Anti-tumor evaluation of a novel methoxyphenyl substituted chlorin photosensitizer for photodynamic therapy

Article abstract of DOI:10.1016/j.jphotobiol.2020.112015

Photodynamic therapy (PDT) is a non-invasive and innovative therapeutic approach which has been increasingly applied in clinical cancer therapy. As the central element of PDT, the development of novel photosensitizers (PSs) with longer absorption wavelength, proper lipophilic/hydrophilic profiles, target tissue selectivity, and higher photo?/lowest dark-cytotoxicity is a challenging task. Previously, we designed and synthesized a series of novel long-wavelength chlorin e6 (Ce6)-based PSs via introducing aromatic groups to the vinyl of Ce6 skeleton. The new formed compounds with π-extension system exhibited improved photodynamic effects and spectral characteristics. Among these π-conjugated chlorin PSs, (E)-3²-(4-methoxyphenyl)-chlorin e6, named A15, was expected to be a potent antitumor candidate as a PDT agent due to its good photobiological properties. Herein, in this work, we evaluated the effectiveness of A15 in cancer PDT. In vitro, a novel rare earth probe, ATTA-Eu³⁺ was applied to detect the singlet oxygen (¹O₂) production of A15 in solution and human hepatoma HepG2 cells, respectively. Moreover, A15 exhibited strong phototoxicity and weak dark cytotoxity to HepG2 cells. In H22 tumor bearing mice, A15 showed excellent tumor accumulation ability via i.v. administration and induced tumor regression, followed by laser treatment. These results indicated that A15 is a potential novel π–extension chlorin-type PS for PDT applications.

Full text of DOI:10.1016/j.jphotobiol.2020.112015

JournalofPhotochemistry&Photobiology,B:Biology211(2020)112015
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Anti-tumor evaluation of a novel methoxyphenyl substituted chlorin
photosensitizer for photodynamic therapy
⁎
⁎
Yi Donga, Guangzhe Lia,b, ,1, Liu Wanga, Lei Caoa, Yueqing Lia,b, Weijie Zhaoa,b, ,1
a Department of Pharmacy, School of Chemical Engineering, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China
b State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Photodynamic therapy (PDT) is a non-invasive and innovative therapeutic approach which has been increasingly
applied in clinical cancer therapy. As the central element of PDT, the development of novel photosensitizers
(PSs) with longer absorption wavelength, proper lipophilic/hydrophilic profiles, target tissue selectivity, and
higher photo−/lowest dark-cytotoxicity is a challenging task. Previously, we designed and synthesized a series
of novel long-wavelength chlorin e6 (Ce6)-based PSs via introducing aromatic groups to the vinyl of Ce6 ske-
leton. The new formed compounds with π-extension system exhibited improved photodynamic effects and
spectral characteristics. Among these π-conjugated chlorin PSs, (E)-32-(4-methoxyphenyl)-chlorin e6, named
A15, was expected to be a potent antitumor candidate as a PDT agent due to its good photobiological properties.
Herein, in this work, we evaluated the effectiveness of A15 in cancer PDT. In vitro, a novel rare earth probe,
ATTA-Eu3+ was applied to detect the singlet oxygen (1O2) production of A15 in solution and human hepatoma
HepG2 cells, respectively. Moreover, A15 exhibited strong phototoxicity and weak dark cytotoxity to HepG2
cells. In H22 tumor bearing mice, A15 showed excellent tumor accumulation ability via i.v. administration and
induced tumor regression, followed by laser treatment. These results indicated that A15 is a potential novel
π–extension chlorin-type PS for PDT applications.
