Nanoscale photosensitizer with tumor-selective turn-on fluorescence and activatable photodynamic therapy treatment for COX-2 overexpressed cancer cells

Article abstract of DOI:10.1039/d0tb02828b

Effective targeting andin situimaging-guided treatment are particularly important for accurate clinical photodynamic therapy (PDT) of malignant tumors. Herein, we propose a single molecule, namedIMC-DAH-SQ, which possesses dual-targeting components, including structure-inherent targeting (SIT) and cyclooxygenase-2 (COX-2) targeting units, and controllable turn-on near infrared (NIR) fluorescence. Due to its amphiphilicity,IMC-DAH-SQassembles into a nanoprobe with low background fluorescence. After incubation with tumor cells, the SIT and COX-2 recognition characteristics ofIMC-DAH-SQendow it with preferential tumor-targeting activity. The strong binding with overexpressed COX-2 can collapse the nanoprobe to monomers after accumulation in tumor cells, leading to turn-on NIR fluorescence that is completely different from normal cells. Additionally, benefiting from the single molecular model tactic, the nanoprobe has the advantages of simple synthesis without ever considering the loading rate and separation between the photosensitizer and targeting unit. Other favorite features, including superior biocompatibility, weak dark toxicity, and mitochondria enrichment capability, are implemented. All these traits not only afford nanoprobe precision tumor cell targeting capability but also provide promising imaging-guided antitumor therapy. We believe that the single molecular protocol will establish a novel strategy for simultaneous diagnosis and anticancer medicine treatment utilizing versatile but small compounds.

Full text of DOI:10.1039/d0tb02828b

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Nanoscale photosensitizer with tumor-selective
turn-on fluorescence and activatable
photodynamic therapy treatment for
COX-2 overexpressed cancer cells†
Cite this: J. Mater. Chem. B, 2021,
9, 2001
Lan Wang, Yuhuan Zhang, Ying Han, Qi Zhang, Zhenfu Wen, Hongjuan Li,
Shiguo Sun, Xin Chen and Yongqian Xu
*
Effective targeting and in situ imaging-guided treatment are particularly important for accurate clinical
photodynamic therapy (PDT) of malignant tumors. Herein, we propose a single molecule, named IMC-
DAH-SQ, which possesses dual-targeting components, including structure-inherent targeting (SIT) and
cyclooxygenase-2 (COX-2) targeting units, and controllable turn-on near infrared (NIR) fluorescence.
Due to its amphiphilicity, IMC-DAH-SQ assembles into a nanoprobe with low background fluorescence.
After incubation with tumor cells, the SIT and COX-2 recognition characteristics of IMC-DAH-SQ endow
it with preferential tumor-targeting activity. The strong binding with overexpressed COX-2 can collapse
the nanoprobe to monomers after accumulation in tumor cells, leading to turn-on NIR fluorescence
that is completely different from normal cells. Additionally, benefiting from the single molecular model
tactic, the nanoprobe has the advantages of simple synthesis without ever considering the loading rate
and separation between the photosensitizer and targeting unit. Other favorite features, including
superior biocompatibility, weak dark toxicity, and mitochondria enrichment capability, are implemented.
All these traits not only afford nanoprobe precision tumor cell targeting capability but also provide
promising imaging-guided antitumor therapy. We believe that the single molecular protocol will
establish a novel strategy for simultaneous diagnosis and anticancer medicine treatment utilizing
versatile but small compounds.
