Purine-based Ir(iii) complexes for sensing viscosity of endo-plasmic reticulum with fluorescence lifetime imaging microscopy

Article abstract of DOI:10.1039/d0cc07867k

Novel purine-based iridium complexes were designed for selective determination of ER viscosity. TheIr-PHpossessed excellent ER targeting ability and could distinguish the viscosity changes under ER stress by fluorescence lifetime image microscopy (FLIM),

Full text of DOI:10.1039/d0cc07867k

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Purine-based Ir(III) complexes for sensing viscosity
of endo-plasmic reticulum with fluorescence
lifetime imaging microscopy†
Cite this: Chem. Commun., 2021,
7, 2265
5
Received 3rd December 2020,
Accepted 22nd January 2021
Xin Liu, Kun Li, * Lei Shi, Hong Zhang, Yan-Hong Liu, Hao-Yuan Wang,
Nan Wang and Xiao-Qi Yu
*
DOI: 10.1039/d0cc07867k
rsc.li/chemcomm
Novel purine-based iridium complexes were designed for selective with existing reports, many diseases are associated with the cellular
determination of ER viscosity. The Ir-PH possessed excellent ER microenvironment. Therefore, FLIM would be highly promising for
targeting ability and could distinguish the viscosity changes under studying alterations in the cellular microenvironment.
ER stress by fluorescence lifetime image microscopy (FLIM), which
Recently, iridium(III) complexes have been widely investi-
1
5
16–18
may accelerate the development of relative quantitative detection gated in oxygen sensors, photodynamic therapy (PDT),
of microenvironment changes at the subcellular level.
and bioimaging because of their excellent large Stokes
shift, long luminescence lifetime and high quantum yield.
Intracellular viscosity is closely linked to cellular processes, Disappointedly, lots of reported iridium(III) ligands often regulate
1
19,20
such as transportation, diffusion, and metabolism. Therefore, obvious cytotoxicity.
Purine that is present in biological
it is of great significance that tracking intracellular viscosity molecules has been used as anticancer agents, antiviral drugs
2
1–23
changes can help us to understand the physiological or patho- and receptor antagonists due to its biological importance.
2
,3
logical processes of cells. The endoplasmic reticulum (ER) is Although the structure of purines contains N atoms and conju-
2
+
an important organelle, which is mainly responsible for Ca
gated planes, few studies have applied it to the construction of
4
storage, protein synthesis and transportion. The accumulation fluorescent probes. Furthermore, purine has not been used to
of unfolded or excessive misfolded proteins will disturb the construct ligands to synthesise iridium(III) complexes until now.
regular function of ER and it may lead to ER stress; which may In our previous work, purine was utilized as the core structure to
eventually lead to some kinds of diseases, including Alzheimer’s build a series of AIE fluorophores performing an excellent effect in
5
,6
24–26
disease, obesity or diabetes and cancers.
Meanwhile, the cell imaging with good biocompatibility.
This also makes it
accumulation of these unfolded or misfolded proteins would possible for us to synthesize iridium(III) complexes for cell
6
certainly change the viscosity of the ER; so it is valuable to imaging.
develop probes to detect the viscosity changes of ER during ER
stress.
On the other hand, the reported iridium(III) complexes for
sensing viscosity were designed with a rotatable bond or by
1
3
27
28
Fluorescent chemosensors have been extensively developed in adding a rotor, such as Ir6, 1, and C10 (Scheme 1). Due to
the last 10 years, and have provided a good method for tracking the introduction of freely rotating rotors or bonds, a problem is
small molecules or microenvironmental changes within living that it would reduce the conjugation of the entire compound,
7–12
cells.
However, as traditional fluorescence-intensity-based and cause large energy dissipation, resulting in intense
probes, we may not completely avoid the influence of concen- responses to subtle changes. Therefore, it is urgent to develop
tration. FLIM is based on a natural property and could solve the a new strategy to solve this problem.