Photodynamic therapy
Photosensitizer
Tumor
π–extension chlorin
1. Introduction
application and oxygen supply in tissue microenvironment are con-
sidered as three key parameters that determine PDT efficacy. Over the
In recent years, photodynamic therapy (PDT) has been emerging as
a promising therapeutic option for cancer treatment owing to its su-
periorities such as minimized systemic toxicity, minimal invasiveness,
high therapeutic efficacy and potential of developing antitumor im-
munity, compared to conventional cancer therapies (surgery, che-
motherapy, and radiotherapy) [1–4]. This modality has been clinically
approved for treating several types of solid tumors such as prostate [5],
head and neck skin and oesophagus [6] as well as currently being
employed in clinical trials for metastatic ovarian cancer [7]. PDT em-
ploys the photosensitizer (PS, a photo-activatable molecule) which
upon irradiation transfers its energy to surrounding oxygen molecules
to generate high cytotoxic singlet oxygen (1O2) species, enabling irre-
versible destruction of selected cells [8–10]. Herein, PS molecule, light
years, despite substantial efforts have been dedicated to manipulate the
components of these critical parameters, the application of PDT for
cancer has not yet been substantially pursued mainly due to its effec-
tiveness limited by poor light penetration depth and lack of ideal PSs
[11]. Majority of the PSs possess unfavorable pharmacokinetic prop-
erties, such as low aqueous solubility, low bioavailability, poor cellular
permeability, which leads to inefficient singlet‑oxygen generation in the
target tumor site. To overcome these shortcomings, although remark-
able studies have been devoted to the construction of different nano-
carriers serving as the PS selective delivery vehicles [2,12], ultimately
the most promising strategy of improving the performance in PDT
process should be placed on the chemical aspects of the PS molecule
itself [13].
Abbreviations: (1) PDT, photodynamic therapy; (2) A15, (E)-32-(4-methoxyphenyl)-chlorin e6; (3) ATTA-Eu3+, (4′-(9-anthryl)-2,2′:6′,2″-terpyridine-6,6″-diyl)bis
(methylenenitrilo) tetrakis (acetate)-Eu3+; (4) 1O2, singlet oxygen; (5) HepG2 cell, human heptatoma cell line; (6) 3D, three-dimensional; (7) H22 cell, mouse
hepatoma cell line; (8) CHC, Chenghai Chlorin; (9) DMSO, dimethyl sulfoxide; (10) PBS, phosphate buffered saline; (11) MTT, 3-(4,5-dimethyl-2thiazolyl)-2,5-
diphenyl-2H-tetrazoliumbromide; (12) DPBF, 1,3-diphenylisobenzofuran; (13) PSs, photosensitizers.
⁎ Corresponding authors.
E-mail addresses: liguangzhe@dlut.edu.cn (G. Li), zyzhao@dlut.edu.cn (W. Zhao).
1 Contributed equally to this work
https://doi.org/10.1016/j.jphotobiol.2020.112015
Received 12 March 2020; Received in revised form 26 August 2020; Accepted 2 September 2020
Availableonline05September2020
1011-1344/©2020ElsevierB.V.Allrightsreserved.

Y. Dong, et al.
JournalofPhotochemistry&Photobiology,B:Biology211(2020)112015
Chlorin e6 (Ce6), an FDA-approved second-generation PS [14],
reflux extracted with acetone (1500 ml) for 5 times, and then the
concentrated extract was obtained by silica gel flash column chroma-
tography (CH2Cl2/ AcOEt). Solution of Et2O/HCl was added to the
crude product, and 3.1 g deep green powders pyropheophorbide-a
(CHP) were gained (3‰). CHP was dissolved in THF (20 ml), and 5%
KOH was dropwise added under N2 at 40 °C for 30 min. The pH of
solution was adjusted to 5–6 with 2 M HCl, and 2.6 g dark green
powder CHC was obtained (98%). CHC (C34H36N4O6): 1H NMR
(400 MHz, DMSO) δ 9.78 (1H, s), 9.71 (1H, s), 9.10 (1H, s), 8.31 (1H,
dd, J = 17.8, 11.6 Hz), 6.46 (1H, d, J = 17.8 Hz), 6.18 (1H, d,
J = 11.6 Hz,), 5.39 (1H, d, J = 19.0 Hz), 5.31 (1H, d, J = 19.5 Hz),
4.61 (1H, q, J = 6.8 Hz), 4.43 (1H, d, J = 10.2 Hz), 3.81 (2H, q,
J = 7.4 Hz), 3.58 (3H, s), 3.52 (3H, s), 3.30 (3H, s), 2.49 (2H, m), 2.25
demonstrates strong clinical utilities in PDT on account of its high
singlet oxygen generation efficiency [15], near-infrared light excitation
allowing deep tissue penetration, and fluorescence imaging capability
[1,16]. Nevertheless, the major obstacle encountered in the application
of Ce6 in vivo is its poor water solubility. It is well known that the
tendency towards aggregation of Ce6 in the biologic medium leads to
the increase of the dark toxicity and nonspecific accumulation in un-
desired sites [17]. To solve this problem, novel chlorin-type PSs have
been exploited extensively, driven by the flexibility of its core structure
for straightforward chemical modification [18]. Many studies have
been focusing on adding hydrophilic moieties such as sugars [19],
amino acids [20] and PEG segments [21] onto the Ce6 skeleton to
improve its bioavailability. However, the enhanced polarity of these
Ce6 derivatives may hamper themselves to diffuse through the various
biological barriers, especially cell membrane, with a compromise of the
cellular permeability. Herein, the lipophilic/hydrophilic balance of a PS
has a significant impact on the outcome of PDT [18].
