Epidermal Growth Factor Receptor-Targeted Delivery of a Singlet-Oxygen Sensitizer with Thermal Controlled Release for Efficient Anticancer Therapy

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00670

Photodynamic therapy (PDT) utilizing light-induced singlet oxygen has achieved attractive results in anticancer fields; however, its development is hindered by limited light penetration depth, skin phototoxicity, tumor hypoxia, and PDT-induced hypoxia. In

Full text of DOI:10.1021/acs.molpharmaceut.9b00670

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Article
Epidermal Growth Factor Receptor-Targeted Delivery of a Singlet Oxygen
Sensitizer with Thermal Controlled Release for Efficient Anticancer Therapy
Juanjuan Chen, Dongyao Li, Beibei Huo, Fengling Zhang, Xuan
Zhao, Gankun Yuan, Dan Chen, Meiru Song, and Jinping Xue
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00670 • Publication Date (Web): 25 Jun 2019
Downloaded from http://pubs.acs.org on June 26, 2019
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Y3-1, which contains a small molecular-targeted agent and a singlet oxygen thermal-controlled releasing
moiety, exhibits excellent erlotinib-based tumor targeting delivery and enhanced PDT- mimetic anticancer
activities.
82x32mm (600 x 600 DPI)
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Epidermal Growth Factor Receptor-Targeted
Delivery of a Singlet Oxygen Sensitizer with
Thermal Controlled Release for Efficient Anticancer
Therapy
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Juanjuan Chen,†,⊥Dongyao Li,†,⊥ Beibei Huo,† Fengling Zhang,‡ Xuan Zhao,† Gankun Yuan,†
†
Dan Chen,† Meiru Song,† and Jinping Xue*,
†National and Local Joint Biomedical Engineering Research Center on Photodynamic
Technologies, College of Chemistry, Fuzhou University, Fuzhou 350116, Fujian, China.
‡College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 310053,
Zhejiang, China.
KEYWORDS: targeted delivery, erlotinib, singlet oxygen release, 2-pyridone, anticancer therapy
ABSTRACT. Photodynamic therapy (PDT) utilizing light-induced singlet oxygen has achieved
attractive results in anticancer fields; however, its development is hindered by limited light
penetration depth, skin phototoxicity, tumor hypoxia and PDT-induced hypoxia. Inspired by our
previous research work and the limitations of PDT, we introduce a small-molecule targeted drug
erlotinib into the singlet oxygen chemical source endoperoxide to achieve an EGFR-targeted
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PDT-mimetic sensitizer (Y3-1) for anticancer therapy. We demonstrated that the erlotinib-based
precise delivery of the singlet oxygen chemical source (in vitro photosensitization) to EFGR-
overexpressing tumor cells and tissues. Moreover, the anticancer assays validated that the
enhanced anticancer efficacy (in vitro and in vivo) of Y3-1 was due to reversible singlet oxygen
thermal release. This study is expected to provide a smart strategy to break through the current
roadblock in targeted PDT and achieve a more efficient anticancer therapy model.
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INTRODUCTION.
Photodynamic therapy (PDT) has been considered as a specific and controllable therapeutic
modality for cancer.1-4 During PDT in cancer, the synergistic action of photosensitizers (PSs),
light and oxygen in the tumor region can generate highly reactive singlet oxygen (1O2), which
results in cell apoptosis or necrosis, shut down the tumor microvasculature and induce the
anticancer immune response.2,5-6 PDT has tremendous therapeutic potential because of its
advantages such as minimal invasiveness, negligible drug resistance, and quick recovery.1-5,7
However, there are still some drawbacks that must be overcome to realize the full promise of
PDT in anticancer treatment.First, the light, which is needed for the generation of singlet oxygen,
cannot penetrate tissues to more than a few millimeters.8-10In addition, photosensitizers
accumulation in skin during PDT will lead to skin phototoxicity upon exposure to light.11-12
Second, tumor hypoxia and PDT-induced hypoxia seriously hinder effective singlet oxygen
generation, which makes PDT a self-limiting method.13-15Finally, the PDT dosimetry problem,
which relates to the photosensitizer, light and oxygen, still limits the clinical application of
PDT.7,16-17 These problems are not only due to the lack of ideal photosensitizers or smart
delivery/activation strategies, but also lie at the core of the PDT paradigm.
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Inspired by the fact that highly reactive singlet oxygen is the critical and ultimate cytotoxic
factor during effective PDT, we propose a smart strategy to circumvent all of these issues.
