Synthesis and characterization of a novel near-infrared fluorescent probe for living cells imaging

Article abstract of DOI:10.1166/jnn.2020.16942

Currently, optical probes with near-infrared (NIR) fluorescence are of great interest in chemical biology. In the present study, we designed and synthesized a novel NIR fluorescent probe, IR789. IR789 has high selectivity and sensitivity for living cells imaging. The stronger excitation and emission characteristics suggested its dominant optical properties over ICG. IR789 also showed a high affinity and inconspicuous cytotoxicity at the cellular level. The results of fluorescent image in living A549 cells (human lung adenocarcinoma epithelial cell line) further demonstrated its potential applications for biomedical diagnosis in biological systems utilization of nanotechnology.

Full text of DOI:10.1166/jnn.2020.16942

Article
Journal of
Nanoscience and Nanotechnology
Vol. 20, 668–672, 2020
Copyright © 2020 American Scientific Publishers
All rights reserved
www.aspbs.com/jnn
Printed in the United States of America
Synthesis and Characterization of a Novel Near-Infrared
Fluorescent Probe for Living Cells Imaging
Jing Xing1ꢀ2, Zhe Wang3, Chunlong Sun4, and Min Ji1ꢀ2ꢀ∗
1School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China
2School of Biological Science and Medical Engineering & Collaborative Innovation Center of Suzhou Nano Science and Technology,
Southeast University, Suzhou 215123, China
3Emergency Department, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China
4College of Life Sciences, Binzhou University, Binzhou 256600, China
Currently, optical probes with near-infrared (NIR) fluorescence are of great interest in chemical
biology. In the present study, we designed and synthesized a novel NIR fluorescent probe, IR789.
IR789 has high selectivity and sensitivity for living cells imaging. The stronger excitation and emis-
sion characteristics suggested its dominant optical properties over ICG. IR789 also showed a high
affinity and inconspicuous cytotoxicity at the cellular level. The results of fluorescent image in liv-
ing A549 cells (human lung adenocarcinoma epithelial cell line) further demonstrated its potential
applications for biomedical diagnosis in biological systems utilization of nanotechnology.
Keywords: Near-Infrared Fluorescence Imaging, Human Lung Adenocarcinoma Epithelial Cell
IP: 146.185.206.14 On: Fri, 20 Dec 2019 04:10:12
Line, Fluorescent Probe, Indocyanine Green, IR789.
Copyright: American Scientific Publishers
Delivered by Ingenta
1. INTRODUCTION
ICG has been used clinically to estimate cardiac output,
liver function, and retinal angiography. Additionally, it has
been intensively investigated as a potential photo-sensitizer
by a number of research groups since its approval by the
U.S. FDA in 1954 [14–17]. However; ICG is accompa-
nied with several major shortcomings in imaging applica-
tion. For example, low quantum yield, optical instability
in the body [18–21], a tendency to aggregate and degrade
rapidly in aqueous solution [22], and unrestrained leakage
in blood vessels [23]. All these defects limit its applica-
tion in in vivo imaging. Therefore, intense investigation is
required to improve the brightness, solubility and optical
stability for in vivo imaging. A number of research groups
have been addressing in the design and synthesis of ICG
derivatives [24–29].
In this paper, we designed and synthesized a new NIR
fluorescent probe called IR789. Our strategy for design-
ing a novel near-infrared fluorescent probe is based on
the optical instability in the body, as it affects NIR imag-
ing of biological systems. Keeping these points in mind,
we chose tricarbocyanine, a near-infrared fluorescent dye
with high extinction coefficients, as the fluorophore that
could provide good photostability when they were applied
to biological systems [31]. Herein, we describe IR789 can
Application of fluorescent imaging techniques in NIR
region has received considerable attention because of their
high practicability in cell biology, pharmacology, disease
diagnosis, and other related fields [1–3]. A satisfactory
NIR fluorescence imaging needs excellent NIR probes
with superior chemical and photophysical properties [4].
The ideal NIR probe must function in NIR spectrum, i.e.,
600–900 nm, because of numerous advantages like there
is minimal inter-fering absorption and fluorescence from
biological samples, a sensitive and noninvasive imaging
effect, and an enhanced tissue penetration depth [5–7].