(2H, m), 1.69 (3H, m), 1.65 (3H, m), −1.65 (1H, s), −1.93 (1H, s). 13
C
NMR (100 MHz, DMSO) δ 175.6, 174.7, 174.1, 170.2, 168.6, 153.9,
148.8, 145.0, 138.7, 136.6, 135.7, 135.2, 134.6, 134.3, 130.8, 129.7,
129.4, 126.6, 122.5, 103.8, 101.7, 98.7, 94.6, 53.0, 48.6, 40.6, 27.3,
23.4, 19.3, 18.2, 12.5, 12.4, 11.4, 9.5. HRMS (ESI) m/z: [M + H]+
calcd 597.2713, found 597.2699.
Given the penetration of light in tissue increases with wavelength in
the range of 600–800 nm, lengthening the absorption wavelength is
another necessary consideration when designing a potent PS. Several
studies demonstrated that extending the chromophoric π-conjugation
by grafting a strong electron-donating group to the core of the chro-
mophore resulted in a redshift of the spectral band as well as the singlet
oxygen generation enhancement [22–24]. These studies suggested that
the physicochemical and pharmacokinetic properties of a PS, encom-
passing longer-wavelength absorption Q band, lipophilic/hydrophilic
profiles, cellular permeability, target tissue selectivity, and photo−/
dark-cytotoxicity etc. could be tuned via selective structural modifica-
tion of its respective parent molecule.
The synthesis of (E)-32-(4-methoxyphenyl)-chlorin e6 (A15) was
reported previously. Briefly, A15 was prepared from the CHC via im-
proved regioselective bromination and Suzuki-Miyaura cross-coupling
reactions (Fig. 1) [25]. A15 (C41H42N4O7): 1H NMR (400 MHz, CDCl3:
MeOD = 3:1) δ 9.80 (s, 1H), 9.75 (s, 1H), 8.98 (s, 1H), 8.28 (d,
J = 17.1 Hz, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 17.1 Hz, 1H),
7.10 (d, J = 8.2 Hz, 2H), 5.61 (m, 1H), 5.34 (m, 1H), 4.55 (m, 2H),
3.95 (s, 3H), 3.81 (m, 2H), 3.61 (s, 3H), 3.55 (s, 3H), 3.31 (s, 3H), 2.66
(m, 1H), 2.26 (m, 2H), 1.78 (d, J = 6.7 Hz, 3H), 1.72 (m, 1H), 1.66 (t,
-
J = 7.4 Hz, 4H). HRMS (ESI) m/z: [M-H] calcd 701.2974, found
701.2971.
We previously designed and synthesized a series of novel long-wa-
velength chlorin e6-based PSs [25]. Our synthetic strategy was in-
troducing aromatic groups to the vinyl of Ce6 via regioselective bro-
mination followed by Suzuki-Miyaura coupling reactions, ultimately
forming π-extension chlorin systems. It turned out that the photo-
dynamic effects and spectral characteristics of these compounds were
finely modulated towards favorable directions, such as the maximum
wavelength redshift, improved phototoxicity, enhanced cellular uptake
and ROS generation. Among these π-conjugated chlorin PSs, (E)-32-(4-
methoxyphenyl)-chlorin e6, named A15, appeared to be a potent an-
titumor candidate for PDT due to its good photobiological properties.