Singlet oxygen can be produced by in vitro photosensitization with sufficient oxygen
concentration and then stored by a chemical method; finally, the trapped singlet oxygen can be
precisely delivered into the tumor region and reversibly release singlet oxygen for anticancer
therapy. This smart strategy is supposed to resolve the problems of oxygen and skin
phototoxicity in PDT. Moreover, this approach is free of the in vivo use of complicated laser
instruments and has potential application in the treatment of deep-seated tumors.
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The ideal chemical sources of singlet oxygen should have high 1O2 yields and no side reactions
in the cell. The endoperoxides of 2-pyridone and its derivatives have been demonstrated to be
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excellent chemical sources with clean cycloreversion reactions with very high O2 yields.18-21
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They can decompose to release O2 under thermal conditions and result in cancer cell death.21-
22In addition, 2-pyridone and its derivatives can trap singlet oxygen to form endoperoxides by
photosensitization.19-22 In fact, there are some reports about photosensitizer endoperoxide
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derivatives22-25 and controlled release of O2 by gold nanorod.26 However, these former
sensitizers still depend on in vivo light and oxygen concentration in tumor region because of self-
photosensitization, and the latter requires 808 nm laser irradiation. More importantly, they all
suffer from poor cancer targeting which may result in damage to normal tissues and organs. Thus,
it is desirable to develop cancer-targeted delivery of singlet oxygen chemical sources.
Erlotinib (Tarceva), a small-molecule targeted anticancer drug, can target the ATP binding
domain of tyrosine kinase in epidermal growth factor receptor (EGFR) overexpressing tumors
and exhibits specific affinity to tumor cells.27-30 Our previous research work showed that it can
successfully deliver phthalocyanine photosensitizers to EGFR-overexpressing tumors with high
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selectivity.31-35 Based on the above consideration, we introduced erlotinib into a 2-pyridone
endoperoxide for precise delivery of the 1O2 provider (generated through an in vitro
photodynamic reaction) for targeted anticancer therapy. A novel compound (Y3-1) was designed
and prepared through a click chemistry reaction. Y3-1 shows the temperature-sensitive release
ability of 1O2 with a t1/2 value of 13 h at 37 °C, high specific affinity and anticancer activities to
EGFR-overexpressing cancer cells and tumor tissue. This work may not only hope to break
through the reliance of PDT on light, oxygen and PS, but also improve the efficacy of small-
molecule targeted anticancer drugs. More importantly,an accuratedosimetric description of
singlet oxygen-based anticancer therapy may become feasible by this strategy, because of the
quantitative supply of 1O2 in this unimolecular system. To the best of our knowledge, this is the
first report of the precise application of EGFR-targeted anticancer drug to precisely deliver the
1O2 chemical sources to the tumor region.
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MATERIALS AND METHODS
Chemicals and Instruments. All solvents and chemicals were purchased from Sinopharm
Chemical Reagent Co., Ltd and were subjected to treatments of deoxygenation and dehydration.
Mito-Tracker Green, Lyso-Track red, DAPI (1,3-diphenylisobenzofuran), ROS (reactive oxygen
species;) detection kits and BCA protein assay kits were purchased from Beyotime
Biotechnology Co., Ltd. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
were purchased from Sigma Chemical Corp. Roswell Park Memorial Institute 1640 (RPMI-1640)
medium and trypsin-EDTA solution were purchased from Gibco Life Technologies. Annexin V-
FITC/PI apoptosis detection kits were purchased from BD Company. All cell lines were
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provided by Institute of Life Sciences, Chinese Academy of Sciences. H NMR and C NMR
spectra were recorded on nuclear magnetic resonance with models of AVANCE III (400 or 500
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MHz). Data of HRMS were detected by LC-QTOF-MS (G6520B). Purity of all final compounds
was 95% or higher. Detecting the ability of Y3-1 to release 1O2 using a Lambda 365-UV-Visible
absorption spectrometer. Other instruments include a microplate reader (PerkinElmer
Corporation), BD AccuriTM C6 Flow cytometer (Becton Dickinson), FV-1000 laser confocal
scanning microscope (Olympus Corporation). The in vivo fluorescence imaging was performed
by in vivo fluorescence molecular tomography (FMT) using the FMTTM 2500 system
(PerkinElmer Corporation).
