Glycine-conjugated porphyrin fluorescent probe with iRGD for live cell imaging

Article abstract of DOI:10.1016/j.cclet.2017.03.001

A porphyrin modified by glycine has been synthesized and developed as a near-infrared (NIR) fluorescence probe to detect tumor. Porphyrins’ longwavelength emission at ～650?nm can efficiently avoid the spectral crosstalk with Spontaneous fluorescence in th

Full text of DOI:10.1016/j.cclet.2017.03.001

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Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Glycine-conjugated porphyrin fluorescent probe with iRGD for live cell
imaging
Qian Zhanga,b, Xue Dong , Kun-Peng Wanga,b, Ting-Ting Zhua,b, Feng-Nan Suna,b
,
a,b
a,b,
a,b
Shu-Xian Meng *, Ya-Qing Feng
a
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 3 January 2017
Received in revised form 13 January 2017
Accepted 2 March 2017
Available online xxx
A porphyrin modified by glycine has been synthesized and developed as a near-infrared (NIR)
fluorescence probe to detect tumor. Porphyrins’ longwavelength emission at ꢀ650 nm can efficiently
avoid the spectral crosstalk with Spontaneous fluorescence in the visible light region. A disulfide-based
cyclic RGD peptide named iRGD c (CRGDKGPDC), a tumor homing peptide, harbors a cryptic C-end Rule
(
CendR) motif that is responsible for neuropilin-1 (NRP-1) binding and for triggering extravasation and
Keywords:
Fluorescence probe
iRGD
Glycine
Porphyrin
Near-infrared
tumor penetration of the peptide to improve the imaging sensitivity and therapeutic efficacy. We used N
À hydroxy succinimide as an activator to introduce the glycine methyl ester to detect tumor. We got a
porphyrin modified by glycine. The affinity between probe and tumor cell entered GLC-82 cells (human
glandular lung cancer cell line) can be observed by Confocal Microscope. The toxicity of probe has been
identified by MTT Assay. The summary has been gotten that the porphyrins were nontoxic to GLC-82 cells
and glycine modified porphyrin has a good affinity with GLC-82 cells under the iRGD function by our
experiment.
©
2017 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
1. Introduction
probes, targeting of tumors. Up to now, the common NIR
fluorophores only have cyanine dyes, such as Cy5 and Cy7 [7,8].
Tumor is a great life threat to human. The past decade we have
Porphyrins [9], due to the unique properties of photophysical
and photochemical, have a special relevance to photodiagnosis
(PD) and in photodynamic therapy (PDT) of oncological and non
oncological diseases [10]. Moreover, we used porphyrins as
fluorescence probes because they are relatively stable and have
good photostability [11].
witnessed the burgeoning of optical imaging and its wide
applications for biomedicines such as genomics, proteomics, cell
biology, and drug discovery [1].
As we all know, early-stage tumor has been detected by the
invention of kinds of probes, especially the invention of
radiationless fluorescence probes [2,3]. Fluorescence probe which
can be used to improve tumor early detection is still a popular topic
among related experts and researchers. Recently, the near-infrared
NIR) fluorescence probes are increasingly popular in biological
imaging and sensing for the reason that longwavelength
650–900 nm) excitation and emission have the advantages of
minimum photodamage to biological samples, deep tissue
penetration, and minimum interference from background to
fluorescence by biomolecules in the living systems [4–6]. However,
the development of NIR fluorescence probes has some troubles,
such as photostabilities and fluorescence quantum yields of
Tumor-penetrating internalizing RGD peptide (iRGD) is known
to have a relatively high and specific affinity for
avb
3 À integrin
receptor [12–14], which is increased vascular and tissue
permeability in a tumor-specific and neuropilin-1-depenendent
manner, allowing co-administered drugs to penetrate into
extravascular tumor tissue. The CendR motif, a class of cell and
tissue-penetrating peptides with a specific R-x-x-R carboxyl-
terminal motif, is not active unless it occupies a C-terminal
position in the peptide after the cleavage, recognizing neuropillin-
1(the receptor for the CendR peptides) and facilitating the
penetration into the tumor of iRGD and co-administered drugs
(
(
[
15].
