An I6P7 peptide modified fluorescent probe for bio-imaging

Article abstract of DOI:10.1039/c8nj04255a

The I₆P₇ peptide was proved to specifically bind to the interleukin-6 receptor, which can be exploited as an effective tumor-targeting moiety. Herein, a novel tumor-targeting fluorescent probe (DMP) conjugated with the I₆P₇ peptide was designed and synthesized. This probe was validated to possess valuable photophysical properties. The biocompatibility was evaluated by investigating its cytotoxicity on HUVEC and U87 cells, respectively. The tumor cell affinity has been confirmed by bio-imaging of this probe on different human cancer cells (U87, A549 and MCF-7 cells) and normal cells (HUVEC cell). The probe demonstrates low cytotoxicity, high tumor cell affinity and favorable mitochondria-targeting capability. Overall, these results revealed that DMP can serve as an important fluorescent probe for tumor-targeted bio-imaging.

Full text of DOI:10.1039/c8nj04255a

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An I6P7 peptide modified fluorescent probe for
bio-imaging†
Cite this:DOI: 10.1039/c8nj04255a
Yuxin Yao,‡a Lijuan Gui,‡a Beike Gao,b Zhenwei Yuan,a Yisha Chen,a Chen Wei,a
a
a
Qing He,a Fei Wang,a Mingjun Xu
and Haiyan Chen
*
The I6P7 peptide was proved to specifically bind to the interleukin-6 receptor, which can be exploited
as an effective tumor-targeting moiety. Herein, a novel tumor-targeting fluorescent probe (DMP)
conjugated with the I6P7 peptide was designed and synthesized. This probe was validated to possess
valuable photophysical properties. The biocompatibility was evaluated by investigating its cytotoxicity on
HUVEC and U87 cells, respectively. The tumor cell affinity has been confirmed by bio-imaging of this
probe on different human cancer cells (U87, A549 and MCF-7 cells) and normal cells (HUVEC cell). The
probe demonstrates low cytotoxicity, high tumor cell affinity and favorable mitochondria-targeting
capability. Overall, these results revealed that DMP can serve as an important fluorescent probe for
tumor-targeted bio-imaging.
Received 20th August 2018,
Accepted 3rd December 2018
DOI: 10.1039/c8nj04255a
rsc.li/njc
IL-6R, ascribed to its high-efficiency of binding to IL-6R.3 Based on
this, I6P7 could be modified as a potential tumor-targeting ligand
Introduction
In recent years, biomarkers have been widely applied in clinics for bio-imaging in vitro and in vivo.10
for cancer diagnosis. Interleukin-6 (IL-6) is a multifunctional
Optical imaging has emerged as an effective tool for bio-
cytokine characterized as a regulator of inflammatory and imaging due to its high spatial resolution, sensitivity, specificity
immune responses.1 Nevertheless, the disregulation of IL-6 and and low-cost.11,12 The major problem, limited tissue penetration,
its receptor interleukin receptor (IL-6R) in human cancers has been could be resolved through designing near-infrared (NIR) fluoro-
validated by recent research studies.2 IL-6R, a membrane-bound phores, under which tissues do not exhibit auto-fluorescence.
glycoprotein, has been detected to be overexpressed in several Thus, NIR optical imaging is fitting for bio-imaging applications
cancer cells, such as human glioma U87 cells, adenocarcinomic in vivo.13,14 In recent years, many fluorophores, which possess
human alveolar basal epithelial A549 cells and cervical cancer strong NIR absorption and emission properties, have been
cells.3–5 The identification of novel carcinoma biomarkers with reported in the literature and applied in bio-imaging, including
higher specificity and sensitivity is eagerly demanded for cancer cyanine dye, coumarin, rhodamine, squaraine, BODIPY and
diagnosis and therapy. IL-6R is composed of a ligand-binding part, dicyanomethylene-4H-pyran (DCM).15–20 In 2015, Tian et al.
which is an 80 kDa molecule related directly to IL-6, and a signal- synthesized a diethylaminophenyl substituted BODIPY-based
transduction component. This component is an IL-6Rb chain, pH-activatable theranostic nanosystem.21 In addition, Choi’s
glycoprotein 130 (gp 130), whose function is to form binding sites group utilized a NIR fluorescent probe to realize tissue-specific
of IL-6 with high affinity and to transduce signals.6,7 Many cellular imaging.22 Compared with others, DCM was capable of exhibiting
functions (e.g. angiogenesis, cell mobility and differentiation) are better photo-stability, which was more suitable for application in
affected by the mediation of IL-6 signaling via gp 130, which are biological samples.16,23–26 However, DCM-type derivatives have
greatly involved in the pathogenesis of several human cancers.8,9 seldom been developed for tumor-targeting.