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Synthesis. Compound Y1. Alpha,alpha'-Dibromo-p-xylene (1.218 g, 4.614 mmol) and NaN3
(0.3019 g, 4.644 mmol) were dissolved in DMF(10 mL) and stirred at room temperature
overnight. The crude products were purified by silica gel column chromatography (PE: CH2Cl2 =
20:1) to give compound Y1 (0.3985 g, 38.2%) as a white crystal. 1H NMR (400 MHz, CDCl3): δ
7.44 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 4.52 (s, 2H), 4.37 (s, 2H). 13C NMR (126 MHz,
DMSO-d6): δ 138.38, 136.24, 130.07, 129.20, 129.14, 53.70, 34.47.
Compound Y2. Compound Y1 (0.3985 g, 1.762 mmol) was dissolved in DMF (10 mL) and 2-
hydroxypyridine (0.2534 g, 2.665 mmol) was added, and then K2CO3 (0.2920 g, 2.113 mmol)
18-Crown and KI were added as catalyst. The mixture was stirred at room temperature overnight.
The crude products were purified by silica gel column chromatography (CH2Cl2:CH3OH =
100:1); Compound Y2 was obtained as a colorless liquid (0.3564 g, 84.2%). 1H NMR (400 MHz,
DMSO-d6): δ 7.80 (d, J = 6.4 Hz, 1H), 7.47 – 7.39 (m, 1H), 7.33 (q, J = 8.1 Hz, 4H), 6.43 (d, J =
9.1 Hz, 1H), 6.25 (t, J = 6.7 Hz, 1H), 5.11 (s, 2H), 4.43 (s, 2H). 13C NMR (126 MHz, DMSO-
d6): δ 161.88, 140.56, 139.58, 137.84, 135.35, 129.08, 128.44, 120.37, 106.02, 53.74, 51.33.
HRMS (ESI): m/z 240.1011, calcd for C13H12N4O [M+H]+: 241.1084, found for [M+H]+:
241.1084, Δ = 0.41 ppm.
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Compound Y2-1. Compound Y2 (0.5477 g, 2.2779 mmol) and MB (0.0544 g, 0.1700 mmol)
were dissolved in CDCl3 (10 mL) and illuminated by 670 nm laser (1 W) in presence of oxygen
at -20 °C for 4 h with stirring. When the reaction was completed, the crude product was purified
by silica gel column chromatography by the frozen CH2Cl2 (0 °C) to give compound Y2-1
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(0.1868 g, 30.1%) as a dark yellow liquid. H NMR (500 MHz, CDCl3): δ 7.30 (d, J = 7.8 Hz,
2H), 7.23 (d, J = 7.8 Hz, 2H), 6.73 (tt, J = 9.1, 6.4 Hz, 2H), 5.55 (dd, J = 5.1, 2.1 Hz, 1H), 5.06
(dt, J = 5.7, 1.8 Hz, 1H), 4.84 – 4.77 (m, 1H), 4.45 (d, J = 15.4 Hz, 1H), 4.33 (s, 2H). 13C NMR
(126 MHz, CDCl3): δ 168.29, 135.53, 135.34, 134.39, 128.87, 128.82, 128.37, 83.47, 78.08,
54.33, 53.58, 46.72. HRMS (ESI): m/z 272.2640, calcd for C13H12N4NaO3 [M+Na]+: 295.0802,
found for [M+Na]+: 295.0811, Δ = 3.05 ppm.
Compound Y3. Compound Y2 (0.3568 g, 1.483 mmol) and erlotinib (0.8754 g, 2.225 mmol)
were dissolved in mixture solvent (THF/H2O/tBu-OH, 8/4/6 mL) with CuSO4·5H2O and sodium
ascorbate was added as a catalyst. The mixture was stirred at room temperature overnight. The
crude product was purified by silica gel column chromatography (CH2Cl2:CH3OH from 100:1 to
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20:1) to give compound Y3 (0.8368 g, 89.0%) as a light-yellow solid. H NMR (400 MHz,
DMSO-d6): δ 9.58 (s, 1H), 8.63 (s, 1H), 8.48 (s, 1H), 8.23 (s, 1H), 7.96 – 7.86 (m, 2H), 7.77 (d, J
= 6.7 Hz, 1H), 7.54 (d, J = 7.7 Hz, 1H), 7.47 – 7.38 (m, 2H), 7.32 (q, J = 8.1 Hz, 4H), 7.23 (s,
1H), 6.40 (d, J = 9.1 Hz, 1H), 6.22 (t, J = 6.6 Hz, 1H), 5.63 (s, 2H), 5.08 (s, 2H), 4.35 – 4.26 (m,
4H), 3.82 – 3.72 (m, 4H), 3.38 (s, 3H), 3.36 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 161.86,
154.05, 148.57, 147.14, 140.58, 140.51, 139.56, 137.93, 135.72, 131.39, 129.46, 128.69, 128.60,
122.27, 122.05, 120.79, 120.35, 119.24, 106.03, 103.73, 70.62, 70.54, 68.84, 68.51, 58.88, 58.82,
53.22, 51.28. HRMS (ESI): m/z 633.2700, calcd for C35H35N7O5 [M+H]+: 634.2772, found for
[M+H]+: 634.2799, Δ = 1.10 ppm.