So far, NIR fluorescence probes have been generally clas-
sified as inorganic and organic molecules. The organic NIR
fluorescent dyes are more attractive candidates for imaging
because of the emerging NIR dyes with improved physical
properties and their availability for large-scale chemical
synthesis in comparison with inorganic dyes [8–11]. How-
ever, only a few organic NIR dyes are available currently.
Heptamethine cyanine dyes are a kind of important and
widely used organic NIR fluorescent dyes [12] and one of
the most representative is indocyanine green (ICG) [13].
∗Author to whom correspondence should be addressed.
668
J. Nanosci. Nanotechnol. 2020, Vol. 20, No. 2
1533-4880/2020/20/668/005
doi:10.1166/jnn.2020.16942

Xing et al.
Synthesis and Characterization of a Novel Near-Infrared Fluorescent Probe for Living Cells Imaging
provide good photostability with high extinction coeffi-
cients and demonstrate a promising application in biomed-
ical diagnosis utilization of nanotechnology.
2. MATERIALS AND METHODS
2.1. Apparatus and Chemicals
All 1H-NMR were recorded on the Bruker Avance
ACF-300/500 MHz instrument in DMSO-d6 or CDCl3.
Chemical shifts (ꢀꢁ are expressed in parts per mil-
lion (ppm) relative to TMS as an internal standard.
Molecular weight was determined through a LC/MS
system (Agilent). Potassium 2,3,3-trimethyl-3H-indole-
5-sulfonate, p-nitrobenzyl bromide, 2-chloro-1-formyl-3-
hydroxymethylenecyclohexene, and ICG were purchased
from Sigma-Aldrich (Shanghai, China). The A549 cells
(human lung adenocarcinoma epithelial cell line) was
obtained from the American Type Culture Collection. All
reactions were monitored by thin-layer chromatography
(TLC) on silica gel GF-254 glass plates. Column chro-
matography was performed with 200–300 mesh silica gel
by flash column techniques. All reagents and solvents were
of commercially available analytical grade. Solvents were
purified and dried by standard methods and distilled prior
to use when necessary.
Figure 1. 1H NMR spectra of IR787.
The compound 3 (0.74 g, 1.98 mmol) and compo-
und 4,2-chloro-1-formyl-3-hydroxymethylenecyclohexene
(0.17 g, 0.99 mmol), were dissolved in 30 mL of the
mixture of butyl alcohol and toluene (7:3, v/v). The
ꢀ
solution was shaken under argon at 75 C for 6 h, cooled
down to room temperature and filtered. The solid material
obtained was washed by 50 mL of toluene. The residue
was subjected to column chromatography on silica gel
using DCM/MeOH (12:1, v/v) as an eluent. The final
2.2. Synthesis of IR789
The synthetic route of IR789 was shown in Scheme 1.
Compound 1, potassium 2,3,3-trimethyl-3H-indole-5-
1
IP: 146.185.206.14 On: Fri,d2ar0kDgerece2n0s1o9lid04w:1a0s:1I2R789 (0.31 g, 35.4%); H-NMR
Copyright: American Scientific Publishers
(400 MHz, DMSO-d6ꢁ: ꢀ (ppm) = 1ꢂ75 (s, 14H), 2.53
Delivered by Ingenta
(t, 4H), 5.71 (s, 4H), 6.34–6.38 (d, J = 14.4 Hz, 2H),
sulfonate (0.89 g, 3.21 mmol) and compound 2,
p-nitrobenzyl bromide (0.9 g, 4.17 mmol) were dissolved
in 10 mL of toluene. The solution was shaken under
7.33–7.35 (d, J = 8 Hz, 2H), 7.52–7.54 (d, J = 8.4 Hz,
4H), 7.63–7.65 (d, J = 8Hz, 2H), 7.88 (s, 2H), 8.22–8.27
(m, 6H). TOF-MS m/z: 884.2 [M–H+]− (1H NMR and
MS spectra of IR789; see Figs. 1 and 2).
ꢀ
argon at 90 C for 16 h and then cooled down to room
temperature. Vacuum filtration was used to isolate the
resulting precipitate and filtrate was evaporated and con-
centrated under vacuum. The residue was subjected to col-
umn chromatography on silica gel using EA/MeOH (1:1,
v/v) as an eluent. The reddish-brown solid obtained was
compound 3,2,3,3-trimethyl-1-(4-nitrobenzyl)-3H-indol-1-
ium-5-sulfonate (0.94 g, yield 78.3%).
2.3. Absorption Spectra
The absorption spectra were measured at room temper-
ature (RT) with a UV-visible spectrophotometer at 600–
900 nm. The probe IR789 (1 mL, 0.01 mM) and ICG
(1 mL, 0.01 mM) were added to 10 mL cuvettes and
Scheme 1. Synthetic route of IR789.
J. Nanosci. Nanotechnol. 20, 668–672, 2020
669

Synthesis and Characterization of a Novel Near-Infrared Fluorescent Probe for Living Cells Imaging
Xing et al.
Figure 2. MS spectra of IR787.
their respective absorption spectroscopy was studied in
methanol solution.
were performed in quintuplicates and every data was plot-
ted as mean values SD.
2.4. Fluorescence Analysis
2.6. Confocal Imaging of IR789 in Living Cells
The A549 cells with good cell viabilꢀity were inoculated
in 10 ꢄM of IR789 and ICG at 37 C for 30 min and
then washed thrice with PBS buffer (pH 7.4). The flores-
cent images were obtained on a confocal microscope with
an objective lens (×20). The excitation wavelength was
789 nm when the cells were incubated with IR789 while
805 nm when the cells were incubated with ICG.
The stock of probe IR789 and ICG used for fluorescence
spectral analysis were prepared in methanol. The prepara-
tion was diluted to 0.01 mM in methanol solution and were
separately added to the 10 mL cuvettes. The fluorescence
intensity of mixture was measured at ꢃex = 824 nm.
2.5. MTT Assay
Human lung adenocarcinoma epithelial cell line (A549
cells) were firstly kept in the RPMI 1640 medium, which
3. RESULTS AND DISCUSSION
3.1. The Absorption and Fluorescence Spectral
IP: 146.185.206.14 On: Fri, 20 Dec 2019 04:10:12
contains 10% (v/v) of fetal bovine serum, 1% of penicillin,
Copyright: American Scientific Publishers
and 1% of streptomycin. Then, the cells were incubated at
Delivered by IngePnrtoaperty of IR789
37 ꢀC, 95% RH and 5% of CO2. Prior to experimentation,
A549 cells were seeded in 6-well culture plates containing
different concentrations of IR789 and allowed to adhere
for 24 h. The plates were further incubated for additional
4 h after the addition of 10 ꢄL of MTT solution (5 mg.
mL−1ꢁ to each well. The supernatant was removed and
100 ꢄL of DMSO was added to each well for 15–20 min.
The absorbance (OD) was measured at 490 nm with the
plate reader (Perkin-Elmer Victor3™ꢁ. All the experiments
The absorption and emission spectra of IR789 (0.01 mM)
and ICG (0.01 mM) were examined in methanol solu-
tion. IR789 has a maximal absorption at 789 nm while
ICG has a maximal absorption at 783 nm (Fig. 3(a)). The
results showed that IR789 had much higher blue exci-
tation peak than ICG. Figure 3(b) showed that the ꢃex
of IR789 and ICG was 824 nm and 832 nm, respec-
tively and both had a similar emission peak in methanol
solution.
Figure 3. Examination of absorption and fluorescence spectra of IR789 in methanol solution. (a and b): The absorption and emission spectra of
IR789 (10 ꢄM) and ICG (10 ꢄM) in methanol solution.
670
J. Nanosci. Nanotechnol. 20, 668–672, 2020

Xing et al.
Synthesis and Characterization of a Novel Near-Infrared Fluorescent Probe for Living Cells Imaging
concluded that probe IR789 had a comparatively low cel-
lular toxicity.
3.3. Fluorescent Confocal Microscopy Image
To confirm the biological applicability of IR789, the con-
focal fluorescence images of A549 cells were measured.
The cells were incubated by IR789 or ICG at pH 7.40
and then washed three times by PBS buffer. Figures 5(a)
and (c) showed that IR789 and ICG could permeate into
the membrane. The fluorescence stability was followed in
30 min (Fig. 5). The viability of A549 cells was measured
by bright field transmission method following IR789 and
ICG (Figs. 5(b, d)) incubation and the results were consis-
Figure 4. The viability of A549 cells. The viability of A549 cells were
incubated with IR789 and ICG at the same dose in different concentra-
tions (0–120 ꢄM) in 37 ꢀC incubator for 24 h. Gray bars represent ICG,
while black bars represent IR789.
tent with Figures 5(a) and (c).
4. CONCLUSION
Near infrared fluorescent probes are of great importance
for biological applications. We developed a modified pro-
cedure for the chemical synthesis of a highly fluorescent,
novel NIR fluorescence probe (IR789) to improve the over-
all yield for potential large-scale preparation along with
its characterization of its structural, cytotoxic and optical
features. The results indicated that IR789 demonstrated
outstanding optical properties and photostability in serum,
which allowed living cells with minimal NIR background
fluorescence from the hosts to achieve excellent signal-to-
3.2. MTT Assay
Biocompatibility is always the first property to consider in
terms of the biological applications in living system [32].
Thus, we determined the cytotoxicity of probe IR789 by
MTT assay. IR789 was evaluated for its in vitro cyto-
toxic activity against A549 cells. As shown in Figure 4,
after 24 h incubation in IR789, A549 cells maintained
more than 90% of their viabilities when IR789 concentra-
tion was 10–80 ꢄM, and nearly 75% of their viabilities
when IR789 concentration was 120 ꢄM. The results of
background ratio. Furthermore, IR789 had low cytotoxic-
IP: 146.185.206.14 On: Fri, 20 Dec 2019 04:10:12
MTT assay indicated that the viability of A549 cells was
not altered even when IR789 concentration was as high
as 120 ꢄM in comparison with ICG. Therefore, it can
Copyright: American Scientific Publishers
ity to living cells and was successfully imaged in cellular
environment. The aforementioned preliminary evaluation
results indicated that IR789 has higher prospects as a bio-
compatible NIR fluorescent sensor for sensitive intracellu-
lar imaging compared with ICG.
Delivered by Ingenta
The new probe for living cells imaging may contribute
well to our understanding in cell biology, pharmacol-
ogy and disease diagnosis of biological systems. There
is opportunity to develop IR789 containing drug delivery
systems to enhance the in vivo stability and biodistribu-
tion profile of this dye allowing tumor accumulation and
resulting in better efficacy.
Acknowledgments: This research was financially sup-
ported by National Natural Science Foundation of China
General Program (81671745) and Science and Technology
Development Program of Suzhou (ZXY201412).
References and Notes
1. Escobedo, J.O., Rusin, O., Lim, S. and Strongin, R.M., 2010. NIR
dyes for bioimaging applications. Current Opinion in Chemical Biol-
ogy, 14(1), pp.64–70.
2. Xu, Y., Tian, M., Zhang, H., Xiao, Y., Hong, X. and Sun, Y.,
2018. Recent development on peptide-based probes for multi-
functional biomedical imaging. Chinese Chemical Letters, 29(7),
pp.1093–1097.
3. Wang, L., Liu, M. and Chen, Y., 2018. Carbon dots and terbium Co-
enhanced fluorescence of europium nanoparticles for cell imaging.
Journal of Biomedical Nanotechnology, 14(11), pp.1898–1905.
Figure 5. Confocal fluorescence images of A549 cells. (a) Confocal
fluorescence images of living cells A549 incubated with 10 ꢄM IR789 at
37 ꢀC for 30 min; (b) bright field image of living A549 cells; (c) confocal
fluorescence images of living cells A549 incubated with 10 ꢄM ICG at
37 ꢀC for 30 min; (d) bright field image of living A549 cells.
J. Nanosci. Nanotechnol. 20, 668–672, 2020
671

Synthesis and Characterization of a Novel Near-Infrared Fluorescent Probe for Living Cells Imaging
Xing et al.
4. Hilderbrand, S.A. and Weissleder, R., 2010. Near-infrared fluores-
cence: Application to in vivo molecular imaging. Current Opinion
in Chemical Biology, 14(1), pp.71–79.
5. Frangioni, J., 2003. In vivo near-infrared fluorescence imaging. Cur-
rent Opinion in Chemical Biology, 7(5), pp.626–634.
6. Pansare, V., Hejazi, S., Faenza, W. and Prud’homme, R.K., 2012.
Review of long-wavelength optical and NIR imaging materials: Con-
trast agents, fluorophores and multifunctional nano carriers. Chem-
istry of Materials, 24(5), pp.812–827.
7. Dip, F., Roy, M., Lo Menzo, E., Simpfendorfer, C., Szomstein, S.
and Rosenthal, R.J., 2015. Technical description and feasibility of
laparoscopic adrenal contouring using fluorescence imaging. Surgi-
cal Endoscopy, 29(3), pp.569–574.
20. Desmettre, T., Devoisselle, J.M. and Mordon, S., 2000. Fluo-
rescence properties and metabolic features of indocyanine green
(ICG) as related to angiography. Survey of Ophthalmology, 45(1),
pp.15–27.
21. Wunder, A., Tung, C.H., Muller-Ladner, U., Weissleder, R. and
Mahmood, U., 2004. In vivo imaging of protease activity in arthri-
tis: A novel approach for monitoring treatment response. Arthritis &
Rheumatology, 50(8), pp.2459–2465.
22. Manchanda, R., Fernandez-Fernandez, A., Nagesetti, A. and
McGoron, A.J., 2010. Preparation and characterization of a poly-
meric (PLGA) nanoparticulate drug delivery system with simulta-
neous incorporation of chemotherapeutic and thermo-optical agents.
Colloids and Surfaces B, 75(1), pp.260–267.
8. Zhang, J., Qiao, Z., Yang, P., Pan, J., Wang, L. and Wang, H., 2015.
Recent advances in near-infrared absorption nanomaterials as pho-
toacoustic contrast agents for biomedical imaging. Chinese Journal
Chemistry, 33(1), pp.35–52.
23. Tang, Y. and McGoron, A.J., 2009. Combined effects of laser-ICG
photothermotherapy and doxorubicin chemotherapy on ovarian can-
cer cells. Journal of Photochemistry and Photobiology B, 97(3),
pp.138–144.
9. Resch-Genger, U., Grabolle, M., Cavaliere-Jaricot, S., Nitsch-Ke, R.
and Nann, T., 2008. Quantum dots versus organic dyes as fluorescent
labels. Nature Methods, 5(9), pp.763–775.
24. Li, P., Xiao, H. and Tang, B., 2012. A near infrared fluorescent probe
for selective simultaneous detection of Fe2+ and Cl− in living cells.
Chinese Journal Chemistry, 30(9), pp.1992–1998.
10. Goncalves, M.S., 2009. Fluorescent labeling of biomolecules with
organic probes. Chemical Reviews, 109(1), pp.190–212.
11. Cho, E.C., Glaus, C., Chen, J., Welch, M.J. and Xia, Y., 2010.
Inorganic nanoparticle-based contrast agents for molecular imaging.
Trends in Molecular Medicine, 16(12), pp.561–573.
25. Sun, C., Cai, J., Chen, J., Wu, Y., Wang, P., Zhou, G., Zong, X.,
Chen, B., Lv, Y. and Ji, M., 2015. The synthesis of a novel
near-infrared fluorescent probe and its application in imaging
of living cells. Applied Biochemistry and Biotechnology, 175(3),
pp.1644–1650.
12. Luo, S., Zhang, E., Su, Y., Cheng, T. and Shi, C., 2011. A review
of NIR dyes in cancer targeting and imaging. Biomaterials, 32(29),
pp.7127–7138.
13. Zheng, C., Zheng, M., Gong, P., Jia, D., Zhang, P., Shi, B., Sheng, Z.,
Ma, Y. and Cai, L., 2012. Indocyanine green-loaded biodegrad-
able tumor targeting nanoprobes for in vitro and in vivo imaging.
Biomaterials, 33(22), pp.5603–5609.
26. Xing, J., Zhou, G., Sun, C., Zhang, H., Chen, B., Zong, X., Cai, J.
and Ji, M., 2016. Synthesis and characterization of a novel near-
infrared fluorescent probe for applications in imaging A549 cells.
Biotechnology Letters, 38(11), pp.1851–1856.
27. Sun, C., Du, W., Wang, P., Wu, Y., Wang, B., Wang, J. and Xie, W.,
2017. A novel mitochondria-targeted two-photon fluorescent probe
for dynamic and reversible detection of the redox cycles between
peroxynitrite and glutathione. Biochemical and Biophysical Research
Communications, 494(3–4), pp.518–525.
28. Sun, C., Du, W., Wang, B., Yao, Z., Liu, J., Wang, J., Xie, W.,
Wu, T., Fan, Y. and Yang, H., 2017. Synthesis of a new deoxyglu-
cose derivative modified near-infrared fluorescent probe for tumor
diagnosis. Biochemical and Biophysical Research Communications,
488(2), pp.340–347.
29. Toyota, T., Fujito, H., Suganami, A., Ouchi, T., Ooishi, A., Aoki, A.,
Onoue, K., Muraki, Y., Madono, T., Fujinami, M., Tamura, Y. and
Hayashi, H., 2014. Near-infrared-fluorescence imaging of lymph
nodes by using liposomally formulated indocyanine green deriva-
tives. Bioorganic & Medicinal Chemistry, 22(2), pp.721–727.
30. Peng, X., Song, F., Lu, E., Wang, Y., Zhou, W., Fan, J. and Gao, Y.,
2005. Heptamethine cyanine dyes with a large stokes shift and
strong fluorescence: A paradigm for excited-state intramolecular
charge transfer. Journal of the American Chemical Society, 127(12),
pp.4170–4171.
14. Ishizawa, T., Saiura, A. and Kokudo, N., 2016. Clinical application
IP: 146.185.206.14 On: Fri, 20 Dec 2019 04:10:12
of indocyanine green-fluorescence imaging during hepatectomy.
Clinical application of indocyanine green-fluorescence imaging
Copyright: American Scientific Publishers
Delivered by Ingenta
during hepatectomy. Hepatobiliary Surgery and Nutrition, 5(4),
pp.322–328.
15. Hao, S., Li, D., Ma, G., Yang, J. and Wang, G., 2013. Application of
intraoperative indocyanine green video angiography for resection of
spinal cord hemangioblastoma: Advantages and limitations. Journal
of Clinical Neuroscience, 20(9), pp.1269–1275.
16. Toru, F., Masataka, S., Tetsuya, A., Isao, H., Eiichi, O. and
Naoyuki, O., 2012. Intraoperative near-infrared fluorescence imaging
with novel indocyanine green-loaded nanocarrier for spinal metasta-
sis: A preliminary animal study. The Open Biomedical Engineering
Journal, 6, pp.80–84.
17. Tan, X., Luo, S., Wang, D., Su, Y., Cheng, T. and Shi, C., 2012.
A NIR heptamethine dye with intrinsic cancer targeting, imaging
and photosensitizing properties. Biomaterials, 33(7), pp.2230–2239.
18. Pande, A.N., Kohler, R.H., Aikawa, E., Weissleder, R. and Jaffer,
F.A., 2006. Detection of macrophage activity in atherosclerosis
in vivo using multichannel, high-resolution laser scanning fluores-
cence microscopy. Journal of Biomedical Optics, 11(2), p.021009.
19. Rudin, M., 2009. Noninvasive structural, functional, and molecular
imaging in drug development. Current Opinion in Chemical Biology,
13(3), pp.360–371.
31. Tang, B., Liu, X., Xu, K., Huang, H. and Yang, G., 2007. A dual
near-infrared pH fluorescent probe and its application in imaging of
HepG2 cells. Chemical Communications, 28(36), pp.3726–3728.
32. Funayama, T., Sakane, M., Abe, T. and Ochiai, N., 2012. Photo-
dynamic therapy with indocyanine green injection and near-infrared
light irradiation has phototoxic effects and delays paralysis in spinal
metastasis. Photomedicine and Laser Surgery, 30(1), pp.47–53.
Received: 18 December 2018. Accepted: 7 March 2019.
672
J. Nanosci. Nanotechnol. 20, 668–672, 2020