Recently, a heptapeptide I6P7 (N to C terminus: LSLITRL) has been
With this goal in mind, a novel DCM derivative fluorescent
designed and synthesized to inhibit the binding between IL-6 and probe (DMP) decorated with I6P7 for bio-imaging was designed
and synthesized. It exhibited a strong absorption band at 420 nm
a Department of Biomedical Engineering, School of Engineering,
China Pharmaceutical University, 24 Tongjia Lane, Gulou District,
Nanjing, 210009, China. E-mail: chenhaiyan@cpu.edu.cn
b JiangSu XinHai Senior High School, No. 58 Cangwu Road, Haizhou District,
Lianyungang, 222006, China. E-mail: 2532267562@qq.com
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8nj04255a
and an emission peak at 705 nm. DMP showed good biocom-
patibility in tumor cells and good solubility in water. The I6P7
peptide acts as a tumor targeting ligand, increasing the ability
of the probe to specifically target tumor cells. Moreover, the
targeting ability was validated using multiple cell lines. The
results revealed the feasibility of DMP for targeting IL-6R and
promising application in bio-imaging.
‡ These authors contributed equally to this work.
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Results and discussion
Design and synthesis of DMP
A novel NIR fluorescent probe capable of bio-imaging was
designed and synthesized (Scheme 1). DMP consists of a fluo-
rescence reporting group (a DCM dye) and a tumor-targeting
group (I6P7). The fluorescence reporting group was conjugated
with I6P7 through monomethyl esterification with glutaric
anhydride with a satisfactory yield of 19.76%. All the structures
1
of the compounds concerned above were validated by H NMR
and MALDI-TOF-MS (Fig. S2–S7, ESI†).
Optical response of I6P7 to DCM-L
Fig. 2 (A) Absorbance and (B) fluorescence spectra of compound DMP
(10 mM) in PBS buffer with different pHs (pH = 4, 5, 6, 7, 8) and saline
containing 1% DMSO. (C) Absorbance and (D) fluorescence spectra of
compound DMP (10 mM) in different solutions (H2O, saline, PBS, CH2Cl2,
CH3OH, DMF) containing 1% DMSO. lex = 420 nm.
A spectroscopic experiment was performed in PBS buffer (pH =
7.4, 10 mM) with 50% DMSO (compound 1 and DCM-L) or 10%
DMSO (DMP) at 37 1C. As shown in Fig. 1, the absorption and
fluorescence spectra of compound 1, DCM-L and DMP were
investigated. The absorbance spectra of compound 1 and DCM-L
show an absorption band from 500 nm to 600 nm with an
absorption peak at 555 nm (Fig. 1A). The fluorescence spectra
were extended to a wide range from 600 nm to 850 nm with a
peak at 705 nm (Fig. 1B). And DMP exhibited a new absorption
at 420 nm and two fluorescence peaks extending from 650 to
DMP is steady at pH values ranging from 4 to 8. The absorbance
and fluorescence spectra of DMP in different solutions further
illustrated that it can present better photophysical properties in
inorganic solutions, which supported the conclusion above.
720 nm. Compared with 1 and DCM-L, DMP exhibits a larger Cytotoxicity assay
Stokes shift, which can improve detection sensitivity. Further-
In order to evaluate the cell affinity of DMP, the biocompati-
more, the difference of dissolution systems between DMP
(10% DMSO) and 1 and DCM-L (50% DMSO) demonstrated
that DMP had better solubility in the aqueous phase. The
changes could be due to the I6P7 peptide.