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Compound Y3-1. Compound Y2-1 (0.2000 g, 0.7346 mmol) and erlotinib (0.2981g, 0.7577
mmol) were dissolved in mixture solvent (CHCl3/H2O/tBu-OH, 8/4/6/2 mL) with CuSO4.5H2O
and sodium ascorbate was added as a catalyst. The mixture was stirred at 3 °C overnight. When
the reaction was completed, the crude product was purified by silica gel column chromatography
3 times by the frozen solvent (CH2Cl2:CH3OH from 100:1 to 20:1, 0 °C). After cold vacuum
distillation (0 °C), compound Y3-1 (0.0467 g, 8.43%) was obtained as the white solid. 1H NMR
(500 MHz, DMSO-d6): δ 9.46 (s, 1H), 8.54 (d, J = 2.5 Hz, 1H), 8.23 (d, J = 1.9 Hz, 1H), 7.95 (d,
J = 1.8 Hz, 1H), 7.91 (d, J = 6.5 Hz, 2H), 7.62 (d, J = 1.8 Hz, 1H), 7.53 (dt, J = 7.7, 1.3 Hz, 1H),
7.42 (t, J = 7.9 Hz, 1H), 7.30 (s, 1H), 7.28 (s, 1H), 7.21 (d, J = 1.7 Hz, 2H), 7.19 (s, 1H), 5.88 (d,
J = 6.5 Hz, 1H), 5.58 (s, 2H), 4.52 (s, 1H), 4.35 (dd, J = 5.7, 4.2 Hz, 2H), 4.31 – 4.27 (m, 2H),
4.10 (dd, J = 6.4, 1.7 Hz, 1H), 3.88 – 3.84 (m, 4H), 3.71 (dd, J = 4.1, 1.7 Hz, 1H), 3.62 (d, J =
4.1 Hz, 1H), 3.48 (d, J = 4.1 Hz, 6H). HRMS (ESI): m/z 665.2598, calcd for C37H40N7O9
[M+CH3COOH]+: 726.2882 , found for [M+CH3COOH]+: 726.2895 , Δ = 1.82 ppm.
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Singlet Oxygen Release Assay. 1,3-Diphenylisobenzofuran (DPBF) was used as the O2
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scavenger to measure the release of O2 at 37 °C or 25 °C.A mixture of DPBF and Y3 or Y3-1
was placed in a water bath (37 °C or 25 °C) and covered with foil. The absorption was monitored
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at 415 nm. The release of O2 can be identified as a first-order reaction, according to the first-
order reaction kinetic formula: ln(A0/A) = kt, where A0 and A are the initial and time-point
absorbance values of DPBF, respectively, and k is the DPBF consuming rate, which is the
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releasing rate of O2. The half-life (t1/2) value can be calculated by the following formula: t1/2
ln2/k.
=
Cellular Uptake. HELF, Hcc827, HepG2and H1975 cells were seeded onto a plate and
incubated overnight (100 μL, 5000 cells/well). Each cell was set up with six wells. Drugs were
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added to the wells (25 μM, 100 μL/well) for 24 h. The plates were gently washed with PBS 3
times. 1% SDS solution (120 μL) was then added to each well to lyse the cells. The fluorescence
intensity of drugs (ex/em: 335/481 nm) in the cell extract was measured on a microplate reader.
Cellular protein content was determined with a BCA Protien Assay Kit. The cellular uptake is
expressed as nmol drug per mg cell protein.