Received 5th December 2020,
Accepted 21st January 2021
DOI: 10.1039/d0tb02828b
rsc.li/materials-b
ideal alternative, integrating multiple targeting and tumor
environment-activated mechanisms into a single molecule with-
Introduction
Photodynamic therapy (PDT) has become a promising treatment out carrier delivery could improve the accuracy and efficiency of
modality because of its noninvasiveness, small side effects and recognition and minimize the side effects of PDT. Due to the
no drug resistance.1 PDT relies on photosensitizers and excita- unique and fast metabolism, the membrane of tumor cells
tion light to produce cytotoxic reactive oxygen species (ROS) to possesses more negative charge than normal cells, leading
kill cancer cells.2 However, ROS are non-targetable and have a them to absorb and swallow positively charged molecules
short lifetime; therefore, photosensitizers must be enriched in preferentially.5–8 Structure-inherent targeting (SIT) provides a
the tumor site to improve the PDT effect availably and to reduce new opportunity to achieve targetable delivery. In the past, SIT
side effects. To achieve targeting capability of photosensitizers, was mainly used for biological imaging; it has rarely been used
strategies such as the bioconjugation of photosensitizers with for tumor-targetable delivery.9
target ligands have been widely utilized.3,4 However, the con-
Cyclooxygenase-2 (COX-2) is closely related to the growth of
jugated molecular synthesis is time-consuming and laborious, various stages of cancer; it is overexpressed in tumor cells and
and the separation of the photosensitizer and targeting unit and has become a specific biomarker of tumor cells.10 As an
a low loading rate of the photosensitizer are unavoidable. As an inhibitor of COX-2, indomethacin (IMC) can closely interact
with COX-2; also, due to the high affinity between IMC and
COX-2, IMC is a useful ligand for tumor targeting.11 Mitochondria,
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of
Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi, 712100,
the powerhouse of cells, regulates the growth and metabolism of
P. R. China. E-mail: xuyq@nwsuaf.edu.cn
cells.12 The dysfunction of mitochondria can easily lead to the
† Electronic supplementary information (ESI) available: Additional spectra such
death of cells.13 If PDT photosensitizers can target and accumulate
as zeta potential distribution, cell viability, fluorescent imaging in cells and NMR
spectra of compounds. See DOI: 10.1039/d0tb02828b
in the mitochondria of tumor cells, they can subsequently produce
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toxic ROS, which would significantly promote the killing of cancer SQ inhibits fluorescence emission and ROS generation. After
cells.14,15 Thus, undoubtedly searching for photosensitizers tar- accumulation in tumor cells, the binding of IMC with COX-2
geting the mitochondria of tumor cells is beneficial to improve disperses the nanoprobe into the monomer. Obvious NIR
the PDT efficiency.
fluorescence appears in the tumor, which can be utilized for
Among many biological imaging technologies, fluorescence the fluorescence recognition of tumor cells. Due to the positive
imaging has become a useful tool because of its high sensitivity, charge of IMC-DAH-SQ, it further targets the mitochondria. As a
real-time imaging with high spatial resolution, noninvasiveness, promising photosensitizer, SQ can produce singlet oxygen in
etc. PDT that is accompanied by the fluorescence imaging of mitochondria under excitation. PDT-induced ablation was espe-
tumor cells in the whole process, called imaging-guided PDT, cially triggered in tumor cells. This multifunctional and intelligent
can visually control and effectively improve the effects of PDT by molecule with carrier-free delivery can realize high selectivity and
limiting the light range and reducing the phototoxicity.16 Therefore, good efficiency of PDT of tumor cells.
ideal PDT photosensitizers should have good biocompatibility, low
dark toxicity and high singlet oxygen production, and they can be
Experimental
Materials
used for imaging-guided PDT with NIR fluorescence.17 Although
the existing photosensitizers, mainly including porphyrin, phthalo-
cyanines, BODIPY, and aggregation-induced emission (AIE) dyes,18
have been widely used in imaging-guided PDT, their ‘‘always-on’’
signal characteristics are not conducive to distinguishing tumor
cells from normal cells. Moreover, they do not have the ability to
only produce ROS in tumor cells and not in normal cells. Therefore,
it is still challenging to develop photosensitizers that meet all the
above requirements.
The chemical reagents used here were purchased from Energy
Chemical Reagents Co. and Mayer Chemical Reagents Co.
(Shanghai, China). All these reagents were used as received
without further purification unless otherwise stated. The water
used was ultrafilter deionized. The Live-Dead cell staining kit
was purchased from Yeasen Biotech Co., Ltd (Shanghai, China).
