI at 37 ºC for 120 min. Furthermore, 50 μM bestatin (a
competitive APN inhibitor) was added to the dishes for a 60 min incubation to study
the effect of APN activity on fluorescence detection, subsequently, the inhibition

groups were treated with 2 μM APN-CyI at 37 ºC for 120 min. After incubation, the
dishes were washed by DMEM without FBS for three times to remove the dye in
solution. And the cells were imaged by a FV1000-IX81 confocal microscopy with a
60 × objective lens (λex = 640 nm, λem = 700-800 nm).
2.8.Imaging singlet oxygen in living cells
HepG-2 cells were seeded to cell culture dishes with a density of about 1 × 104
cells/ml and incubated for 24 h at 37 ºC under a humidified atmosphere containing 5%
CO2. Then all the cells were treated with 3 μM APN-CyI at 37 ºC for 120 min.
DCF-DA (10 μM) as a singlet oxygen capture agent was added to the medium for 20
min incubation, in addition, NAC (N-acetyl-L-cysteine, 50 e.q) was added to one
group to quench singlet oxygen. Then the irradiation group and the NAC group were
exposed under 660 nm (100 mW/cm2) irradiation for 5 min and imaged by the
confocal microscopy with a 60 × objective lens immediately. The dark group received
no treatment prior to imaging (λex = 488 nm, λem = 500-550 nm).
2.9.Cell viability imaging experiment
Calcein-AM and Propidium Iodide (PI) were employed to detect the cell viability
with fluorescence imaging. HepG-2 cells were seeded to cell culture dishes with a
density of about 1 × 104 cells/mL and incubated for 24 h at 37 ºC under a humidified
atmosphere containing 5% CO2. Then all the cells were treated with 3 μM APN-CyI
at 37 ºC for 120 min. Subsequently, the cells were exposed to 660 nm irradiation (20
mW/cm2) for 0, 10, 20 min and were incubated for another 12 h. The DMEM medium
was replaced by 2 ml PBS and Calcein-AM and PI (1 μL) were carefully added by a
pipetting gun. After a 20 min incubation, the three groups were imaged by the
confocal microscopy with a 10 × objective lens (Calcein-AM: λex = 488 nm, λem
500-550 nm; PI: λex = 561 nm, λem = 590-640 nm).
=
2.10.
Cell apoptosis imaging
Annexin V-FITC and Propidium Iodide (PI) were used to measure cell apoptosis
with fluorescence imaging. HepG-2 cells were seeded to cell culture dishes with a
density of about 1 × 104 cells/mL and incubated for 24 h at 37 ºC under a humidified
atmosphere containing 5% CO2. Then all the cells were treated with 1 μM APN-CyI
at 37 ºC for 120 min. Subsequently, the cells were exposed to 660 nm irradiation (20
mW/cm2) for 20 min and were incubated for 15 min or 3 h. The DMEM medium was
replaced by 2 mL binding buffer and Annexin V-FITC and PI (5 μL) were carefully
added by a pipetting gun. After a 10 min incubation, the cells were imaged by the
confocal microscopy with a 10 × objective lens (Annexin V-FITC: λex = 488 nm, λem
= 500-550 nm; PI: λex = 561 nm, λem = 590-640 nm).
2.11.
Fluorescence imaging on 4T1-tumor mice model