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Cellular ROS Assay. Cellular ROS were determined by using a probe DCFH-DA (2',7'-
dichlorodihydrofluorescein diacetate) which can form the fluorescent compound DCF (2,7-
dichlorofluorescein) in the presence of ROS. The cells were seeded onto a plate and incubated
overnight (100 μL, 5000 cells/well). Six replicates per concentration were set for each drug (25
μM, 100 μL/well). A positive control group and a negative control group were used. After 24 h,
the plate was washed with PBS, and DCFH-DA (10 μM, 40 μL/well) was added. After 30 min,
the plate was washed 3 times with PBS, and 120 μL of 1% SDS lysis buffer was added. The
DCF fluorescence was measured by a microplate reader (ex/em: 488/525 nm).
Cytotoxicity Experiment. Hcc827, HepG2 or H1975 cells were seeded onto 96-well plates at
4000 cells per well and incubated overnight. The dissolved drugs (erlotinib, Y3, or Y3-1) were
diluted in culture medium to a series of concentrations. Six replicates per concentration were set
for each compound. After 24 h of incubation, 10 μL of MTT solution (5 mg/mL) was added to
each well and then the plate was incubated for 4 h. Then, 100 μL of DMSO was added to each
well, followed by 20 min incubation. The OD value of each well was measured by a microplate
reader (λabsmax = 570 nm).
Flow Cytometric Assay. HepG2 cells were grown to approxiamately 80% confluence and
incubated with different concentrations of Y3-1 for 24 h. Then, the cells were trypsinized and
washed three times with PBS. After incubation with the reagents in the Annexin V-FITC/PI
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Apoptosis Kit for 20 min in the dark, the cells were analyzed on a BD AccuriTM C6 flow
cytometer.
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Establishment of H22 Tumor-bearing Mouse Model. Female Kunming mice (4 weeks old,
18-20 g) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd, Shanghai, China.
These mice were maintained and handled in accordance with the recommendations of the
Institutional Animal Care and Use Committee (IACUC). 200 μL of H22 ascites tumor cell
suspension (approximately 5000000 cells/mL) was subcutaneously inoculated into the right side
of the mice. In vivo imaging and therapy experiments started when the tumor reached 80-100
mm3 7 days after inoculation.
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Fluorescence Molecular Tomography Imaging. H22 tumor-bearing mice were randomly
divided into two groups: the erlotinib group (200 μL 0.1 mg/kg erlotinib), and the Y3-1 group
(200 μL 0.1 mg/kg Y3-1). Fluorescence molecular tomography imaging was carried out at
different time points after the administration.
In Vivo Anticancer Assay. H22 tumor-bearing mice were randomly divided into three groups:
the control group (200 μL PBS), erlotinib group (200 μL 0.1 mg/kg erlotinib), and Y3-1 group
(200 μL 0.1 mg/kg Y3-1). Tumor growth was monitored by measuring the tumor volume (TV),
which is calculated from the length (L) and width (W) of the tumor using the formula (TV =
0.5×L×W2) every day. Body weight was also recorded daily. After 11 days of treatment, the
mice were sacrificed and the main organs (heart, liver, spleen, lung, kidney and tumor) were
collected and embedded in paraffin and stained with hematoxylin and eosin (H&E).
RESULTS AND DISCUSSION
Design and Synthesis. The synthesis of the title compound Y3-1 and its related compounds
are shown in Scheme 1. Initially, a substitution reaction of alpha,alpha'-dibromo-p-xylene with
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sodium azide in N,N-dimethylformamide (DMF) overnight at room temperature yielded a white
crystal of Y1. Next, Y2 was obtained through an electrophilic addition reaction of 2-
hydroxypyridine with Y1 using 18-crown-6 and potassium iodide as a catalyst. After irradiation
by a 670 nm laser in the presence of methylene blue and oxygen, compound oY2 then underwent
nucleophilic substitution to give compound Y2-1. Subsequently, Y2-1 performed 1,3-dipolar
cycloaddition with erlotinib in the presence of sodium ascorbate and CuSO4·5H2O to give Y3-1.
For comparison, Y2, which has only a 2-pyridone moiety, also performed 1,3-dipolar
cycloaddition with erlotinib to give Y3 without a 1O2 generation moiety. All these compounds
were characterized by 1H NMR, 13C NMR and HRMS (see Supporting Information Figures S1-
S12)
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Scheme 1. Synthesis of Y3-1 and its related compounds.
Singlet Oxygen Release Investigation. The 1O2 release ability of Y3-1 was first investigated
using 1,3-diphenylisobenzofuran (DPBF) as a selective 1O2 quencher at 37 °C or 25 °C in DMF.