The mitochondrial membrane potential fluorescent probe JC-1
was purchased from Solarbio Science & Technology Co., Ltd
(Beijing, China).
Herein, we report a small molecule, IMC-DAH-SQ, which can
be easily synthesized as a PDT reagent (Scheme 1). It has a tumor
cell targeting function via SIT and interacts with COX-2. Mean-
while, the tumor cell-induced turn-on fluorescence imaging can
be used for recognition and possible imaging-guided PDT.
Specifically, we connected IMC and a NIR fluorescence squaraine
(SQ) dye through a short flexible chain. This simple connection
avoids the separation of the photosensitizer and the recognition
unit, and it ensures a high loading rate of the photosensitizer.
The positively charged feature of SQ endows IMC-DAH-SQ with
preferential access to tumor cells via SIT.19 Due to the amphi-
philicity, IMC-DAH-SQ can aggregate in aqueous solution to
form nano-photosensitizers. The nanoscale architecture endows
IMC-DAH-SQ with a low background fluorescence signal due to
the aggregation-caused quenching (ACQ) characteristic of SQ
(Scheme 1). The molecular self-assembly system of IMC-DAH-
General methods
1H NMR and 13C NMR data were obtained from a Bruker
AVANCE III 500 spectrometer (Germany) (NMR data were
referenced to TMS used as the internal standard). Mass spectra
were obtained with an LCQ Fleet mass spectrometer (USA) and
an AB SCIEX LC-30A + TripleTOF5600 + mass spectrometer
(Singapore). Fluorescence spectra and UV-Vis spectra were
carried out on a RF-6000 fluorescence spectrometer and a
Shimadzu 1750 UV-visible spectrometer (Japan), respectively.
Confocal fluorescence imaging of cells was performed using an
Olympus FV1000MPE laser scanning confocal microscope
(Japan). Dynamic light scattering (DLS) and zeta potential were
performed with a Malvern Instruments Limited ZEN3600 (Britain).
The quantum yields of fluorescence of the samples were measured
using Rhodamine B in ethanol (F = 0.49) as a standard and
calculated according to the method below:
2
ðFsÞðIuÞðAsÞðlexsÞðZu Þ
Fu ¼
ðIsÞðAuÞðlexuÞðZs2Þ
where F represents the fluorescence quantum yield. I represents
the integrated emission intensities. A is the absorbance at the
excitation wavelength. lex represents the excitation wavelength. Z is
the refractive index of the corresponding solution. The subscripts u
and s represent the sample and the standard, respectively.
Synthesis of IMC-DAH-Boc
IMC-DAH-Boc was synthesized according to a previously
reported method with slight modification.20 To a 100 mL
single-necked flask, IMC (0.59 g, 1.65 mmol), HATU (0.94 g,
2.5 mmol), DAH-Boc (0.425 g, 1.95 mmol), and TEA (0.505 g,
Scheme 1 The structure of IMC-DAH-SQ and the cascade processes of
aggregation, disaggregation, lighting up and ROS production of IMC-DAH-SQ. 5 mmol) were dissolved in 10 mL DMF. Then, the mixture was
2002 | J. Mater. Chem. B, 2021, 9, 2001ꢀ2009
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stirred for 4 h at room temperature. After completion of energy conformations and good hydrogen-bond geometries were
the reaction, the mixture was poured into excess ice water. The considered. Molecular-protein docking results and small-molecule-
precipitate was collected by filtration and dried to obtain the protein co-crystal structures were shown using Pymol.22
crude compound IMC-DAH-Boc as a milk yellow solid, and pure
Cell viability assay
IMC-DAH-Boc was obtained by column chromatography (514 mg,
56.05% yield). 1H NMR (500 MHz, CDCl3) d 7.64 (d, J = 8.5 Hz, 2H), The cell viabilities of HepG2 and HL7702 cells were evaluated
7.46 (d, J = 8.5 Hz, 2H), 6.86 (m, 2H), 6.67 (dd, J = 9.0, 2.5 Hz, 1H), by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide
5.70 (s, 1H), 4.51 (s, 1H), 3.80 (s, 3H), 3.61 (s, 2H), 3.16 (q, J = (MTT) assays. In short, the different cell lines were seeded in
6.5 Hz, 2H), 3.01 (d, J = 6.0 Hz, 2H), 2.36 (s, 3H), 1.42–1.32 96-well plates (NEST) with an intensity of 8 ꢁ 104 cells per mL,
(m, 13H), 1.25–1.13 (m, 4H). ESI-MS: m/z calcd for [M + H]+: respectively. After 24 h incubation, the medium was replaced with
578.10, found: 578.26.