1
3
problem, which is often closely related to the environment, such
In the present study, we introduce purine derivatives into
14
as temperature, dielectric constant, and viscosity. Combined the iridium(III) complex Ir-PH for the first time, which can
aggregate in ER owing to its lipophilic and cationic char-
Key Laboratory of Green Chemistry and Technology, Ministry of Education,
College of Chemistry, Sichuan University, Chengdu, 610064, China.
E-mail: kli@scu.edu.cn, xqyu@scu.edu.cn
acteristics. This complex could possess viscosity-sensitive properties
without a plane or bond that could rotate individually. We specu-
lated that the reason for viscosity response may be the changes of
torsion angles between the phenyl ring and purine, and between the
pyridine ring and b-carboline; so we designed Ir-PMe-1 and Ir-PMe-2
to confirm our conjecture. We deduced that Ir-PMe-1 had a weaker
†
Electronic supplementary information (ESI) available: Synthetic schemes,
experimental procedures, characterization of all compounds, and supplementary
data. CCDC 2014009 and 2014010. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/d0cc07867k
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assigned to spin-allowed intraligand ( IL) transitions and less
intense absorption bands at 360–500 nm assigned to a mixture
of spin-allowed and spin-forbidden metal-to-ligand charge
1
3
29
transfer transitions ( MLCT/ MLCT) (Fig. S2, ESI†). Upon
excitation at 370 nm, Ir-PH and Ir-PMe-2 display strong emis-
sion at ca. 550 nm, and Ir-PMe-1 has a 20 nm red shift and
exhibits weaker emission compared with Ir-PH. This phenom-
enon confirmed our conjecture that the methyl of Ir-PMe-1
restricted the change of torsion angles between the phenyl ring
and purine and led to an increased degree of conjugation.
We firstly investigated the fluorescence spectra of all com-
plexes in various solvents. The results revealed that Ir-PH and
Ir-PMe-2 had much stronger fluorescence intensity in 99%
glycerol than other solvents, while for Ir-PMe-1 it was not
Scheme 1 Chemical structures of iridium(III) complexes for sensing obvious. As we know, glycerol–water mixtures are frequently
viscosity.
used to adjust the viscosity of a solution. To investigate the
influence of viscosity on the emission intensity of all complexes,
viscosity response and Ir-PMe-2 was consistent with Ir-PH, because fluorescence spectra were recorded in a series of viscosity
the methyl of Ir-PMe-1 would restrict the change of torsion angles gradient mixtures composed of glycerol and water at room
between the phenyl ring and purine. Actually, the experimental temperature. As shown in Fig. 2, Ir-PH exhibited very weak
results were consistent with our conjecture. We confirmed that Ir-PH fluorescence in low viscosity, while increasing the proportion
could localize to ER and Ir-PH could reflect the viscosity of dysfunc- of glycerol, the fluorescence intensity of Ir-PH enhanced drama-
tional ER using FLIM. In conclusion, our work demonstrates tically and increased 22.6-fold at 550 nm as the viscosity
the potential of revealing microenvironmental changes at the increases from 1.01 cP to 76.78 cP, which could be easily
subcellular level.
observed by the naked eye. A linear correlation was obtained
The synthetic routes for Ir-PH, Ir-PMe-1 and Ir-PMe-2 are between emission intensity and solvent viscosity. Ir-PMe-1 emission
shown in the ESI,† (Scheme S1). Ir-PH is also characterized by was enhanced 11.8-fold at 570 nm (Fig. S5, ESI†) and Ir-PMe-2 was
X-ray crystal diffraction analysis (Fig. 1C and Fig. S1, Tables S1, enhanced by 37.5-fold at 550 nm (Fig. S6, ESI†). This conclusion
S2, ESI†). We systematically studied the photophysical proper-
ties of Ir-PH, Ir-PMe-1 and Ir-PMe-2 (Table S3, ESI†). In PBS at
298 K, they showed intense absorption bands at 270–360 nm
Fig. 2 (A) Fluorescence emission intensities and (C) lifetimes of Ir-PH
(10 mM) in mixtures of water and glycerol at different viscosities (1.01, 1.11,
1.31, 1.86, 2.86, 4.48, 7.50, 14.70, 22.84, 45.38, and 76.78 cP); (B) fluores-
cence emission intensity–viscosity curves; (D) fluorescence lifetime–
Fig. 1 (A) The single crystal structure of compound PH; (B) the single
29
crystal structure of 1-Py-bC; (C) the single crystal structure of Ir-PH, the
H atoms, counterions, and solvent have been omitted for clarity; (D) the
molecular packing of Ir-PH. Carbon, hydrogen, nitrogen and iridium atoms
are shown in grey, white, blue, and red, respectively.