All the experiments and operations were carried out with guide for the care and
use of laboratory animal resources and Dalian Medical University animal care and use
committee and approved by the institutional animal care and use committee of the
Dalian Medical University. Mice breast cancer cells (4T1 cells) were employed to
establish the tumor mice models. When female Balb/c mice weighed about 20 g, the
4T1 cells were inoculated to their armpits. After two weeks of growth, when the
volume of 4T1 tumors reached 100 mm3, the mice were subjected to the intratumoral
injection of 100 μL APN-CyI (100 μM) within the period of anesthesia. And the
fluorescence imaging of 4T1-tumor mice was carried out on a NightOWL II LB983
small animal in vivo imaging system (German).
2.12.
Tumor suppression experiments of APN-CyI on 4T1-tumor mice model
4T1 cells were inoculated to female Balb/c mice weighed about 20 g. when the
volume of 4T1 tumors reached 100 mm3, they were divided into 4 groups, the dark
group was treated with 200 μL saline, the light groups was treated with 200 μL saline
and exposed under 670 nm irradiation for 20 min, the A-dark group was treated with
200 μL APN-CyI (100 μM) and the A-light group was treated with 200 μL APN-CyI
(100 μM) and exposed under 670 nm irradiation for 20 min. All the groups were given
saline or photosensitizer through intratumoral injection within the period of mice
anesthesia and the irradiation was conducted after injection for approximately 100
min. The weight of mice and the volume of tumors were measured every two days
until three weeks. The volume of tumors was calculated by the following formula:
V = 1/2 × a × b2
V represents tumor volume, a represents the longest diameter of the tumor site,
and b represents the diameter perpendicular to a.
3. Results and discussion:
3.1. Design and synthesis of photosensitizer APN-CyI
The hemicyanine fluorophore possessed an obvious D-π-A structure, such
structure was widely used in NIR fluorescence imaging. And by introducing halogen
heavy atoms into the indole ring, the singlet oxygen yield of the hemicyanine
fluorophore was significantly increased. Hence, by concatenating the hemicyanine
fluorophore CyI-OH and the L-alanine modification ligand through a 4-aminobenzyl
alcohol linkage, we successfully synthesized an APN enzyme-triggered
photosensitizer APN-CyI, which was accurately characterized by high resolution
mass spectrometry (ESI-HRMS), nuclear magnetic resonance hydrogen spectroscopy
(1H NMR) and carbon spectroscopy (13C NMR). The response mechanism of
APN-CyI to APN is shown in Scheme 2.

Scheme 2 Response mechanism of APN-CyI with APN.
3.2. Spectral properties and responses to APN
Firstly, we studied the absorption and emission spectra of APN-CyI in different
solvents (Fig. S1). Enzymatic hydrolysis of substrates required the aqueous
environment, hence we investigated the spectral properties of photosensitizer
APN-CyI in aqueous solution (PBS/DMSO = 9/1, v/v, 10 mM, pH = 7.4). In order to
confirm that the L-alanine moiety of APN-CyI could be cleaved by APN enzyme, the
APN-CyI solution (5 μM) was treated with 100 ng/mL APN at 37 ºC. With the
reaction processed, an obvious red shift from 610 nm to 685 nm in absorption spectral
was observed accompanied by the color change of solution from blue to green in 120
minutes (Fig. 1a and Fig. S2a). Meanwhile, the fluorescence intensity at 717 nm was
significantly enhanced (Fig. 1b and Fig. S2b), which demonstrated that the -alanine
L
modification ligand could be hydrolyzed by APN and released CyI-OH.
Consequently, the fluorescence quantum yield increased from 0.005 (APN-CyI) to
0.024 (CyI-OH) in aqueous solution (PBS/DMSO = 9/1, v/v, 10 mM, pH = 7.4).
Moreover, in order to test the sensitivity of APN-CyI, the concentration titration
experiment was conducted (Fig. S2c), and what gratifying was that there was a good
linear relationship between the concentration of APN and the fluorescence intensity,
despite the extremely low enzyme concentration (0-20 ng/mL) (Fig. 1c). The
detection limit for APN is 0.058 ng/mL, which is lower than most reported molecules
that respond to APN[43-45]. This fact testified that APN-CyI could be used to detect
APN quantitatively in vitro.
Good specificity is a key point for activated photosensitizer to achieve imaging
in vivo. To evaluate the selectivity of APN-CyI to common biochemical molecules,
metal salts (NaCl; KBr; CaCl2; NiCl2; MgCl2; NH4Cl; NaI; NaClO4; NaOAc; Na2CO3;
Na2S; NaNO3; NaNO2), reactive oxygen species (NaClO; H2O2), reductive species
(GSH; AA) and tumor-related enzymes (NTR; GGT) were treated with our
photosensitizer in aqueous solution for 120 minutes at 37 ºC (Fig. S2c). Only APN
could induce an extremely strong fluorescence enhancement of APN-CyI, which
verified APN-CyI was highly selective to APN under physiological condition.
In order to study the response mechanism of APN-CyI reacted with APN, the
solution before and after the response was analyzed by high resolution mass
spectrometry. A distinct peak at m/z = 700.2034 (calcd. 700.2031 for [M-I-]+) was
found in the solution before response attributed to APN-CyI (Fig. S3a). While a peak
at m/z = 524.1085 (calcd. 524.1081 for [M-I-]+) was observed after a treatment with
APN (Fig. S3b), which strongly implied that the alanine modified group was
hydrolyzed to generate CyI-OH. Moreover, an APN activity inhibition experiment