IMC-DAH-SQ solution with different concentrations (0–20 mM in
1% DMSO). Incubation continued for 24 h at 37 1C; then, the
medium of IMC-DAH-SQ solution was removed and incubation
continued for certain time intervals in fresh RPMI-1640 at 37 1C.
Synthesis of IMC-DAH-SQ
SQ was prepared according to the reported literature.21
To a 100 mL flask in an ice bath, IMC-DAH-Boc (166.8 mg, After that, the cells were washed twice with PBS buffer, and 100 mL
0.3 mmol), 5 mL DCM and 5 mL TFA were added in turn. The freshly prepared MTT solution (0.5 mg mLꢀ1) was added. After
mixed solution was stirred at room temperature for 10 h and 4 h, the MTT solution was removed and 100 mL DMSO was added
then concentrated by a rotary evaporator. The residue was to each well. Then, the plate was gently shaken to dissolve the
dissolved with 20 mL ethyl acetate, washed with 5 M NaOH precipitates produced. The absorbance of MTT at 490 nm was
solution (3 ꢁ 20 mL) and saturated NaCl solution (2 ꢁ 20 mL), measured by a microplate reader.
dried with anhydrous Na2SO4 and concentrated by rotary
evaporator to obtain the desired compound IMC-DAH
Cell imaging
(123 mg, 89.9% yield). The compound IMC-DAH was used Cancer cell lines (HepG2, A549, MCF-7) and normal cell lines
directly in the next reaction without further purification. (HL7702 and 293T) were seeded on 35 mm glass-bottomed
IMC-DAH (123 mg, 0.27 mmol), SQ (130 mg, 0.216 mmol), dishes (NEST) with culture medium at 37 1C and incubated for
DMAP (10 mg, 0.08 mmol) and TEA (80 mL, 0.575 mmol) were 24 h. The culture medium was carefully removed, and the cells
mixed in 10 mL DCM. The resulting solution was stirred at were further incubated with 1 mL IMC-DAH-SQ (5 mM) medium
room temperature for 10 h under nitrogen protection. The solution at room temperature for 0.5 h. Then, the excess
mixture was concentrated by a rotary evaporator to afford the nanoprobe was removed by washing with PBS buffer three
crude compound. The crude compound was purified with silica times, and the obtained cells were imaged.
gel column chromatography to obtain a cyanic solid IMC-DAH-SQ
(67 mg, 30.39% yield). 1H NMR (500 MHz, DMSO-d6) d 8.73 (t, J =
Inhibitor control experiment
5.5 Hz, 1H), 8.06 (t, J = 5.5 Hz, 1H), 7.99 (d, J = 7.5 Hz, 1H), 7.92 HepG2, A549 and MCF-7 cells were seeded on 35 mm glass-
(d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.65–7.58 (m, 5H), 7.54 bottomed dishes (NEST) and incubated in culture medium at
(d, J = 7.5 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 37 1C for 24 h. The cells were pretreated with 50 mM inhibitor
7.33 (d, J = 8.0 Hz, 1H), 7.12 (s, 1H), 6.84 (d, J = 9.0 Hz, 1H), 6.63 indomethacin and celecoxib at 37 1C for 16 h. The unabsorbed
(dd, J = 9.0, 2.5 Hz, 1H), 6.18 (s, 1H), 5.76 (s, 1H), 4.31 (q, J = 6.5 Hz, inhibitor was carefully removed by washing with PBS buffer
4H), 3.73 (s, 3H), 3.51 (m, 4H), 3.09 (q, J = 6.5 Hz, 2H), 2.23 (s, 3H), three times. Then, the cells were incubated with 5 mM IMC-
1.60 (m, 2H), 1.45 (m, 2H), 1.38–1.27 (m, 6H), 1.23 (t, J = 7.0 Hz, DAH-SQ for 0.5 h at room temperature and washed with PBS
4H). 13C NMR (125 MHz, DMSO-d6) d 173.5, 169.3, 167.7, 163.1, buffer before imaging.