The stability of probe DMP at different pH values and in
different solutions was further investigated (Fig. 2). As indicated
in Fig. 2A, the absorbance spectra and fluorescence spectra dis-
play a weak change at pH values ranging from 4 to 8. At a higher
pH value, the intensity decreases. Based on the above results,
bility was investigated on HUVEC cells and U87 cells firstly. The
cytotoxicity of DMP was evaluated using conventional MTT
assays (Fig. 3 and Fig. S8, ESI†). As shown in Fig. 3A, even after
being treated with a relatively high concentration of the probes
(80 mM) for 72 h, the viability of the HUVEC cells was still
maintained above 95%, which means that the probe did not
show any conspicuous cytotoxicity and had good biocompati-
bility. Excitingly, Fig. 3B illustrates that when DCM-L was con-
jugated with the I6P7 peptide, the viability of U87 cells incubated
with DMP for 72 h began to display a downward trend.
Cell affinity evaluation of DMP
For efficient tumor targeting of the NIR probes, compound
DCM-L was conjugated to the I6P7 peptide to target IL-6R. The
I6P7 sequence has high affinity for IL-6R, which is over-expressed
in many tumor cells. The schematic diagram of tumor cell
imaged by the probe DMP was shown in Scheme 2. The cell
affinity of DMP was further investigated on IL-6R over-expressed
cells (A549, MCF-7 and U87) using confocal fluorescence micro-
scopy.27–29 As shown in Fig. 4, the green channel enabled
Scheme 1 Synthesis of probe DMP.
Fig. 1 Absorbance (A) and fluorescence (B) spectra of compound 1 (10 mM)
and DCM-L (10 mM) in a DMSO/PBS solution (50/50, v/v, pH 7.4, 10 mM) and
DMP (10 mM) in PBS buffer (pH = 7.4) containing 10% DMSO. lex (1, DCM-L) =
555 nm; and lex (DMP) = 420 nm.
Fig. 3 Cell viability of HUVEC (A) and U87 (B) cells incubated with DCM-L
and DMP for 72 h, respectively. Data are expressed as mean Æ S.D. (n = 4).
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DMP showed a gradual increase with increasing time. The fluores-
cence is strongest at 2 h after incubation with DMP (Fig. 4A). To
validate the tumor targeting ability of DMP in other tumor cells, the
confocal fluorescence imaging of U87 cells and MCF-7 cells was
further performed. Similar results can be observed on U87 and
MCF-7 cells, as shown in Fig. 4D and G. The semi-quantitative
analysis of the fluorescence intensity at three different time periods
(0.5 h, 1 h and 2 h) manifests a more intuitive presentation (Fig. 4B,
E and H). Furthermore, colocalization experiments were performed
by co-staining the tumor cells to evaluate the subcellular localiza-
tion. The punctate distribution of fluorescence is consistent with
fluorescence activation occurring in the mitochondria. When the
fluorescence signal from DMP (red) showed substantial overlap
with the mitochondria stain (green), the resulting image is
yellow or orange. The fluorescent signal of the linear regions
of interest (ROI) showed a tendency to synchronize with a
Scheme 2 The schematic diagram of tumor cell imaged by the probe DMP.
Fig. 4 LCFM fluorescence images of DMP in A549, U87 and MCF-7 cells.
(A, D, and G) Fluorescence imaging of A549, U87 and MCF-7 cells stained
with DMP (80 mM, lex = 405 nm). The cells were incubated with DMP at
37 1C for 0.5 h, 1 h and 2 h. (B, E, and H) Semi-quantitative fluorescence
intensity analysis. (C, F, and I) Linear profile and scatter plot were used to
characterize the overlap degree of red fluorescence from DMP and green
fluorescence from MitoTracker Green. Scale bar: 20 mm.
Fig. 5 LCFM fluorescence images of the probes in A549, U87 and MCF-7
cells. (A, C and E) Fluorescence imaging of A549, U87 and MCF-7 cells
stained with DMP (80 mM, lex = 405 nm) and DCM-L (80 mM). The cells
were incubated with DMP and DCM-L at 37 1C for 2 h. The blocking
experiments were carried out by adding a free I6P7 peptide (80 mM) into the
cells prior to DMP incubation. (B, D and F) Semi-quantitative fluorescence
visualization of the mitochondria using MitoTracker Green and the
red channel enabled visualization using DMP. A time-dependent
investigation was further carried out. The fluorescence intensity of intensity analysis. Scale bar: 20 mm.