was also performed to indirectly illustrate the response mechanism. When APN was
pretreated with 25 μM bestatin (a competitive inhibitor of APN), the fluorescence
intensity significantly decreased after the response, which indicated that the catalytic
activity of APN was significantly inhibited (Fig. 1d). Hence, these facts proved that
APN activated APN-CyI by hydrolyzing the alanine amide bond to form CyI-OH.
Good photostability is a prerequisite for effective use of photosensitizer.
Therefore, the solution of APN-CyI (10 μM) was exposed at 660 nm irradiation (20
mW/cm2) for 10 minutes. As depicted in Fig. S4a, the absorption intensity of
APN-CyI decreased only slightly in 10 minutes, which proved that APN-CyI had a
good photostability and was suitable for photodynamic therapy.
To verify whether the photosensitizer was activated in response to APN, the
singlet oxygen (1O2) was detected by employing DPBF (1, 3-diphenlisobenzofuran) as
a capture agent. Compared to APN-CyI, a dramatic drop at 415 nm was observed
when the DPBF solution mixed with CyI-OH was exposed at an irradiation (690 nm,
3.0 mW) for 1800 s, which mightily demonstrated that the hydrolytic product
CyI-OH possessed a considerable singlet oxygen yield compared with APN-CyI (Fig
1e and f). To further shed light on this phenomenon, Electron Paramagnetic
Resonance (EPR) was provided to perceive 1O2 by utilizing TEMPO
(2,2,6,6-tetramethylpiperidine-1-oxyl) as a trapping agent. Compared with APN-CyI,
1
an evident characteristic O2 signal was observed in CyI-OH aqueous solution,
verified the enhancement of singlet oxygen yield after activation (Fig S4b). As to the
cause of this phenomenon, we deem that the Intramolecular Charge Transfer (ICT)
process might be restrained and the energy of the excited state be mainly released
through the vibration relaxation pathway in APN-CyI, so the ability of intersystem
crossing (ISC) is restricted.[33] However, after being activated, the ICT process is
recovered, therefore CyI-OH shows high singlet oxygen yield.
Fig.1 (a) UV-vis absorption and (b) fluorescence spectra changes of APN-CyI (5 μM) with APN in
120 min. (c) Linear relationship between the fluorescence intensity of APN-CyI (5 μM) at 717 nm

and various concentrations of APN (0-15 ng/mL). (d) Fluorescence spectra of APN-CyI (5 μM)
inhibition tests. λex = 685 nm, slit: 10/5 nm. (e) (f) Attenuation curve of DPBF treated with
APN-CyI (e) and CyI-OH (f) under 690 nm irradiation in 0-1800 s in MeOH.
3.3. Cell imaging and cytotoxicity
Stimulated by the favorable response of APN-CyI in vitro, we tested its response
in cancer and normal cells. Hepatoma carcinoma cells (HepG-2 cells), mice breast
cancer cells (4T1 cells), normal liver cells (LO2 cells) and kidney fibroblasts cells
(COS-7) were chosen to investigate the endogenous APN activity. After incubation
with 2 μM APN-CyI for 120 min, a distinct NIR fluorescence signal was discovered
in HepG-2 cells and 4T1 cells compared to LO2 cells, which certified the high level
endogenous APN in HepG-2 cells and 4T1 cells. Furthermore, when the cells were
pre-incubated with 50 μM bestatin for 60 min, the NIR fluorescence signal was
inhibited obviously (Fig. 2a and Fig. S5a). Fluorescence intensity was detected and
the average fluorescence value was taken to quantify the results (Fig. 2b and Fig. S5b).
Furthermore, to test the ability of APN-CyI to distinguish cancer cells from normal
cells, a cell co-incubation experiment was conducted by employed HepG-2 cells and
COS-7 cells due to the significant morphological differences. After incubation with
APN-CyI, a significant fluorescence difference was observed and the average
fluorescence value of each region was taken as a quantitative result, which illustrated
that APN-CyI was activated only in cancer cells. As a contrast, the cells incubated
with CyI-OH exhibited undifferentiated fluorescence intensity whether it was cancer
cell or normal cell (Fig. 2c and 2d), since the photosensitizer in the activated state
(CyI-OH) was always at “on” state in all cells. These facts demonstrated that
APN-CyI could be used to monitor endogenous APN in living cells and distinguish
cancer cells from normal cells.