As shown in Figure 1a, the absorbance of DPBF in Y3-1 group decreased significantly at 37 °C,
while the absorbance values in the control group and Y3 group did not show obvious changes,
indicating that only Y3-1 can release 1O2 at 37 °C. In addition, the absorbance of DPBF exhibits
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a small downtrend at 25 °C in Y3-1group (Figure 1b), showing that the 1O2 release ability of Y3-
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1 is greatly reduced at 25 °C. These results demonstrated that Y3-1 can release O2 through
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thermal decomposition. Kinetic stability at body temperature (37 °C) is important. Assuming that
the release of 1O2 follows a first-order reaction kinetics mode, the ln(A0/A) vs. time is shown in
Figure 1c. According to the half-life formula of the drugs, t1/2 = 0.693/k, the half-life of Y3-1 can
be calculated to be 13.0 h.
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Figure 1. a) Decrease in the absorbance of DPBF at 415 nm with time in the presence of no drug
(control group), Y3 or Y3-1 in DMF at 37 °C . b) Decrease in the absorbance of DPBF at 415 nm
with time in the presence of Y3-1 in DMF at 37 °C and 25 °C . c) Plot of Ln(A0/A) of Y3-1 vs.
time, A0 is the absorbance value of DPBF at 0 h. The concentration of Y3 or Y3-1 is 1.47 mM.
In Vitro Cancer Targeting. To demonstrate the cancer targeting ability of the erlotinib moiety
in Y3-1, three cancer cells: Hcc827 cells (erlotinib-sensitive human non-small cell lung cancer
cells with high EGFR expression), HepG2 cells (Cancer cells with high differentiation and
proliferation) and H1975 cells (erlotinib-resistant human non-small cell lung adenocarcinoma
cells) were selected. The cellular uptake of Y3-1 in these three cancer cells was examined by
measuring the fluorescence emission intensity of Y3-1 and the cell protein concentration in the
cell extract. The average relative cellular uptake (nmol/mg protein) data are shown in Figure 2.
The cellular uptake of Y3-1 in Hcc827 cells was very significantly higher than that in H1975
cells (p < 0.001), and significantly higher than that in HepG2 cells (p < 0.01). The differences in
the cellular uptake of Y3-1 are consistent with the erlotinib sensitivity of these cancer cells. The
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results demonstrate that the presence of the erlotinib moiety in Y3-1 results in targeted delivery
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Figure 2. The cellular uptake of Y3-1 in different cancer cells with different erlotinib sensitivities
(six replicates for each group, ** indicates p < 0.01, *** indicates p < 0.001).
In Vitro Anticancer Assay. The cellular generation efficiency of 1O2 is a critical parameter for
the anticancer effect of Y3-1. Therefore, the ROS probe DCFH-DA (2',7'-
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dichlorodihydrofluorescein diacetate) was used to detect intracellular ROS, including O2 in
Hcc827 cells after 25 μM drug treatment. Compared with the two control drugs Y3 and erlotinib,
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our targeted drug Y3-1 could release significantly more O2 in Hcc827 cells (Figure 3a). There
was no significant difference in ROS production efficiency between erlotinib and Y3, which
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contains no endoperoxide moiety to store and release O2. In addition, the ROS production
efficiency of Y3-1 positively correlated with the concentrations of Y3-1 in Hcc827 cells (Figure
S13, Supporting Information). Therefore, all these results indicate that the combination of
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reversible singlet-oxygen storage modules and erlotinib can deliver and release O2 into cancer
cells.
The anticancer effects of Y3-1, Y3 and erlotinib were studied by MTT assays in Hcc827 cells,
HepG2 cells and H1975 cells. The dose-dependent survival curves and corresponding IC50
values are shown in Figure 2b and Table S1 (Supporting Information). No significant difference
was observed in the cellular antiproliferative ability of Y3 (with IC50 = 37.99-68.82 μM) and
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erlotinib (with IC50 = 33.68-76.67 μM) towards these tree cancer cell lines, indicating that the
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introduction of the pyridone moiety to the erlotinib unit does not obviously influence its
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anticancer effects. As expected, Y3-1, Y3-1 armed with O2, showed approximately 2-fold
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higher cytotoxicity (IC50 values of 13.52-38.16 μM) than Y3 or erlotinib, even in erlotinib-
resistant cancer cells. The anticancer effect of Y3-1 in different tumor cells is consistent with its
cellular uptake in different cells, which is related to the erlotinib differences in these cancer cells.