In this study, the iRGD [16], which having tumor-penetrating
ability and good cell membrane permeability, was used to improve
*
Corresponding author.
E-mail address: msxmail@tju.edu.cn (S.-X. Meng).
http://dx.doi.org/10.1016/j.cclet.2017.03.001
001-8417/© 2017 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
1
Please cite this article in press as: Q. Zhang, et al., Glycine-conjugated porphyrin fluorescent probe with iRGD for live cell imaging, Chin. Chem.
Lett. (2017), http://dx.doi.org/10.1016/j.cclet.2017.03.001

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CCLET 3996 No. of Pages 5
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Q. Zhang et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
the permeability of cell membrane firstly, and then the fluorescent
2.4. MTT assay
probe TPP-glycine was apt to enter into cells.
MTT assay [19] can be used to detect the toxicity of cells. The
results in Fig. 4a–c showed that the cell viability decreased with
the increase of concentration of probe. Compared with TPP-glycine
and TPP, the toxicity of TPP-COOH is obviously bigger than the
2
. Results and discussion
2.1. The synthesis of TPP-glycine
others when the concentration of probes is higher than 5 mmol/L.
The synthetic process of TPP-glycine was shown in Scheme 1.
The date has showed that added iRGD has no significant influence
The TPP and TPP-glycine were identified by analyzing the
on cells. In a practical application, the safe concentration of probes
absorption of hydrogen in Fig. 1.
for cells are lesser than 1
viability are greater than 90% when the concentration of probes is
mmol/L. From Fig. 4, the values of cell
1
3
H NMR (400 MHz, CDCl ): d 8.91 (6H), 8.83 (d, 2H, J = 4.7 Hz),
8
7
.48 (d, 2H, J = 8.0 Hz), 8.35 (d, 2H, J = 8.0 Hz), 8.26 (3H), 8.25 (3H),
.85–7.80 (m, 6H), 7.80–7.76 (m, 4H), 4.15 (s, 3H), À2.73 (s, 2H).
MALDI-TOF MS C46 32N4O calcd. for 672.25, found 672.21.
H NMR (400 MHz, CDCl ): 8.91–8.74 (m, 8H), 8.34–8.17
m, 10H, ArH), 7.83–7.70 (m, 9H, ArH), 4.44 (d, 2H, CH ), 3.89 (s, 3H,
CH calcd. for 729.27,
), À2.78 (s, 2H). MALDI-TOF MS C48
found 730.26.
lesser than 1
tumor cells.
mmol/L. Therefore, the probes can be applied to detect
H
2
1
3
d
3. Conclusion
(
2
3
35
H N
5
O
3
In this paper, TPP-glycine was synthesized and characterized by
UV–spectrophotometer, fluorescence spectrophotometer, fluores-
cence microscope, and MTT assay. Experimental results showed
that the porphyrins modified with glycine methyl ester had a good
affinity with GLC-82 cells than other porphyrins when interacted
with iRGD. And it also indicated that iRGD can penetrate GLC-82
cell membranes contribute to enter into cells of fluorescent probe
TPP-glycine.
2.2. The optical properties of TPP, TPP-COOH, TPP-glycine
The absorption, emission intensity and fluorescence quantum
yield results were displayed in Fig. 2 and Table 1, respectively.
These results indicate that introduced carboxyl group and
further glycine methyl ester has no significant influence on the
optical properties of the porphyrin core.
4
. Experimental
2.3. In vitro imaging
4
.1. Materials
As shown in Fig. 3, the aggregation degree of TPP and TPP-
glycine had no obvious change. However, aggregation degree of
TPP-glycine D4 is much stronger than TPP D2 when interacted with
iRGD [17]. Therefore, iRGD has the function which promotes probes
to penetrate GLC-82 cytomembrane easily. The results were
identical with those reported in literature [18].