Fig.2 (a) Fluorescence imaging of APN-CyI (2 μM) in HepG-2 cells and LO2 cells. The inhibition
groups were pre-incubated with 50 μM bestatin. (b) Average fluorescence intensity of APN-CyI in
various treated cells. (c) The co-incubation assays of HepG-2 cells (yellow region) and COS-7
cells (green region) with APN-CyI or CyI-OH. (d) Average fluorescence intensity of APN-CyI or
CyI-OH in various cells. λex = 640 nm, λem = 700-800 nm. Scale bar = 20 μm.
In order to explore the subcellular organelle distribution of APN-CyI in HepG-2
cells, various commercial subcellular organelle dyes including Hoechst 33342
(nucleus), Mito-Tracker Green (mitochondria) and Lyso-Tracker Green (lysosome)
were co-incubated with APN-CyI (Fig. 3). The NIR fluorescence channel overlaid
well with Mito-Tracker Green channel, leading to a high colocalization coefficient (P
= 0.94). In contrast, a weak colocalization fluorescence signal was obtained between
NIR fluorescence channel and Hoechst 33342 channel (P = 0.04) or Lyso-Tracker
Green (P = 0.60). These facts indicated that the hydrolytic product CyI-OH was
mainly distributed into mitochondria and we hypothesized the reason was that the
intramolecular positive charge of CyI-OH was attracted by the negative membrane
potential of the mitochondria and thus concentrated in it.

Fig.3 Subcellular localization experiments of APN-CyI in HepG-2 cells. MitoTracker Green
channel was excited at 488 nm and collected at 500-550 nm. LysoTracker Green channel was
excited at 488 nm and collected at 500-550 nm. Hoechst 33342 channel was excited at 405 nm and
collected at 440-480 nm. APN-CyI channel was excited at 640 nm and collected at 700-800 nm.
Scale bar = 20 μm.
To verify whether the photosensitizer could produce singlet oxygen in living
cells, HepG-2 cells were incubated with 2,7–dichlorodihydrofluorescin diacetate
(DCFH-DA), a singlet oxygen inspection agent. After reacting with singlet oxygen,
DCFH-DA would be oxidized to DCF (2,7-dichlorofluorescin) and thus producing
strong fluorescence signal. As depicted in Fig. S6, an evident fluorescence signal was
obtained in cells after being incubated with APN-CyI under a 660 nm irradiation for 5
min. In contrast, only APN-CyI or irradiation could not induce an obvious
fluorescence enhancement. Moreover, when the cells were pretreated with
N-acetylcysteine (NAC), the fluorescence signal was significantly reduced due to a
redox reaction between NAC and singlet oxygen, which mightily demonstrated that
the singlet oxygen could be triggered by APN-CyI in living cells.
Encouraged by this result, cell viability imaging experiment was performed to
test the cytotoxicity of APN-CyI. Herein, Calcein-AM and Propidium Iodide (PI)
were employed to detect the cell viability with fluorescence imaging. Calcein-AM can
be hydrolyzed by esterase of living cells to produce Calcein, resulting in a strong
green fluorescence and PI can penetrate dead cell membranes and enter their nucleus
to produce red fluorescence. As seen in Fig. 4a, after being treated with APN-CyI for
120 min, almost no cytotoxicity was observed because of the intense fluorescence at
Calcein-AM channel. Nevertheless, when the cells were exposed to NIR irradiation
(660 nm, 20 mW) for 10 min, about 80% of cells were killed by singlet oxygen due to
the strong fluorescence signal in PI channel. Furthermore, after being irradiated for 20
min, almost no cells could survive. These experiments proved that only triggered by