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from Nanjing Peptide Co. Ltd (Jiangsu, China). Mito-Tracker
Green was purchased from KeyGen BioTech (Jiangsu, China).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum
(FBS), penicillin, streptomycin, and trypsin–EDTA were purchased
from the Institute of Biochemistry and Cell Biology, SIBS, CAS
(China). All other chemicals were supplied by J&K Scientific Ltd
(Beijing, China) or Sinopharm chemical reagent Co. Ltd (Shanghai,
China) and utilized without further purification. Ultrapure water
from a Milli-Q reference system (Millipore) was utilized in all
experiments. Silica gel (100–200 mesh) was used for silica column
chromatography.
Fig. 6 LCFM fluorescence images of DMP in HUVEC cells. (A) Fluores-
cence imaging of HUVEC cells stained with DMP (100 mM, lex = 405 nm).
The cells were incubated with DMP at 37 1C for 0.5 h, 1 h and 2 h. (B) Semi-
quantitative fluorescent intensity analysis. Scale bar: 20 mm.
Nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker Avance III-300 instrument (Bruker, Billerica, MA, USA)
1
at 500 MHz for H NMR, and TMS was utilized as an internal
Pearson co-localization coefficient of 0.70 for A549 cells, 0.70 for
U87 and 0.68 for MCF-7 cultivated with DMP (Fig. 4C, F and I). The
results shown above indicate the capability of DMP to selectively
target the mitochondria in tumor cells rather than other organelles.
To further verify the tumor cell affinity of the probe, different
tumor cells were incubated with DMP and DCM-L. As shown in
Fig. 5, the fluorescence images of A549, U87 and MCF-7 cells
incubated with DMP and DCM-L for 2 hours were acquired.
A549, U87, and MCF-7 cells incubated with DMP emitted strong
fluorescence signals, compared with the DCM-L group, which
indicated that the introduction of the I6P7 peptide increased
the tumor cell affinity of DMP. Furthermore, when the free I6P7
peptide was added before incubation with DMP, the tumor cells
exhibited slight fluorescence, which indicated that the free I6P7
peptide competed with DMP for binding IL-6R.
Furthermore, the fluorescence signals of DMP within the
mitochondria were also investigated in HUVEC cells. The result
depicted in Fig. 6 indicates that fairly weak fluorescence was
observed in HUVEC cells after incubation with DMP for 2 hours.
The semi-quantitative analysis also indicated that no distinct fluores-
cence enhancement appeared as the time extended (Fig. 6B). These
results also verified the important role of the I6P7 peptide in terms of
the tumor-targeting capability of the probe.
reference. Mass spectra were collected on a tandem quadrupole
mass spectrometer (Waters, Milford, MA, USA) with a MALDI-
TOF-MS resource and an AB SCIEX 5800 matrix-assisted
laser desorption ionization time-of-flight (MALDI-TOF) mass
spectrometer (SCIEX, Beijing, China), respectively. Absorption
spectra were recorded on a Lambda 25 UV-vis spectrophoto-
meter (PerkinElmer, USA), and fluorescence spectra were obtained
by using a PerkinElmer-LS55 spectrophotometer (PerkinElmer, USA).
Synthesis of DMP and DCM-L
Compound 1, (E)-2-(2-(4-hydroxystyryl)-4H-chromen-4-ylidene)
malononitrile, was synthesized according to the reported
method.30 The synthesis routine is shown in Fig. S1 (ESI†).
Compounds DCM-L and DMP were fabricated by the following
method (Scheme 1). Compound DCM-L: compound 1 (800.00 mg,
2.56 mmol, 1 equiv.) was dissolved in 10 mL anhydrous
N,N-dimethylformamide (DMF). Then dihydro-2H-pyran-2,6
(3H)-dione (1.46 g, 12.81 mmol, 5 equiv.) and N,N-dimethyl-
pyridin-4-amine (DMAP) (625.86 mg, 5.12 mmol, 2 equiv.) were
added followed by dropwise addition of triethylamine (TEA)
(1.30 g, 12.81 mmol, 5 equiv.) under nitrogen protection at
room temperature. The resulting mixture was stirred overnight.