160.9, 159.3, 157.2, 155.6, 155.5, 140.3, 140.2, 137.6, 135.1, 134.2,
131.1, 130.8, 130.2, 129.0, 125.0, 124.5, 123.0, 122.7, 114.5, 114.4,
Lipopolysaccharide up-regulates the COX-2 level
113.2, 112.8, 111.1, 101.9, 86.2, 86.1, 55.4, 43.5, 41.6, 41.0, 38.4, HL7702 and 293T cells were seeded on 35 mm glass-bottomed
31.2, 30.9, 29.2, 25.9, 25.9, 13.4, 12.6, 12.0. TOF MS: m/z calcd for dishes (NEST) and incubated in culture medium at 37 1C for
[M ꢀ CF3SO3ꢀ]+: 870.2909, found: 870.2929.
24 h. The cells were pretreated with 1 mg mLꢀ1 lipopolysac-
charide (LPS) at 37 1C for 24 h. The unabsorbed LPS was
carefully removed by washing with PBS buffer three times,
Molecular modeling
The X-ray crystal structures of COX-2 binding with indomethacin- and the cells were further incubated with 5 mM IMC-DAH-SQ
ethylenediamine-dansyl conjugate (PDB code: 6BL4) was retrieved for 0.5 h at room temperature and washed with PBS buffer
from the Protein Data Bank. The chemical structure of the nano- before imaging.
probe IMC-DAH-SQ was prepared using the protein preparation
wizard in Maestro with standard settings. The grids of COX-2 were
Co-localization experiment
generated using Glide version 10.2, following the standard HepG2 cells were seeded on 35 mm glass-bottomed dishes
procedure recommended by Schrodinger. The conformational (NEST) and incubated in culture medium at 37 1C for 24 h.
ensembles were docked flexibly using Glide with standard settings The cells were pretreated with 5 mM IMC-DAH-SQ and Rhoda-
in both standard and extra precision modes. Only poses with low mine 123 at room temperature for 0.5 h. The excess nanoprobe
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and Rhodamine 123 were carefully removed by washing with Calcein-AM/PI staining experiment
PBS three times before imaging.
HepG2 cells were seeded on 35 mm glass-bottomed dishes
(NEST) and incubated in culture medium at 37 1C for 24 h.
Then, the cells were pretreated with different concentrations of
IMC-DAH-SQ at 37 1C for 4 h. The glass-bottomed dishes were
irradiated with a 630 nm LED light for 12.5 min at a power
density of 25 mW cmꢀ2 and further incubated for 24 h.
A Calcein-AM/PI Double Stain Kit was used according to the
instructions.
In vitro ROS detection
9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) was
employed to detect ROS in vitro. The typical procedure for
detection of ROS is as follows: IMC-DAH-SQ and 50 mM ABDA
were mixed in DMSO in a cuvette. Then, the solution was
irradiated with a 630 nm LED lamp (25 mW cmꢀ2) at room
temperature.
Flow cytometry
Determination of the ROS quantum yield
Cells were gathered in each group, centrifuged for 5 min, and
washed twice with PBS. 1–10 ꢁ 105 cells were suspended in PBS.
In the assay, cell death was detected by the Annexin V-EGFP/PI
Apoptosis Detection Kit (Yeasen Biotechnology Co., Ltd China).
In short, HepG2 cells treated with IMC-DAH-SQ were suspended
in 100 mL buffer mixed with 5 mL propidium iodide (PI) and
5 mL Annexin V-EGFP, incubated at room temperature for 20 min
in the dark, and detected via flow cytometer (BD Biosciences, FACA
CA, USA). The flow cytometry was qualified within 1 h after the dye
solution was added.