In addition, the flow cytometric assay of Annexin V-FITC/PI was utilized to further validate the
mode of cell death caused by Y3-1. As shown in Figure 3c and Figure S14 (Supporting
Information), the populations of apoptotic cells (upper right and low right phases) and necrotic
cells (upper left phase) increased significantly with increasing Y3-1 concentration. These results
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clearly show that Y3-1 can induce apoptosis and necrosis in tumor cells by mimicking the O2-
dependent anticancer mechanismof PDT in the absence of light and oxygen.
Figure 3. a) Cellular ROS generation in Hcc827 cells by erlotinib, Y3 and Y3-1 (the drug
concentration is 25 μM, six replicates are used for each group, ** indicates p < 0.01, ***
indicates p < 0.001, ns indicates p > 0.05). b) Cytotoxic effects of erlotinib, Y3, and Y3-1
towards Hcc827 cells, HepG2 cells or H1975 cells (data are expressed as the means ± SD from
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three experiments, each performed in quadruplicate). c) Histogram of cell populations after 0.0,
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12.5 and 50 μM Y3-1 treatment by Annexin V-FITC/PI detection.
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In Vivo Cancer Targeting. To further confirm the precise delivery of Y3-1 to the tumor by
the erlotinib moiety, in vivo fluorescence imaging was also performed by using the FMT
(fluorescence molecular tomography) TM 2500 system (PerkinElmer Inc.). For this purpose, 0.1
mg/kg Y3-1 (labeled with Cy5 NHS ester) or erlotinib (labeled with Cy5 Azide) was injected
into the tail vein of Kunming mice bearing H22 tumors. As shown in Figure 4a-b, Y3-1 exhibited
an increasing accumulation in tumor tissue within 8 h and then gradually decreased. The highest
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accumulation time was much shorter than the half-life of Y3-1, enabling Y3-1 release O2 and
achieve excellent anticancer effects. Skin phototoxicity, which results from drug accumulation in
skin tissue, is still a fundamental barrier to the clinical application of PDT. Therefore, we also
studies Y3-1 accumulation in normal skin tissues. As shown in Figure 4a-b, no visible
accumulation appeared in normal skin tissues over time. Additionally, the total amount of Y3-1
or erlotinib in the tumor tissue was obviously higher than that in normal skin tissue (Figure 4).
This result indicates the high specificity of Y3-1 to tumors, which can be attributed to the
erlotinib moiety with its high targeting ability towards tumor tissues.
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Figure 4. In vivo cancer targeting investigation by using Kunming mice bearing subcutaneous
H22 tumors. 3D fluorescence images based on FMT after administration with (a) Y3-1 or (b)
erlotinib. Comparison of the average fluorescence density of drugs in normal skin tissue and
tumor after administration with (c) Y3-1 or (d) erlotinib (** indicates p < 0.01, *** indicates p <
0.001).
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In Vivo Anticancer Assay. To further investigate the in vivo anticancer effect of Y3-1, H22
tumors were established in Kunming mice. When the tumor volume reached 50-80 mm3, 0.1
mg/kg Y3-1 or erlotinib was injected into the mouse tail vein in the test group, while the control
group was treated with PBS. As shown in Figure 5a and c, erlotinib could partially decrease
tumor volume compared with that in the control group. Moreover, we found that Y3-1 induced a
complete tumor inhibition with a significant difference (p < 0.01) compared with erlotinib group
and an extremely significant difference (p < 0.001) compared with the control group. We also
examined hematoxylin-eosin (H&E) staining on tumor sections (Figure 5d). Compared with the
control group and erlotinib group, Y3-1 group contained more deformed nuclei (karyopyknosis,
karyorrhexis, and karyolysis), which indicates severe necrosis of cancer cells. The enhanced in
vivo anticancer effect of Y3-1 may be attributed to its singlet oxygen chemical source
endoperoxide acting like PDT-based mechanisms supported by accumulating evidence: directly
killing cancer cells, shutting down the tumor microvasculature and inducing antitumor immunity.
These data provide support for the excellent anticancer performance of Y3-1 through the EGFR-
targeted delivery of the thermal-controlled release singlet oxygen sensitizer inspired by targeted
PDT-based mechanisms.