The solvent was removed under vacuum and further purified by
silica gel column chromatography to afford the compound
1
DCM-L as a yellow solid: yield 352 mg, 44%. H NMR (500 MHz,
Conclusions
DMSO-d6) d 12.09 (s, 1H), 8.75 (s, 1H), 7.93 (d, J = 10 Hz, 1H),
7.89–7.72 (m, 3H), 7.65 (m, 3H), 7.28 (d, J = 16 Hz, 1H), 6.97
(d, J = 16 Hz, 1H), 6.88 (d, J = 5 Hz, 1H), 2.73 (t, J = 5 Hz, 2H),
2.41–2.32 (m, 2H), 1.94–1.85 (m, 2H). 13C NMR (125 MHz,
DMSO-d6), d (ppm): 174.45, 171.75, 158.38, 153.32, 152.44,
152.32, 138.00, 135.92, 133.10, 129.82, 126.65, 125.11, 123.00,
120.22, 119.52, 117.52, 116.20, 107.31, 61.01, 33.24, 33.08, 20.28.
MALDI-TOF-MS calculated for C25H18N2O5: 426.43, found 425.0754.
Compound DMP. Compound DCM-L (100.00 mg, 234.51 mmol,
1 equiv.) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo
[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (183.05 mg,
351.77 mmol, 1.5 equiv.) were dissolved in 5 mL DMF with
N-ethyldiisopropylamine (DIPEA) (45.46 mg, 351.77 mmol,
In summary, we have developed an effective fluorescent probe
(DMP) with a targeted peptide. The probe shows low cytotoxicity
and excellent photophysical properties. Moreover, the targeting
ability is also verified in several kinds of cells. These experimental
results show the feasibility of targeting IL-6R and its potential use as
a small molecular probe in future. However, as IL-6/IL-6R exists in
many diseases, the probe cannot target tumors exactly. An in vivo
experiment should be carried out to verify the target ability in future.
Experimental
Reagents and instruments
1-(2-Hydroxyphenyl)ethanone, malononitrile and 4-hydroxy- 1.5 equiv.). The peptide I6P7 was added after the mixture was
benzaldehyde were purchased from Shanghai Macklin Bio- stirred at room temperature for 10 min. The resulting mixture
chemical Co. Ltd (Shanghai, China). Peptide I6P7 was obtained was stirred at room temperature for 30 min. Then the reaction
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mixture was recrystallized with diethyl ether three times to cytotoxicity analysis. The following formula was used to calcu-
generate compound DMP: yield 56.70 mg, 19.76%. 1H NMR late the viability of cell growth: viability (%) = (mean absorbance
(500 MHz, DMSO-d6) d 12.80 (s, 1H), 8.76 (s, 1H), 8.67 (s, 1H), of the test wells À mean absorbance of the medium control
8.45 (s, 1H), 8.13 (s, 2H), 8.03 (s, 1H), 7.97–7.92 (m, 1H), 7.87– wells)/(mean absorbance of the untreated wells À mean absor-
7.77 (m, 4H), 7.64 (d, J = 7.5 Hz, 1H), 7.58–7.40 (m, 3H), 7.27 (d, bance of the medium control wells) Â 100%.
J = 15 Hz, 2H), 7.06 (d, J = 16 Hz, 1H), 6.76 (s, 2H), 4.99 (s, 1H),
Fluorescence imaging cell analysis using the probes
4.43–4.14 (m, 7H), 3.96 (s, 1H), 3.59 (m, 4H), 3.09 (m, 2H), 2.71
(d, J = 25 Hz, 2H), 2.61 (m, 1H), 2.51 (s, 1H), 2.28 (s, 2H), 1.88
(m, 2H), 1.80–1.32 (m, 15H), 1.27–1.01 (m, 6H), 0.95–0.62 (m,
21H). 13C NMR (125 MHz, DMSO-d6) d 174.30, 173.02, 172.36,
172.33, 171.78, 171.51, 170.45, 169.92, 162.7, 158.44, 157.13,
153.44, 152.51, 152.35, 138.05, 135.97, 133.12, 130.83, 129.82,
126.70, 123.03, 120.27, 119.56, 117.58, 116.53, 116.38, 116.24,
107.35, 106.17, 67.02, 61.89, 61.01, 58.49, 57.49, 55.58, 53.97,
52.29, 51.76, 50.80, 42.25, 41.13, 40.98, 38.72, 36.71, 36.26,
34.43, 33.36, 31.26, 25.24, 24.70, 24.57, 23.58, 23.51, 23.33,
21.96, 21.74, 19.71, 15.81, 11.44. MALDI-TOF-MS calculated
for C62H86N12O14: 1222.64, found 1223.6.