Silica gel (200–300 mesh, 300–400 mesh), Liquid NMR (Bruker
AVANCE III 400 M), MALDI-TOF-MS (Bruker Autoflex tof/tof III),
UV–vis spectrophotometer (Shimadzu UV-1800), Fluorescence
spectrophotometer (Varian Cary Eclipse), Transient Fluorescence
(Horiba JY Fluorolog-3), Flow Cytometry (FACScalibur).
Scheme 1. Synthesis of the probe.
Please cite this article in press as: Q. Zhang, et al., Glycine-conjugated porphyrin fluorescent probe with iRGD for live cell imaging, Chin. Chem.
Lett. (2017), http://dx.doi.org/10.1016/j.cclet.2017.03.001

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3
Fig. 1. 1H NMR spectra of TPP (left) and TPP-glycine (right).
Fig. 2. Absorption (a) and fluorescence (b) spectra of TPP, TPP-COOH, TPP-glycine in CH
2
Cl
2
solution (3 Â10À6 mol/L).
Table 1
Absorption wavelength, B band-excited fluorescence maxima of the porphyrins,
fluorescence quantum yield and fluorescence lifetime in CH Cl solution.
(0.9 mmol, 200 mg) was added. After the solution was further
stirred at room temperature for 1 h. NaHCO (106 mg, 1 mmol) was
added and the solvent was removed from reduced pressure. The
product was isolated by column chromatography (silicagel) using
2
2
3
em (nm) a
b
Porphrins
labs(nm)
l
F
f
t (ns)
TPP
TPP-COOH
TPP-glycine
417
418
418
651
651
652
0.11
0.102
0.103
8.42
8.50
8.49
2 2
CH Cl /hexane = 1/2 as eluent. Next, A mixture of TPP (0.1 mmol)
and KOH (2 mmol) in THF (5 mL), EtOH (1 mL) and water (1 mL) was
refluxed for 2 h. After the solution was cooled to room temperature,
the residue was dissolved in water (50 mL) and acidified to pH 3
a
Excitation wavelength/nm: TPP 417, TPP-COOH 418, TPP-glycine 418.
Measured at room temperature.
b
with 1 mol/L HCl solution. CH
was collected. The solvent was removed in vacuo. Recrystallization
from CH Cl gave TPP-COOH (0.95 mmol, 95%) C45 calcd.
2 2
Cl was added and the organic layer
Pyrrole(AR, Nanjin Tianzun Chemical Ltd.), methyl p-formyl-
2
2
30 4 2
H N O
benzoate (AR, Nanjin Tianzun Chemical Ltd.), trifluoroacetate (AR,
Northern Chemical Reagent Chemical Ltd), benzaldehyde (AR,
Tianjin Guangfu Chemical Limited Corporation), DMF (AR, Tianjin
Guangfu Chemical Ltd.), iRGD (96%, GL Biochem Ltd.).
for 658.24, found 659.20.
ꢁ
The EDC HCl (1.53 mg, 8 mmol) and NHS (1.15 mg, 10 mmol)
were added to the solution of TPP-COOH (2.63 mg,5 mmol) in
anhydrous DMF (2 mL). The mixture was stirred at room
temperature for 12 h. And then glycine (2.4 mg, 4 equiv.) was
4
.2. Synthesis of TTP-glycine
dissolved in anhydrous DMF (2 mL) and a large excess of K
added. The reaction was kept for 12 h at room temperature and
then poured into CH Cl (30 mL), washed with water. The product
was collected by filtration and purified by flash chromatography
CH Cl ) to obtain the final TPP-glycine as a solid.
2 3
CO was
A solution of phenyl pyrrolidine (146.2 mg, 1 mmol), methyl
-formylbenzoate (82.1 mg, 0.5 mmol), and aromatic aldehyde
2
2
4
(
4
0.5 mmol) in CH
2
Cl
2 3
(100 mL) were added F CCOOH (0.6 mmol,
(
2
2
4 mL). The mixture was stirred in the dark for 3 h and then DDQ
Please cite this article in press as: Q. Zhang, et al., Glycine-conjugated porphyrin fluorescent probe with iRGD for live cell imaging, Chin. Chem.