For the ROS quantum yield (F) measurement, 1,3-diphenyl-
benzofuran (DPBF) was used as a probe, and methylene blue
(MB, F = 0.52 in DMSO) was used as the reference. The mixture
solutions were irradiated at 630 nm (25 mW cmꢀ2) for 60 min. The
absorbance of the solutions was measured for different periods of
time. The following equation was used to calculate the ROS
quantum yield:23
mðPSÞ FðMBÞPðMBÞ
F ¼ FðMBÞ
mðMBÞ FðPSÞ PðPSÞ
where m is the slope of the absorption change of DPBF at 415 nm
against the irradiation time. F is the absorption correction factor,
which is given as F = 1 ꢀ 10ꢀOD. P is the absorbed photonic flux.
Results and discussion
Photophysical properties of IMC-DAH-SQ
The synthesis and detailed characterization of IMC-DAH-SQ are
listed in the ESI† (Scheme S1 and Fig. S10–S16). The spectra of
IMC-DAH-SQ in different solutions were studied (Table S1,
ESI†). For the emission spectra, IMC-DAH-SQ has a strong
NIR fluorescence peak in DMSO solution and a weak one in
aqueous solution (Fig. 1a). The weak fluorescence intensity in
aqueous solution can be attributed to ACQ, affording low
background fluorescence of IMC-DAH-SQ. As shown in Fig. 1b
and Table S1 (ESI†), IMC-DAH-SQ showed an absorption peak in
organic solutions. In aqueous solution, IMC-DAH-SQ demon-
strated two prominent absorption peaks at 654 and 607 nm,
assigned to the monomer and aggregates. To check the morphology
of the IMC-DAH-SQ aggregates in aqueous solution, scanning
electron microscopy (SEM) and dynamic light scattering (DLS)
spectroscopy were performed. Regular and spherical nanoparticles
with sizes in the range of 130–150 nm were found (Fig. 2),
supporting the formation of the IMC-DAH-SQ nanoprobe. The
surface zeta potential of the IMC-DAH-SQ nanoparticles is
Intracellular ROS detection
HepG2 cells were seeded on 35 mm glass-bottomed dishes
(NEST) and incubated in culture medium at 37 1C for 24 h.
The cells were pretreated with 5 mM IMC-DAH-SQ at room
temperature for 0.5 h. The unabsorbed nanoprobe was carefully
removed by washing with PBS buffer three times, and the cells
were further incubated with DCFH-DA in fresh culture medium
for 0.5 h at room temperature. After that, the glass-bottomed
dishes were irradiated with a 630 nm LED light for 12.5 min at a
power density of 20 mW cmꢀ2. Then, the cells were imaged by a
fluorescence confocal microscope.
Mitochondrial membrane potential detection
HepG2 cells were seeded on 35 mm glass-bottomed dishes
(NEST) and incubated in culture medium at 37 1C for 24 h.
The cells were pretreated with different concentrations of IMC-
DAH-SQ at 37 1C for 4 h. Then, the cells were irradiated with a
630 nm LED light for 12.5 min at a power density of 20 mW cmꢀ2
and further incubated for 24 h. The mitochondrial membrane
potential probe JC-1 was used according to the instructions.
Photodynamic therapy in vitro
Cancer cells HepG2 in 96-well plates (NEST) were seeded with
culture medium at 37 1C and incubated for 24 h. Fresh medium
with IMC-DAH-SQ was used to replace the culture medium,
followed by incubation at 37 1C for 0.5 h. Then, the cells were
irradiated with a 630 nm LED light for 12.5 min at a power
density of 25 mW cmꢀ2 and further incubated for 24 h. For
detection of the cell viability, MTT assays were carried out.
Fig. 1 (a) Emission spectra and (b) UV-vis absorption spectra of IMC-
DAH-SQ (5 mM) in different solutions.
2004 | J. Mater. Chem. B, 2021, 9, 2001ꢀ2009
This journal is The Royal Society of Chemistry 2021