A549, MCF-7 and U87 cells, which are known to overexpress
IL-6R under hypoxic conditions, are selected to assess the applic-
ability of DMP in intracellular IL-6R monitoring. The cells were
seeded in laser scanning confocal microscope (LSCM) culture
dishes at a density of 5 Â 105 cells per well and subsequently
incubated at 37 1C in a humidified atmosphere containing 5%
CO2. When the whole cells took up 70–80% space of the culture
dishes, the cells were treated with DMP (100 mM) in DMSO/PBS
buffer (1 : 99, v/v) for 30 min, 60 min and 90 min, respectively.
For mitochondrial staining, the cells were stained with Mito-
Tracker Green (1.0 mM) for 30 min and then washed with PBS
buffer three times to remove the free dye. Blocking experiments
were carried out, in which the cells were incubated with free
I6P7 peptide for 0.5 h prior to incubation with DMP. Fluores-
cence images were recorded on an FV1000 confocal fluores-
cence microscope (Olympus, Japan). The fluorescence signal
was collected at the NIR fluorescence channel (680 Æ 30 nm,
lex = 420 nm), and green fluorescence channel (516 Æ 30 nm,
lex = 490 nm), respectively.
In vitro characterization
UV-vis and fluorescence measurements of compound 1 and
DCM-L were carried out in a PBS solution containing 50%
dimethyl sulfoxide (DMSO), pH 7.4. Fluorescence spectra were
recorded in the range of 500 to 800 nm under irradiation with a
laser. Stock solutions of 1, DCM-L and DMP (1 mM) in DMSO
were initially prepared. In a 3.0 mL tube, PBS buffer (10 mM,
pH 7.4, 2 mL) and the above stock solutions were mixed. The
final solution volume was adjusted to 3.0 mL with PBS buffer.
After rapid mixing of the solution, it was transferred to a 10 Â
10 mm quartz cell and incubated at 37 1C for in vitro detection.
Conflicts of interest
There are no conflicts to declare.
Cell culture
A549 cells (adenocarcinomic human alveolar basal epithelial
cells), MCF-7 cells (human breast adenocarcinoma cells), U87
cell (human glioma cells) and HUVEC cells (human umbilical
vein endothelial cells) were obtained from American Type
Culture Collection (ATCC, USA). The cell lines were cultivated
on glass-bottom culture dishes in an atmosphere of 5% CO2 at
37 1C in DMEM supplemented with 10% FBS and 1% (v/v)
penicillin–streptomycin.
Acknowledgements
The authors are grateful to the Natural Science Foundation
Committee of China (NSFC 81671803), the National Key Research
and Development Program (Grant No. 2017YFC0107700), the Out-
standing Youth Foundation of Jiangsu Province (GX20171114003,
BK20170030), the Fok Ying Tung Education Foundation (161033),
the ‘‘Double First-Class’’ University project (CPU2018GY06) and
the Priority Academic Program Development of Jiangsu Higher
Education Institutions for their financial support.
Cytotoxicity assay
The in vitro cytotoxicity of the probe to HUVEC and U87 cell
lines was measured by MTT assay. Briefly, the cells were loaded
in 96-well culture plates at 7000 cells per well and subsequently
incubated for 24 h in a CO2 culture box. After culture for 24 h,
the cells were further maintained at 37 1C for 24 h under 5%
CO2 after treatment of probes DCM-L and DMP (100 mL per well)
at a wide concentration range from 0 to 80 mM. Then to each
well was added MTT (20 mL, 5.0 mg mLÀ1) and the cells were
incubated for another 4 h. The medium containing MTT was
then carefully removed and 150 mL of DMSO was added into
each well. The plates were gently shaken for 15 min at room
temperature before the absorbance measurements. The absor-
bance at 490 nm was measured in a 96-well microplate reader.
Seven independent experiments were carried out for in vitro
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