Herein in this work, we further exploited a relatively convenient and
economic approach to generate A15 starting from Chenghai Chlorin
(CHC) as a precursor molecule. CHC, structurally same as Ce6, was
derived from the crude chlorophyll extracts of the spirulina from China
Chenghai Lake. More importantly, we reported on the detail evaluation
of in vitro and in vivo anti-tumor efficiency of A15 as a PDT agent.
2.3. Photosensitizer solution
In vitro study, CHC and A15 were dissolved in DMSO at a con-
centration of 10 mM, while as were dissolved in normal saline at 8 mg/
ml for in vivo evaluation. All samples were kept in −4 °C away from
light.
2.4. Cell culture
Human liver hepatocellular carcinoma cell line (HepG2) was ob-
tained from ATCC and murine hepatoma H22 cell line was purchased
from National Infrastructure of Cell line Resource (Beijing, China). Both
of the cell lines were cultured in high-glucose DMEM and 1640 medium
supplemented with with 10% (v/v) FBS, 100 IU/ml penicillin and
100 mg/ml streptomycin, respectively. Cultures were maintained in a
humidified incubator at 37 °C with 5% (v/v) CO2.
2. Materials and methods
2.5. Singlet oxygen measurments
2.1. General information
1O2 measurments were carried out in aqueous solution and in cells.
For the comparison of 1O2 generation kinatics of A15 and CHC in
aqueous solution, a lanthanide probe, ATTA-EU3+ was used, which
could generate long-life fluorescence signal with large stokes shift after
binding with 1O2 [26,27]. The mixture solution of A15 or CHC with
ATTA-EU3+ was prepared in PBS and the final concentrations were
50 μM (A15 or CHC) and 10 μM (ATTA-EU3+), respectively. One
hundred microliters of A15-ATTA-EU3+, CHC-ATTA-EU3+ or ATTA-
EU3+ solution were added to the 96-well plate (n = 4), respectively.
The time-resolve fluorescence intensity of the solution was detected
with microplate reader (Tecan, US) after 0, 1, 3, 5 and 10 min of illu-
mination with LED light (660 nm, 25 mW/cm2).
All reactions were under nitrogen in air-free solvents with protec-
tion from direct light. Silica gel (200–300 mesh) was used for flash
column chromatography. 1H NMR and 13C NMR spectra were recorded
on a Bruker Avance 400 MHz spectrometer. The chemical shifts are
reported in ppm (δ) and coupling constant (J) values are given in hertz
(Hz). HRMS were obtained on a LTQ Orbitrap XL high resolution mass
spectrometer (Thermo Fisher Scientific). All the chemicals and reagents
obtained from commercial suppliers were used without purification.
2.2. Chemistry
For 1O2 detection in cells, HepG2 cells were seeded in 24-well plate
at a density of 1 × 106 cells/well. After incubation with 50 μM of A15
or CHC for 2 h, the cells were further incubated with 10 μM of ATTA-
EU3+ active ester (cell-membrane permeable) for 20 min. Then the cells
Chenghai Chlorin (CHC), with same chemical structure as chlorin e6
(Ce6), was extracted from the spirulina powers derived from Chenghai
Lake in Yunnan Province of China. Spirulina powders (980 g) were
2

Y. Dong, et al.
JournalofPhotochemistry&Photobiology,B:Biology211(2020)112015
Fig. 1. Synthetic approaches for the preparation of CHC and A15. Reagents and conditions: (a) acetone, reflux, 2 h; (b) Et2O/HCl; (c) (i) 5% KOH, THF, 40 °C, (ii) 2 M
HCl, 97%; (d) CH3I, K2CO3, DMF, R.T.; (e) Br2, CH2Cl2/Et2O, −97 °C; (f) 4-methoxyphenylboronic acid, Pd/PPh3/base, 1,4-dioxane; (g) 1 M NaOH, acetone, R.T.,
12 h.
were washed two times with PBS and added with fresh DMEM medium.