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Fig. 3. Fluorescence microscope images of living GLC-82 cells. (B1) GLC-82 cells; (B2) GLC-82 cells were incubated with TPP for 1 h; (B3) GLC-82 cells were incubated with
TPP-COOH for 1 h; (B4) GLC-82 cells were incubated with TPP-glycine for 1 h. (D1–D4): GLC-82 cells were pre-incubated with iRGD for 1 h, and then treated with the probe
(
D1-none, D2-TPP, D3- TPP-COOH, D4-TPP-glycine) for 1 h. For DAPI and BODIPY, the excitation wavelength is set as 488 nm and 633 nm, respectively.
Fig. 4. The MTT assay results of TPP (a), TPP-COOH (b) and TPP-glycine (c).
4
.3. Measurement of fluorescence quantum yield
wells, added anti-fade fluorescence mounting medium and fixed
on the glass slides with transparent nail polish. The made-samples
were measured with Confocal Microscope (Ultra View Vox).
À6
The concentration 3 Â10 mol/L of TPP, TPP-COOH, or TPP-
2 2
glycine were prepared which the porphyrins dissolved in CH Cl
solution. And then the values of UV absorption and emission
intensity were tested by UV–spectrophotometer and fluorescence
spectrophotometer.
4.5. MTT assay
Cell viability measurements were performed by MTT analysis.
4
Cells were plated at a density of 5 Â10 cells each well into
ꢂ
4.4. Cell culture and in vitro imaging
96-multiwell plate for 24 h in the incubator (37 C, 5% CO
2
). Making
up 2
TPP–glycine. Concentrations were as follows: 10
mol/L, 10 nm, 100 nm. First, adding 100 L iRGD into cells and
m
mol/L iRGD and different concentration TPP, TPP À COOH,
The GLC-82 cells were cultured with RPMI 1640 medium in the
m
mol/L, 5 mol/L,
m
ꢂ
incubator (37 C, 5% CO
2
). The cultured cells were washed by PBS,
1
m
m
and added trypsin. Then put them into the 15 mL centrifuge tube.
Finally, centrifuging (500 rpm, 5 min), making suspension of cells
and counting cells.
cultured for 1 h. Next, different concentration TPP, TPP À COOH,
TPP À glycine were added to the wells which including iRGD and
wells without iRGD, respectively. After 48 h, 10 mL MTT (5 mg/mL)
Cover glasses were plated into 48-multiwell plate, and then a
was added into every single well and incubation was continued for
4
ꢂ
density of 5 Â10 cells each well were plated and grown on cover
4 h at 37 C. Then, 90
m
L DMSO was added to single wells to
ꢂ
glasses, cultured 24 h in the incubator (37 C, 5% CO
2
). After 24 h,
dissolve the blue formazan in the live cells. Finally, the value of OD
were measured at 490 nm using a microplate reader (Thermo
Scientific).
the iRGD (2
firstly. Next 2
wells which including iRGD and no iRGD, respectively and cultured
h. Then the cells were washed by PBS three times and added
m
mol/L) was plated in single well and cultured 1 h
m
mol/L TPP, TPP-COOH, TPP-glycine was plated in
1
Acknowledgments
paraformaldehyde to fix cells for room temperature 5 min and
washed by PBS three times again. And then cellular nuclei were
stained by DAPI for room temperature 5 min. The DAPI was washed
by PBS three times. Finally, the cover glasses were taken out from
This work is supported by National Natural Science Foundation
of China (No. 21076147). We are grateful to Prof. H. Huang and Prof.
L. Yang for providing GLC-82 cells and some support.
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5
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Lett. (2017), http://dx.doi.org/10.1016/j.cclet.2017.03.001