To study the biosafety of Y3-1, we examined the body weight and the HE staining of other
normal organs. As shown in Figure 5b and d, no loss of body weight or other signs of toxicity
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manifested following the drugs administration (Figure 5b) and no obvious pathological
alterations were observed in the HE staining of the heart, liver, spleen, lung and kidney in all
mice after 11 days of treatment (Figure 5d). These results indicate that Y3-1 has a high biosafety
index.
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Figure 5. a) Tumor volume curves of mice treated with solvent, erlotinib and Y3-1. b) Body
weight curves of mice treated with solvent, erlotinib and Y3-1. c) Photos of mice and excise
tumors after treatment on the 11th day. d) HE staining of tumor sections and major organs (heart,
liver, spleen, lung and kidney) from H22 tumor bearing mice on the 11th day. The scale bars are
100 μm.
In conclusion, we successfully developed a proof of principle concept for targeted anticancer
therapy by introducing an EGFR targeting moiety into a PDT-mimetic sensitizer, which does not
require in vivo oxygen or light. We demonstrated that singlet oxygen, the key factor in PDT, can
be stored in Y3-1 by in vitro photosensitization and be precisely delivered into EGFR-
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overexpressing cancer cells/tissues. Additionally, our results clearly confirmed the excellent
anticancer efficacy of Y3-1 achieved by the delivery of thermal-release singlet oxygen. As the
first demonstration, this smart strategy might provide a new concept and model for the further
development of more efficient anticancer drugs. Other drugs based on this strategy are currently
ongoing in our lab and further results will be reported soon.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Cellular antiproliferative activities, ROS production, Flow apoptotic results, 1H NMR, 13C NMR
and HRMS spectra characterization (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: xuejinping66@fzu.edu.cn. Phone: +86-0591-2286-7923. Fax: +86-0591-2286-7923.
ORCID
Juanjuan Chen: 0000-0002-9148-2206
Jinping Xue: 0000-0001-6952-0475
Author Contributions
⊥J. C. and D. L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
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This work was supported by the National Natural Science Foundation of China (81703345,
81803346, 51672046), and the National Health and Family Planning Commission jointly
established a scientific research fund (WKJ2016-2-14).
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ABBREVIATIONS
PDT, photodynamic therapy; EGFR, epidermal growth factor; PSs, photosensitizers; DMF, N,N
dimethylformamide; DPBF, 1,3-diphenylisobenzofuran; ROS, reactive oxygen species; DCFH-
DA, 2’,7’-dichlorodihydrofluorescein diacetate; H&E, hematoxylin-eosin.
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(35) Chen, J.-J.; Huang, Y.-Z.; Song, M.-R.; Zhang, Z.-H.; Xue, J.-P. Silicon phthalocyanines
axially disubstituted with erlotinib toward small-molecular-target-based photodynamic therapy.
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Scheme 1. Synthesis of Y3-1 and its related compounds.
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Figure 1. a) Decrease in the absorbance of DPBF at 415 nm with time in the presence of no drug (control
group), Y3 or Y3-1 in DMF at 37 °C. b) Decrease in the absorbance of DPBF at 415 nm with time in the
presence of Y3-1 in DMF at 37 °C and 25 °C. c) Plot of Ln(A /A) of Y3-1 vs. time, A0 is the absorbance
0
value of DPBF at 0 h. The concentration of Y3 or Y3-1 is 1.47 mM.
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Figure 2. The cellular uptake of Y3-1 in different cancer cells with different erlotinib sensitivities (six
replicates for each group, ** indicates p < 0.01, *** indicates p < 0.001).
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a) Cellular ROS generation in Hcc827 cells by erlotinib, Y3 and Y3-1 (the drug concentration is 25 μM, six
replicates are used for each group, ** indicates p < 0.01, *** indicates p < 0.001, ns indicates p > 0.05).
b) Cytotoxic effects of erlotinib, Y3, and Y3-1 towards Hcc827 cells, HepG2 cells or H1975 cells (data are
expressed as the means ± SD from three experiments, each performed in quadruplicate). c) Histogram of
cell populations after 0.0, 12.5 and 50 μM Y3-1 treatment by Annexin V-FITC/PI detection.
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In vivo cancer targeting investigation by using Kunming mice bearing subcutaneous H22 tumors. 3D
fluorescence images based on FMT after administration with (a) Y3-1 or (b) erlotinib. Comparison of the
average fluorescence density of drugs in normal skin tissue and tumor after administration with (c) Y3-1 or
(d) erlotinib (** indicates p < 0.01, *** indicates p < 0.001).
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