the irradiation, APN-CyI could induce cell death by producing singlet oxygen.
In order to further study the cell killing mode of APN-CyI, a cell apoptosis
experiment was conducted by utilizing Annexin V-FITC and Propidium Iodide (PI)
after an incubation with APN-CyI. In the early stage of apoptosis, the
phosphatidylserine located on the inner surface of the cell membrane will be
evaginated to the outer surface and bind with Annexin V-FITC specifically, hence
result in an intense fluorescence on cytomembrane. As apoptosis proceeds, increased
cell membrane permeability leads to PI entering the nucleus, producing red
fluorescence. As depicted in Fig. 4b, neither APN-CyI nor irradiation could induce
cell apoptosis. Nevertheless, when the cells were incubated for just 20 min after a
photoinduction with APN-CyI, a distinct fluorescence was obtained at Annexin
V-FITC channel rather than PI channel accompanied by the obvious changes in cell
morphology, which was illustrated the appearance of apoptosis. Whereafter, after 3 h
incubation, obvious fluorescence signals were detected at both Annexin V-FITC
channel and PI channel, which indicated that APN-CyI could induce cell apoptosis
under NIR irradiation. To further prove this result, flow cytometry assay was
conducted on HepG-2 cells stained with Annexin V-FITC and PI. As shown in Fig. S7,
compared with the other three control groups, only APN-CyI + Light group was
observed significant fluorescence signal in first quartile and fourth quartile,
demonstrated the positive signals in both Annexin V-FITC and PI channel, which
powerfully testified the occurrence of cell apoptosis.
In order to further investigate the cytotoxicity of APN-CyI to different cells
(HepG-2, 4T1 and LO2 cells), MTT method was used to evaluate it under dark and
light conditions. As depicted in Fig. 4c, APN-CyI showed negligible cytotoxicity to
both cancer cells (HepG-2 and 4T1 cells) and normal cells (LO2 cells) under dark
conditions with a concentration less than 4 μM. However, obvious cytotoxicity was
observed after 12 h of photo-induced incubation under a 660 nm irradiation (20
mW/cm2) for 20 min. More significantly, the photo-toxicity of APN-CyI to LO2 cells
was overtly lower than that of HepG-2 and 4T1 cells, which could be explained by the
fact that APN-CyI was activated by APN in cancer cells to produce CyI-OH, leading
to an increase in singlet oxygen yield and cytotoxicity. It was worth noting that when
the concentration of APN-CyI was about 2 μM, it had a considerable killing effect on
cancer cells (cell viability = 15%) and relatively small damage to normal cells (cell
viability = 70%). These experiment results verified that APN-CyI had an effective
photo-toxicity on tumor cells while a comparatively mild toxicity on normal cells.

Fig.4 (a) Fluorescence imaging of Calcein-AM and PI stained HepG-2 cells after different
treatments. Calcein-AM: λex = 488 nm, λem = 500-550 nm. PI: λex = 561 nm, λem = 590-640 nm.
Scale bar = 200 μm. (b) Annexin V – FITC and PI fluorescence imaging experiments in HepG-2
cells after different treatments. Annexin V – FITC: λex = 488 nm, λem = 500-550 nm. PI: λex = 561
nm, λem = 590-640 nm. Scale bar = 20 μm. (c) Cytotoxicity of APN-CyI in various cells under
dark conditions or light conditions.
3.4. Tumor imaging and tumor suppression experiments on tumor mice model
Inspired by the commendable experiment results of APN-CyI on imaging and
PDT effect in vitro, in vivo imaging and tumor suppression experiments were
conducted on 4TI-tumor Balb/c mice model. After intratumoral injection of APN-CyI,
the fluorescence signal of 4TI-tumor Balb/c mice was measured by a NightOWL II
LB983 small animal in vivo imaging system (λex = 665 nm, λem = 700-740 nm). As
seen in Fig. 5a, with the passage of time, the NIR fluorescence intensity at tumor site
gradually increased and reached its maximum value at about 90 minutes, which
suggested that our dye could be activated by APN in tumor site to enhance NIR
fluorescence intensity. The same experiment was carried out three times in different
mice, and the average fluorescence value was taken to quantify the results (Fig. 5b).
The results suggested that APN-CyI could be utilized to detect APN by NIR
fluorescence imaging in vivo.
Furthermore, we evaluated the photodynamic effect of APN-CyI in vivo through