After 0, 2, 5, 10 min of illumination with LED light (660 nm, 25 mW/
cm2), the 1O2 level in live cells was determined by time-resolved
fluorescence microscopy.
right side of the mice back. Measurement of the tumor size was started
on day 8 after inoculation and only the mice with tumor size around
200 mm3 were used for the evaluation of antitumor effect. Tumor vo-
lume was calculated as following formula.
Tumor volume = ab2/2 (a = the length of tumor; b = the width of
tumor) [28].
2.6. Cellular uptake
All animal experiments were approved by the China Committee for
Research and Animal Ethics in compliance with the law on experi-
mental animals.
HepG2 cells were resuspended at densities of 3 × 105 cells/ml in
24-well plates. After 24 h of culturing, the cells were then exposed to a
25 μM solution of different chlorins in serum-free medium for 2 h in the
dark. After incubation, the cells were washed two times with 1 ml
phosphate-buffered saline (PBS) and finally lysed with 1 ml methanol
was added to release uptaken drugs and quantitatively test the con-
centration using UV–vis according to the standard curve of concentra-
tion-dependent UV–Vis absorbance intensity.
2.9. PDT programs in vivo
Twenty tumor-bearing mice were randomly divided into four
groups, shown as below. Treatment was started on Day 8 and followed
every other day. Mice in the group IV were treated with laser light at a
density of 6 J cm−2 (660 nm, 20 mW cm−2, 5 min) at 4 h after i.v.
injection of A15 at a dose of 8 mg/kg body wt. Before PDT, the accu-
mulation of A15 in tumor site was monitored by in vivo live imaging
system. Mice in the group III were only treated with the same density
laser light as group IV, while mice in the group II were given the same
dose of A15 without laser treatment. Throughout the experiment
(20 days), all mice were kept out of the light. The weight and tumor
volume of the mice in each group were measured regularly. Two days
after the last treatment, the mice were anesthetized with intraperitoneal
injection of avertin (tribromoethanol), and subcutaneous tumors were
extracted and weighed. Representative mouse from each group was
photographed to show the appearance of the tumor.
2.7. Cytotoxicity
HepG2 cells were seeded in 96-well plates at a density of 5 × 103
cell/well. After 24 h of culturing, cells were treated with CHC and A15,
respectively, at different concentrations from 0 to 25 μM for 2 h. Then
the cells were further exposed to LED light and the light intensity was
6 J/cm2 (660 nm, 25 mW/cm2) for 4 min. Meanwhile, the CHC or A15
treated cells without light treatment under the same experimental
conditions were conducted for the dark cytotoxicity. After incubation
for 22 h post-treatment, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide tetrazolium (MTT) reduction assay was used to
monitor the cell viability. Each experiment was repeated three times.
Group I: Control (light-);
2.8. Mouse xenograft tumor model
Group II: A15 (8 mg A15/kg body wt, light-);
Group III: Light;
ICR male mice aged 6–8 weeks with a body weight of 25–30 g were
purchased from Liaoning Changsheng Biotechnology (Benxi, China).
H22 cells (1 × 106 in 50 μL PBS) were injected subcutaneously into
Group IV: PDT (8 mg A15/kg body wt treated, light+).
3

Y. Dong, et al.
JournalofPhotochemistry&Photobiology,B:Biology211(2020)112015
2.10. Statistical analysis
intensity.
For detecting 1O2 in PBS solution (Fig. 2A), it should be noted that
in the presence of CHC and ATTA-EU3+, the relative fluorescence in-
tensity dramatically increased by extending irradiation time, whereas
A15 exhibited suppressed 1O2 generation ability. This result was con-
sistent with what we previously reported [25]. 1O2 generation effi-
ciency of a photosensitizer in the buffer doesn't always correlate well
with its photocytotoxicity. For exmaple, Jim M. Fernandez, etc. reported
the singlet oxygen quantum yield of photofrin in phosphate buffer (PB)
plus 1% Triton X-100 (pH 7.4) was 0.89 (scaled to 0.52 for methylene
blue), while the singlet oxygen quantum yield of Chlorin e6 in PB was
0.64 [36]. However, Chlorin e6 showed better photocytotoxicity than
photofrin against human chronic myelogenous leukemia cell line
(K562) [37].
All data shown represented average values, and the error corre-
sponded to the standard deviation error of independent experiments
performed in triplicate. The values were expressed as the mean
SD.
Unpaired two-tailed Student's t-test were used to determine the statis-
tically significant difference among different groups when appropriate
and the analysis of variance (two-way ANOVA) by means of the
GraphPad Prism 8.2.1 (p
< 0.05 was considered statistically sig-
nificant).
3. Results and discussion
3.1. 1O2 generation capability
Thus to further validate the potential of A15 acting as a PDT agent,
intracellular 1O2 assessment was carried out in HepG2 cells as well. As
depicted in Fig. 2C, upon increasing the irradiation time, brighter
imaging intensity was observed with respect to increased ROS stress
levels. The quantification analysis demonstrated that the group in-
cubated with A15 had a significantly higher 1O2 level than that of CHC
(Fig. 2B). The results of 1O2 generated by CHC or A15 measured in
aqueous solution and in cytoplasma were discrepant. The reason might
be the substitution of methoxyphenyl group to the CHC increased the
the lipophiliciy of the precursor molecule, pointing out the importance
of lipophiliciy for a PS to diffuse through cell membrane and to localize
intracellularly.
Singlet oxygen generation capability is an important parameter for
determining the photodynamic killing effect of a PS. As reported in
extensive literature, organic fluorescent probe 1,3-diphenylisobenzo-
furan (DPBF) is used to be applied for detecting the 1O2 in biology
system [29–33]. However, there is the concern about the non-specifi-
city of the probe to 1O2, since DPBF was illustrated as a good candidate
for superoxide-anion detection as well [34,35]. Herein, a novel lan-
thanide fluorescence probe ATTA-EU3+ as a 1O2 detector was adopted
in this work owing to its superior advantages of high efficiency and
specificity in 1O2 assessment [26]. The probe of ATTA-EU3+ itself does
not have fluorescence unless it binds with 1O2, transforming into
fluorescent EP-ATTA-EU3+, which probes the 1O2 level via fluorescence
Fig. 2. Detection of 1O2 generation levels of A15 and CHC by ATTA-EU3+ active ester in PBS solution (A) or in HepG2 cells (B and C). (C) is the time-resolved
fluorescence microscopy images of cells incubated with the PS and the ATTA-EU3+ active ester, followed by the irradiation under LED light. The data shown are the
mean
SD of three independent experiments.
4

Y. Dong, et al.
JournalofPhotochemistry&Photobiology,B:Biology211(2020)112015
Fig. 3. (A) Evaluation of phototoxicity and dark toxicity of CHC, Npe6 and A15 against HepG2 cells by MTT assay. (B) The cellular uptake of CHC, Npe6 and A15 by
HepG2 cells. (A15 vs CHC: p < 0.0001(****), Npe6 vs CHC: p < 0.001(***)). The data shown are the mean SD of three independent experiments.
Fig. 4. (A) tumor growth curves of different groups of tumor-bearing mice during treatment. (B) A plot of body weight versus the numbers of days post different
treatments. (C) Image of tumors excised from each group of mice after treatment. (D) Tumor weight of different groups of tumor-bearing mice after treatment (PDT vs
Control: p < 0.05(*), PDT vs Drug: p < 0.005(**), PDT vs Light: p < 0.05(*)). (E) In vivo live fluoresence imaging of the accumulation of A15 in tumor site after i.v.
injection. (F) Representative mouse from each group was photographed to show the appearance of the tumor.
5

Y. Dong, et al.
JournalofPhotochemistry&Photobiology,B:Biology211(2020)112015
3.2. Phototoxicity and dark toxicity
biosafety of A15 for in vivo applications. This data suggested that the
A15 molecule possessed enormous clinical translational potential as a
PDT agent for the precise treatment of cancer.
The phototoxity and dark toxicity of A15 on tumor cells were
evaluated by MTT assay with mono-L-aspartyl‑chlorine e6 (NPe6, a
second-generation PS) as a positive control. HepG2 cells were exposed
to the increasing concentrations (0–25 μM) of CHC, Npe6 or A15 for 2 h
and the cell viability was determined after additional 22 h incubation in
the absence of the PSs. As shown in Fig. 3B, without irradiation, dif-
ferent PSs treated groups remained up to 80% of the cell viability, in-
dicating that all chlorins had negligible intrinsic cytotoxicity in the
applied concentration range. However, after irradiation, the survival
rates of the HepG2 cells dramatically decreased as a function of PS
concentration. It was worthy to note that the cell viability was only
10% at 1 μM of A15, whereas the viability was over 80% when treated
with the same concentration of CHC. A15 exhibited significantly en-
hanced cell killing effect even at a very low concentration (1 μM), de-
monstrating the chemical modification in chlorin structure improved its
cellular internalization and phototoxicity. These results confirmed our
previous observations on the intracellular 1O2 measurement.
4. Conclusion
In summary, the PDT efficacy of A15 was evaluated in vitro and in
vivo. A15 holds robust intracellular 1O2 generation capacity, highly
photo-toxicity, biocompability, tumor tissue selectivity and excellent
inhibitory of tumor growth. These above traits have paved the way to
make A15 as a promising candidate for PDT clinical translation.
Declaration of Competing Interest
None.
Acknowledgments
This work was funded by the Key Projects of the National Science
and Technology Pillar Program (2015BAD17B05). We acknowledge
EEC Bio-Tech Limited (Guangzhou, China) for technical support. We
thank Prof. Bo Song from Department of Chemistry, Dalian University
of Technology, for providing the lanthanide fluorescence probe ATTA-
EU3+. We thank Prof. Kun Shao from Dalian University of Technology
for technical contributions and advising on the manuscript preparation.
As we know, an ideal PS needs to possess minimal dark toxicity, be
cytotoxic in the presence of light at a defined wavelength [38]. Apart
from that, higher cellular accumulation rate and intracellular ROS
generation capacity of the PS was expected as well since equal or en-
hanced PDT efficacy could be achieved by giving lower dose of the PS,
in attempt to avoid non-tumoral cell uptake and damage [39].
3.3. Cellular uptake
Declaration of Competing Interest
The authors declare no competing financial interest.
References
The cellular uptake of CHC, Npe6 and A15 was determined at a
concentration of 25 μM by measuring the fluorescence intensity of the
intracellular chlorins, and the standard curve was obtained by plotting
fluorescence intensity versus concentration to calculate the intracellular
level of each compound. As shown in Fig. 3B, all the compounds ac-
cumulated successfully in HepG2 cells. Expectedly, A15 possessed a
remarkably higher uptake in comparison with CHC. The observed least
accumulation of Npe6 among the tested chlorins coincided with its
poorest phototoxicity (Fig. 3A).
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3.4. PDT efficacy in vivo
Encouraged by the superior intracellular 1O2 generation efficiency,
satisfactory photo-cytotoxicity as well as negligible dark cytotoxicity, in
vivo PDT evaluation of A15 was carried out on tumor bearing mice. The
mouse hepatocellular carcinoma H22 cell line was used to construct
subcutaneous xenograft tumor model for in vivo studies. When the
tumor volume reached 200 mm3, they were randomly divided into four
groups: control, A15 (light−), light alone and PDT (A15 treated, light
+) group. Before laser treatment, the uptake of A15 by tumor tissue
was confirmed using an in vivo fluorescence imaging system after i.v.
injection of A15 (8 mg/kg body wt). As shown in Fig. 4E, strong
fluorescence signal centered in the tumor region 4 h post-injection, with
negligible biodistribution in other major organs, indicating the possi-
bility of reducing the risk of off-target effects in vivo. The excellent
tissue permeability of the A15 molecule within the tumor site might be
attributed to the inclusion of aromatic groups to the CHC molecule. The
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