2
viscosity curves. (E) Pictures of Ir-PH in the mixtures of H O–glycerol of
various viscosities under 365 nm UV light.
2266 | Chem. Commun., 2021, 57, 2265À2268
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demonstrates that our complexes had a greater increase and a
higher sensitivity for viscosity. On top of that, the lifetimes (t) of
complexes were measured in the presence of various glycerol–water
mixtures. The viscosity extended gradually from 1.01 cP to 76.78 cP,
and the lifetime of Ir-PH increased from 380 ns to 1120 ns (Fig. 2C).
Furthermore, a good linear relationship was obtained between
lifetime and viscosity. (Fig. 2D). At the same time, the lifetime of
Ir-PMe-2 (341–893 ns, Fig. S6C and D, ESI†) also had a linear
relationship with viscosity, but its lifetime was shorter than that of
Ir-PH. Besides, the lifetime of Ir-PMe-1 increased from 74 ns to
406 ns, whereas lifetime–viscosity was without linear correlation
(Fig. S5C and D, ESI†), unexpectedly. In general, the lifetime
exhibited satisfactory specificity and sensitivity toward the viscosity.
Moreover, the luminescence quantum yield (F) (Table S3, ESI†) and
photostability (Fig. S14, ESI†) were measured.
The single crystal structures of PH and Ir-PH are shown in Fig. 3 (A1–A4) Confocal images of A549 cells incubated with Ir-PH
Fig. 1. Ir-PH adopted a slightly distorted octahedral geometry (2 mM, lex = 405 nm, lem = 500–600 nm) and ER-Tracker Red (500 nM,
l
ex = 543 nm, lem = 600–650 nm,). (B1–B4) Cellular colocalization
microscopy images of A549 cells incubated with Ir-PMe-1 (2 mM,
ex = 405 nm, lem = 500–600 nm) and ER Tracker Red (500 nM,
lex = 543 nm, lem = 600–650 nm). (C1–C4) Cellular colocalization
and four molecules make up a unit cell (Fig. S1, ESI†). The Ir–N
bonds (Ir–Nav = 2.029 Å) of C^N ligands were shorter than
the Ir–C bonds (Ir–Cav = 2.061 Å), while the Ir–N bonds
l
(
Ir–Nav = 2.133 Å) of N^N ligands were longer than the Ir–C bonds, microscopy images of A549 cells incubated with Ir-PM-2 (2 mM,
30
matching with the effect of carbon donors. The hexafluoropho-
sphate would have weak interaction with C–H on two adjacent
molecules (Fig. 1D). To our surprise, the torsion angles were
l
ex = 405 nm, lem = 500–600 nm) and ER Tracker Red (500 nM,
l
ex = 543 nm, lem = 600–650 nm).
different between ligands alone and ligands in complex fluorescence image between Ir-PH and ER-Tracker Red demon-
Fig. 1A–C). The torsion angle was 5.601 between the phenyl ring strated that Ir-PH exhibited specific ER staining and the Pearson
(
and purine of PH. While PH was coordinated with Ir(III), the co-localization is 0.99. On the contrary, little co-localization for Ir-PH
torsion angle changed into 10.021 and 11.791. The single crystal with Mito-Tracker Red and Lyso-Tracker deep Red (Fig. S9, ESI†)
structure of 1-Py-bC, which is a ligand of Ir-PH has been reported was observed, and the co-localization values were 0.76, and 0.59,
3
1
previously, and the torsion angle between the pyridine ring and respectively. Ir-PMe-1 and Ir-PMe-2 had analogous results, and
b-carboline was changed from 2.311 to 20.641 before and after co-localization values of Ir-PMe-1 were 0.90, 0.73, and 0.34 with
reacting with Ir(III). These changes may cause the tendency for ERTG, MTG, and LTG (Fig. S10, ESI†), respectively. Co-localization
the torsion angles of ligands interacting with Ir(III) to change to a values of Ir-PMe-2 were 0.94, 0.45, and 0.73 with ERTR, MTDR,
3
2
single ligand’s torsion angles, which may explain why the Ir-PH and LTDR (Fig. S11, ESI†), respectively. These results indicated the
has a viscosity response. Furthermore, Ir-PMe-1 and Ir-PMe-2 ER-targeting ability of Ir-PH, Ir-PMe-1 and Ir-PMe-2, especially Ir-PH.
also could verify our conjecture.
In order to confirm the specificity of viscosity response of cancer cells (HepG 2) and liver normal cells (HL-7702) (Fig. S8, ESI†).
three complexes, we tested the effects of other factors on the
In order to explore the response of Ir-PH to ER viscosity
The excellent ER-targeting of Ir-PH was also confirmed in liver
fluorescence emission intensity of Ir-PH. To eliminate the changes, we also tested the fluorescence intensity of Ir-PH
calcium ion interference effect, the metal ion interference test under ER stress in A549 cells. Tunicamycin (Tm) is a glycosyla-
was conducted, and no obvious changes of the emission tion inhibitor that obstructs the cellular biosynthesis of the
3
3
intensity were observed (Fig. S3, ESI†). To eliminate polarity- N-linked oligosaccharide, which is usually used to stimulate
effect, after measuring in various solvents, we carried out a ER. As the concentration of Tm increased, the ER stimulation
fluorescence spectrum test in water-1,4-dioxane systems of was more obvious, resulting in more unfolded protein and
different fractions (Fig. S4, ESI†), and the results revealed that greater viscosity, and so the higher the fluorescence intensity of
there was no trend between polarity and intensity.
Ir-PH became (Fig. S12, ESI†), and the result was consistent
To utilize Ir-PH, Ir-PMe-1 and Ir-PMe-2 as a cellular viscosity with the corresponding results of in vitro viscosity-sensitivity
À1
sensor, we firstly investigated their cytotoxicity in A549 cells tests. Besides, we also tested Ir-PH with Tm (50 mg mL ) at
(
MTS assay). As shown in Fig. S7 (ESI†), when A549 cells were different times (Fig. S13, ESI†), and we got the same conclusion.
treated with the three complexes at 1.25–5 mM for 24 h, a high
In our previous experiments, there was a correction between
cell viability (490%) was obtained, which indicated that they lifetime and environment viscosity of Ir-PH. For the purpose of
did not interfere with the cell proliferation and physiology monitoring the change of viscosity in ER, we used FLIM to
within the tested concentrations. After that, we desperately record the results and investigated the correction in vivo, which
wanted to know whether the compounds will target the ER, may improve the drawback of images based on intensity.
so we investigated their intracellular location by a confocal A seismic color scale was assigned to images Fig. 4; the blue
microscopy experiment in cells (Fig. 3). The overlapped color represented around or below 10 ns, while lifetimes
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2
3
4
X. Liu, W. Chi, Q. Qiao, S. V. Kokate, E. P. Cabrera, Z. Xu, X. Liu and
Y. Chang, ACS Sens., 2020, 5, 731–739.
L. Li, K. Li, M. Li, L. Shi, Y. Liu, H. Zhang, S. Pan, N. Wang, Q. Zhou
and X. Yu, Anal. Chem., 2018, 90, 5873–5878.
W. H. Chen, G. F. Luo and X. Z. Zhang, Adv. Mater., 2018,
3
1, 1802725.
5
6
T. Hosoi and K. Ozawa, Clin. Sci., 2009, 118, 19.
C. Hetz, J. M. Axten and J. B. Patterson, Nat. Chem. Biol., 2019, 15,
7
64–775.
7
8
9
K. Bi, R. Tan, R. Hao, L. Miao, Y. He, X. Wu, J. Zhang and R. Xu,
Chin. Chem. Lett., 2019, 30, 545–548.
S. Long, Q. Qiao, L. Miao and Z. Xu, Chin. Chem. Lett., 2019, 30,
573–576.
H. He, T. He, Z. Zhang, X. Xu, H. Yang, X. Qian and Y. Yang, Chin.
Chem. Lett., 2018, 29, 1497–1499.
Fig. 4 FLIM of Ir-PH with different concentrations of Tm (A) blank;
À1
À1
À1
(
B) 20 mg mL ; (C) 30 mg mL ; (D) 50 mg mL ; and (E) calculated lifetimes
1
0 H. Qin, L. Li, K. Li and X. Yu, Chin. Chem. Lett., 2019, 30,
of Ir-PH in A549 cells.
71–74.
1
1
1
1 K. K.-W. Lo, Acc. Chem. Res., 2020, 53, 32–44.
2 R. Zhang and J. Yuan, Acc. Chem. Res., 2020, 53, 1316–1329.
3 L. Hao, Z. Li, D. Zhang, L. He, W. Liu, J. Yang, C. Tan, L. Ji and
Z. Mao, Chem. Sci., 2019, 10, 1285–1293.
around or above 20 ns were red. It was obvious that with the
addition of Tm, the Ir-PH displayed slightly longer lifetimes.
The calculated average fluorescence lifetime of Ir-PH was 14 M. Zhang, M. Wen, Y. Xiong, L. Zhang and C. Tian, Chin. Chem.
Lett., 2018, 29, 1509–1512.
5 X. Li, Y. Yin, P. Gao, W. Li, H. Yan, C. Lu and Q. Zhao, Dalton Trans.,
1
1.15 Æ 0.55 ns in the A549 cells with untreated Tm. With
1
the addition of Tm, the lifetime becomes longer and longer;
2017, 46, 13802–13810.
À1
when the concentration of Tm was 50 mg mL , the calculated 16 J. S. Nam, M. Kang, J. Kang, S. Park, S. J. C. Lee, H. Kim, J. K. Seo,
O. Kwon, M. H. Lim, H. Rhee and T. Kwon, J. Am. Chem. Soc., 2016,
average lifetime was 17.3 Æ 1.2 ns. The lifetime of Ir-PH was
138, 10968–10977.
calculated using three exponentials in A549 cells. However, the
cellular environment is pretty complicated compared with
water–glycerol mixtures. Therefore, Ir-PH might be appropriate
for relative quantitative tests of ER viscosity and for the absolute
quantitative detection but a standard curve was measured firstly.
In conclusion, we have designed and successfully synthe-
sized three purine-based iridium complexes for imaging of ER.
We offered an effective method to design viscosity-sensitive
1
1
1
7 X. Li, J. Wu, L. Wang, C. He, L. Chen, Y. Jiao and C. Duan,
Angew. Chem., Int. Ed., 2020, 132, 6482–6489.
8 C. Li, Y. Wang, Y. Lu, J. Guo, C. Zhu, H. He, X. Duan, M. Pan and
C. Su, Chin. Chem. Lett., 2020, 31, 1183–1187.
9 C. Caporale, C. A. Bader, A. Sorvina, K. D. M. MaGee, B. W. Skelton,
T. A. Gillam, P. J. Wright, P. Raiteri, S. Stagni, J. L. Morrison,
S. E. Plush, D. A. Brooks and M. Massi, Chem. – Eur. J., 2017, 23,
15666–15679.
20 K. Zhang, T. Zhang, H. Wei, Q. Wu, S. Liu, Q. Zhao and W. Huang,
Chem. Sci., 2018, 9, 7236–7240.
iridium(III) complexes by using the change of torsion angles. 21 J. Calder ´o n-Arancibia, C. Espinosa-Bustos,
´
A.
Ca n˜ ete-Molina,
R. Tapia, M. Fa u´ ndez, M. Torres, A. Aguirre, M. Paulino and
C. Salas, Molecules, 2015, 20, 6808–6826.
Between them, the fluorescence intensity and lifetime of Ir-PH
displayed a regular response to the environmental viscosity.
The subcellular localization experiment results indicated that
Ir-PH could exclusively accumulate in the ER. Besides, due to its
viscosity-sensitive lifetimes, Ir-PH could distinguish the
2
2 C. Lambertucci, M. Buccioni, D. Dal Ben, S. Kachler, G. Marucci,
A. SpinaciM, A. Thomas, K. Klotze and R. Volpini, Med. Chem.
Commun., 2015, 6, 963–970.
23 M. Welsch, S. Snyder and B. Stockwell, Curr. Opin. Chem. Biol., 2010,
347–361.
changes under ER stress of cells by fluorescence lifetime image 24 L. Shi, K. Li, L. Li, S. Chen, M. Li, Q. Zhou, N. Wang and X. Yu,
microscopy, which provides a new clue for relative quantitative
Chem. Sci., 2018, 9, 8969–8974.
2
5 L. Shi, Y.-H. Liu, K. Li, A. Sharma, K.-K. Yu, M. Ji, L.-L. Li, Q. Zhou,
H. Zhang, J. S. Kim and X.-Q. Yu, Angew. Chem., Int. Ed., 2020, 59,
9962–9966.
detection of microenvironment changes at the subcellular level.
This work was financially supported by the National Natural
Science Foundation of China (No. 22077088, and 21877082)
and the Foundation from Science and Technology Department
of Sichuan Province (2020JDJQ0017). We also thank Cheng-Hui
2
2
6 L. Shi, K. Li, Y.-H. Liu, X.-Y. Liu, Q. Zhou, Q. Xu, S.-Y. Chen and
X.-Q. Yu, Chem. Commun., 2020, 56, 3661–3664.
7 P. Zhang, H. Chen, H. Huang, K. Qiu, C. Zhang, H. Chao and
Q. Zhang, Dalton Trans., 2019, 48, 3990–3997.
Li and Peng Wu from the Analytical & Testing Center at Sichuan 28 F. Liu, J. Wen, S. Chen and S. Sun, Chem. Commun., 2018, 54,
1
371–1374.
University for their helpful discussion about the FLIM imaging.
2
3
3
9 L. He, C. Tan, R. Ye, Y. Zhao, Y. Liu, Q. Zhao, L. Ji and Z. Mao,
Angew. Chem., Int. Ed., 2014, 53, 12137–12141.
0 G. Li, C. Gao, H. Xie, H. Chen, D. Liu, W. Sun, G. Chen and Z. Niu,
Chin. Chem. Lett., 2016, 27, 428–432.
1 C. Tan, S. Lai, S. Wu, S. Hu, L. Zhou, Y. Chen, M. Wang, Y. Zhu,
W. Lian, W. Peng, L. Ji and A. Xu, J. Med. Chem., 2010, 53,
Conflicts of interest
There are no conflicts to declare.
7
613–7624.
3
2 M. J. Leitl, V. A. Krylova, P. I. Djurovich, M. E. Thompson and
Notes and references
H. Yersin, J. Am. Chem. Soc., 2014, 136, 16032–16038.
1
A. B. Goins, H. Sanabria and M. N. Waxham, Biophys. J., 2008, 95, 33 D. Lee, I. Y. Kim, S. Saha and K. S. Choi, Pharmacol. Ther., 2016, 162,
5362–5373.
120–133.
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