tumor inhibition experiment. Tumor volume and body weight were measured every
two days after treatment within 20 days. As shown in Fig. 5c, whether the mice
received 660 nm irradiation (100 mW/cm2, 20 min) or not, the tumor volume of PBS
groups showed explosive growth in 20 days, which were 10-folds as large as when the
treatment just started. In addition, the group treated with APN-CyI without irradiation
showed a slightly tumor suppressive effect (8-folds) because of the low cytotoxicity
of APN-CyI under dark conditions. However, compared with control groups, the PDT
group showed superb tumor inhibition, which was embodied in the fact that the tumor
volume barely increased during 20 days. The tumor pictures were shown in Fig. S8a,
and the quality of tumors were measured, as depicted in Fig. S8b, the tumor quality of
APN-CyI + Light group was significantly less than the other groups with an
inhibitory rate of 77%. These facts strongly proved that the tumor could be inhibited
by the PDT effect induced by APN-CyI under irradiation conditions. Moreover, the
body weight of mice had no significant fluctuation in both control groups and PDT
groups (Fig. 5d), which indicated that APN-CyI had negligible damage to the mice.
To further attest this conclusion, the mice were sacrificed and dissected at the end of
the treatment then the vital internal organs (heart, liver, spleen, lung and kidney) and
tumors were washed with saline and kept in formalin. Subsequently, these organs
were sectioned by paraffin method and stained with hematoxylin and eosin (H&E)
and observed through an optical microscope (Fig. 5e). No significant physiological or
pathological tissue damage and inflammatory lesion were observed of organs and
tumors in three control groups, indicating the low cytotoxicity of APN-CyI under
dark conditions. Besides, organs in PDT group were not significantly different from
those in control groups, however, conspicuous tumor tissue damage was observed in
PDT group, which confirmed an ideal PDT effect of APN-CyI on solid tumors. All
the results suggested that APN-CyI could be used as an effective photosensitizer to
inhibit tumor growth under irradiation conditions.

Fig.5 (a) Fluorescence imaging of endogenous APN in tumor Balb/c mice in 150 min. The mice
were intratumorally injected with 100 μL APN-CyI (100 μM) and were observed with excitation at
665 nm and emission at 700-740 nm. (N = 3) (b) Relative average fluorescence intensity of tumor
site after intratumorally injection with 200 μL APN-CyI (100 μM) in 150 min. (c) Relative tumor
volume after different treatments in 20 days. (d) The body weight of mice after different
treatments in 20 days. (e) H&E staining of organs and tumors in different groups after 20 days.
4. Conclusions:
In summary, we synthesized an iodine substituted NIR hemicyanine
APN-activatable photosensitizer APN-CyI for tumor imaging and photodynamic
therapy in this study. APN-CyI could be activated by APN via hydrolysis of alanine
modified group to form CyI-OH, which displayed a distinct fluorescence emission at
717 nm and a dramatic improvement of singlet oxygen yield compared with
APN-CyI. APN-CyI showed high selectivity and low detection limit (DL = 0.058
ng/ml) to APN and the response mechanism was confirmed by high resolution mass
spectrometry and inhibition experiment. Cell imaging and cytotoxicity experiments
confirmed that APN-CyI could distinguish cancer cells from normal cells and induce
cells apoptosis under 660 nm light irradiation, moreover, APN-CyI had more

phototoxicity to cancer cells compared with normal cells. The in vivo tumor imaging
and tumor suppression experiment testified that APN-CyI were qualified to be an
effective photosensitizer imaging intra-tumor APN and performing photodynamic
therapy for solid tumors after activation. To sum up, this study developed a novel
activatable photosensitizer for APN imaging and photodynamic therapy in vitro and in
vivo. We expect this work to provide a reference for the development of safer and
more effective anti-cancer drugs in the future.
5. Conflicts of interest
The authors declare no competing financial interests.
6. Acknowledgements
This work was supported by the National Natural Science Foundation of China
(project 21421005) and NSFC-Liaoning United Fund (U1608222 and U1908202).
7. Notes and reference
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Declaration of interests
√The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: