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A mitochondrial targeting fluorescent probe based on the covalently linked anti-inflammatory drug dexamethasone and Cy7
Accepted Manuscript Title: A mitochondrial targeting fluorescent probe based on the covalently linked anti-inflammatory drug dexamethasone and Cy7 Authors: Siyuan Liu, Guangbo Ge, Weibing Dong, Xiaojun Peng, Fengyu Liu, Su-Shing Chen, Shiguo Sun PII: DOI: Reference:
S0925-4005(17)31384-9 http://dx.doi.org/doi:10.1016/j.snb.2017.07.170 SNB 22829
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
19-6-2017 20-7-2017 24-7-2017
Please cite this article as: Siyuan Liu, Guangbo Ge, Weibing Dong, Xiaojun Peng, Fengyu Liu, Su-Shing Chen, Shiguo Sun, A mitochondrial targeting fluorescent probe based on the covalently linked anti-inflammatory drug dexamethasone and Cy7, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.07.170 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A mitochondrial targeting fluorescent probe based on the covalently linked antiinflammatory drug dexamethasone and Cy7
Siyuan Liu,a Guangbo Ge,b Weibing Dong,c Xiaojun Peng,a Fengyu Liu,a* Su-Shing Chen,d* Shiguo Sune,a*
a
State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Ling gong Road, Dalian 116024, P.R.
China. E-mail: [email protected] b
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
c
School of Life Science, Liaoning Normal University, Dalian 116081, China
d Systems Biology Laboratory,
Department of Computer Information Science and Engineering, University of Florida,
Gainesville, Florida, United States of America e Key
Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education, School of Pharmacy, Shihezi
University, Shihezi 832000, China E-mail: [email protected]
Graphical Abstract
Dex was proposed as a mitochondrial targeting group and covalently linked with Cy7 to facilitate the penetration of Cy7 into cells thus achieving mitochondrial imaging. Highlights
Dex was proposed as a mitochondrial targeting group and covalently linked with Cy7 to form Cy7-Dex, which facilitated the penetration of Cy7 into cells thus achieving mitochondrial imaging. 1
The probe, Cy7-Dex, could not only target mitochondria, but also exhibit the antiinflammatory effect.
Different cyanine groups had different influences on the hydrolysis rates of Cyn-Dexs and anti-inflammatory effect of Dex, which implied, in drug labeling and monitoring, the appropriate fluorophore should be selected to avoid possible false conclusions.
Abstract:
As one of the most important organelles, mitochondria not only produces energy but is also involved in cell metabolism and many pathological processes. Thus, targeting and monitoring mitochondria is of great importance. In this study, a commonly utilized antiinflammatory drug, dexamethasone (Dex), whose action position is mitochondria, was employed as a mitochondrial targeting functional group, and a near-infrared fluorescent dye, Cy7, was covalently linked with Dex to form Cy7-Dex, for fluorescent visualization of mitochondria. The corresponding Cy5-Dex and Cy3-Dex were synthesized in control experiments to explore the effects of different cyanine groups on the parent drug of Dex. Although all these Dex derivatives can fluorescently visualize mitochondria, different enzyme hydrolysis results and anti-inflammatory effects can be observed among them, thereby illustrating that different fluorophores might induce different effects on the properties of the same parent drug. Thus, only the appropriate fluorophore can be utilized to modify the drug to avoid false conclusions.
Keywords: mitochondrial targeting; fluorescent visualization; dexamethasone; cyanine dyes; drug labeling
1. Introduction
2
As one of the most important organelles, mitochondria plays the key role of producing energy, which is involved in cell metabolism and many pathological processes, from Alzheimer’s disease to cancer.1 Thus, targeting and monitoring mitochondria is of great importance. Fluorometry is highly sensitive, selective, nonradiative, rapidly performed, and can provide real-time information on the localization and quantity of the targets of interest.2 To provide such information, triphenylphosphonium salt or quaternary ammonium salt are commonly utilized to target the mitochondria and the corresponding fluorescent dye for visualization.3 Unfortunately, triphenylphosphonium is quite hydrophobic, and quaternary ammonium salt, such as pyridinium salt, tends to interact with negatively charged substances.4
Dexamethasone (Dex), a synthetic glucocorticoid (GC), is widely used to treat inflammatory and autoimmune diseases, including asthma, rheumatoid arthritis, inflammatory bowel disease and other systemic diseases. 5 The mitochondria is the target of Dex, which combines with the GC receptor (GR) in mitochondria.6 The fluorescent technique provides a comprehensive tool for investigating drug delivery in single cells and whole tissue. Many prodrugs have been developed to comprise drug portion and fluorescence group such as heptamethine cyanine, coumarin, and rhodamine dyes, to achieve drug delivery and monitoring. 7 Drug moiety accounts for delivery and the fluorescence group is in charge of monitoring. In previous studies, an anti-inflammatory drug, indomethacin, has been utilized as a targeting group that connects with fluorophores for cyclooxygenase-2 marking because indomethacin is the substrate of cyclooxygenase-2.8 The above findings prompted us to consider that 3
Dex might be employed as a functional group for mitochondrial targeting, which could carry fluorescent dye for mitochondrial visualizing and monitoring. However, whether the prodrug would work exactly like the original one, such as taking the same route, reaching the same target, and achieving the same therapeutic effect, or whether some influence exists after covalently linking with different fluorescent groups on the same parent drug needs to be determined.
Scheme 1 Structure of the proposed Cy7-Dex
Considering cyanine fluorescent probes have received broad attention for their tissue permeability, compatibility with biological systems and strong fluorescence intensity,9 a near-infrared cyanine dye Cy7 was covalently linked with Dex through carboxylic ester bond to form Cy7-Dex (Scheme 1). Results demonstrated that Cy7Dex located at the mitochondria in the cellular localization experiment. However, in the control experiments, neither Cy7 alone nor the mixture of Cy7 and Dex (the physical mixture of cyanines and Dex was represented as Cyn+Dexs) could penetrate into cells thereby showing the acceleration ability of Dex to facilitate cyanine into cells. Furthermore, the influence of different linked cyanine groups, Cy7, Cy5, and Cy3, on Dex was determined by investigating the difference on cellular localization, 4
enzyme hydrolysis and anti-inflammatory effects, respectively. Although these Dex derivatives can fluorescently visualize mitochondria, different enzyme hydrolysis results and anti-inflammatory effects can be observed among them.
2. Experimental section
2.1. Materials and methods
Dex was purchased from State Food and Drug Administration, P. R. China. MitoTracker Green FM was purchased from Invitrogen. Human liver microsome (HLM), rat liver microsome (RLM) and mouse liver microsome (MLM) were obtained from Research Institute for Liver Diseases (shanghai) CO. Ltd. Other reagents and chemical were of analytical reagent grade and used without further purification. Silica gel 60 (Merck, 0.063-0.2 mm) was used for column chromatography. Analytical TLC was performed using Merck 60 F254 silica gel (precoated sheet, 0.25 mm thick). 1
H NMR spectra and 13C NMR spectra were recorded with a VAIAN DLG-500
spectrometer with chemical shifts reported as ppm. Mass spectra data were obtained on a Q-Tof MS spectrometer (Micromass, Manchester, England). Absorption spectra were measured on a Perkin-Elmer Lambda35 UV-Vis spectrophotometer. Fluorescence measurements were performed on an Aglilent Cary Eclipse fluorescence spectrophotometer. Fluorescence imaging was performed using an OLYMPUS FV1000 inverted fluorescence microscope with a 60× objective lens. High performance liquid chromatogram (HPLC) analyses and purification were performed using an
5
Agilent 1100 HPLC system with a diode array UV detector. The UV spectra of the HPLC peaks were obtained using on-line diode array detection.
2.2. Synthesis
2.2.1. Cy7-Dex
Cy7-Dex: Cy7 (200 mg, 0.26 mmol) and N,N-4-dimethylaminopyridine (DMAP, 38 mg, 0.31 mmol) were added into a 25 mL round-bottom flask with 10 mL dehydrated dichloromethane dissolved. The solution was stirred for 1 h under nitrogen. Then Dex (204 mg, 0.52 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 57 mg, 0.31 mmol) were added into the solution above which was stirred 12 h. The solvent was removed by reduced pressure distillation. The solid mixture was divided by column chromatography. The purified product weighed 267 mg, 68% yield. 1H NMR (500 MHz, CDCl3) δ 8.34 (d, J = 13.9 Hz, 2H), 7.47 (d, J = 10.1 Hz, 2H), 7.41 – 7.33 (m, 4H), 7.24 (dd, J = 13.8, 6.3 Hz, 2H), 7.15 (d, J = 8.0 Hz, 2H), 6.27 – 6.23 (m, J = 10.6 Hz, 4H), 6.03 (s, 2H), 5.09 (d, J = 17.3 Hz, 2H), 4.84 (d, J = 17.3 Hz, 2H), 4.40 (d, J = 8.8 Hz, 2H), 4.30 (s, 1H), 4.13 (d, J = 4.5 Hz, 4H), 3.46 (s, 1H), 3.22 (s, 1H), 3.08 – 2.94 (m, 2H), 2.72 (s, 4H), 2.56 (dd, J = 17.3, 12.2 Hz, 2H), 2.43 (d, J = 3.8 Hz, 5H), 2.34 – 2.22 (m, 6H), 2.15 (dd, J = 19.1, 11.1 Hz, 7H), 1.96 (d, J = 4.5 Hz, 2H), 1.90 – 1.80 (m, 4H), 1.75 (dd, J = 15.7, 10.2 Hz, 7H), 1.70 (s, 12H), 1.55 – 1.46 (m, 10H), 1.18 (dd, J = 7.2, 4.2 Hz, 2H), 1.02 (s, 6H), 0.88 (d, J = 7.1 Hz, 6H).. HRMS: M+, calcd for C86H106ClF2N2O12 +, m/z =1431.7397, found: m/z =1431.7395. 13C NMR (125 MHz, CDCl3) δ 205.54, 187.16, 173.28, 172.47, 167.44, 153.99, 150.75, 144.61, 142.24, 141.21, 129.36, 129.01, 127.99, 125.45, 124.84, 122.37, 110.98, 101.70, 100.30, 91.28, 71.50, 71.20, 68.41,
6
49.43, 48.65, 48.60, 44.53, 44.13, 36.11, 35.91, 34.49, 33.58, 32.57, 31.30, 28.32, 27.64, 27.10, 26.71, 26.21, 24.53, 23.25, 16.69, 15.13
2.2.2. Cy5-Dex
Cy5-Dex: Cy5 (172 mg, 0.26 mmol) and N,N-4-dimethylaminopyridine (DMAP, 38 mg, 0.31 mmol) were added into a 25 mL round-bottom flask with 10 mL dehydrated dichloromethane dissolved. The solution was stirred for 1 h under nitrogen. Then Dex (204 mg, 0.52 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (57 mg, 0.31 mmol) were added into the solution above which was stirred 12 h. The solvent was removed by reduced pressure distillation. The solid mixture was divided by column chromatography. The purified product weighed 223 mg, 61% yield. 1H NMR (500 MHz, CDCl3) δ 8.29 (t, J = 16.1 Hz, 2H), 7.60 (d, J = 9.1 Hz, 2H), 7.46 (d, J = 10.1 Hz, 2H), 7.25 (t, J = 8.9 Hz, 2H), 7.11-7.07 (m, 2H), 6.61 (dd, J = 34.1, 19.2 Hz, 1H), 6.26 (d, J = 15.1 Hz, 2H) 5.97 (s, 2H), 5. 26 (t, J =12.6 Hz, 2H), 5.02 (d, J = 17.3 Hz, 2H), 4.80 (d, J = 17.3 Hz, 2H), 4.35 (d, J = 8.8 Hz, 2H), 4.24 (s, 1H), 4.07 (t, J = 9.3 Hz, 4H), 3.40 (s, 1H), 3.18 (s, 1H), 2.97 – 2.94 (m, 2H), 2.72– 2.66 (m, 4H), 2.60 (t, J = 8.3 Hz, 2H), 2.41– 2.10 (m, 17H), 1.90 – 1.85 (m, 2H), 1.64 (s, 12H), 1.51 – 1.42 (m, 10H), 1.34-1.31 (m, 4H), 1.13-1.08 (m, 2H), 0.96 (s, 6H), 0.81 (d, J = 7.1 Hz, 6H). 0.73-0.70 (m, 4H). HRMS: M+, calcd for C81H101ClF2N2O12 +, m/z =1331.7317, found: m/z =1331.6270. 13C NMR (125 MHz, CDCl3) δ 206.12, 187.74, 180.87, 180.06, 168.02, 154.58, 145.20, 142.83, 141.79,138.15,130.11, 129.94, 129.59, 128.57, 126.04, 125.43, 122.95, 111.56, 106.58, 102.17, 100.88, 91.86, 72.09, 71.79, 68.99, 50.02, 49.42, 49.18, 45.11, 44.71, 36.69, 36.49, 34.92, 34.16, 33.15, 31.89, 28.84, 28.23, 26.80, 25.11, 17.28, 15.71. 7
2.2.3. Cy3-Dex
Cy3-Dex: Cy3 (161 mg, 0.26 mmol) and N,N-4-dimethylaminopyridine (DMAP, 38 mg, 0.31 mmol) were added into a 25 mL round-bottom flask with 10 mL dehydrated dichloromethane dissolved. The solution was stirred for 1 h under nitrogen. Then Dex (204 mg, 0.52 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (57 mg, 0.31 mmol) were added into the solution above which was stirred 12 h. The solvent was removed by reduced pressure distillation. The solid mixture was divided by column chromatography. The purified product weighed 202 mg, 57% yield. 1H NMR (500 MHz, CDCl3) δ 8.40 (t, J = 12.8 Hz, 1H), 7.38 (dd, J = 16.9, 7.4 Hz, 8H), 7.13 (d, J = 7.9 Hz, 2H), 6.83 (d, J = 13.0 Hz, 2H), 6.32 (d, J = 10.1 Hz, 2H), 6.08 (s, 2H), 5.30 (s, 2H), 4.97 (dd, J = 42.2, 17.3 Hz, 4H), 4.40 (d, J = 10.0 Hz, 2H), 4.16 (s, 4H), 3.07 (s, 4H), 2.62 (d, J = 9.4 Hz, 4H), 2.47 (d, J = 6.6 Hz, 4H), 2.38 – 2.21 (m, 6H), 1.84 (d, J = 13.0 Hz, 16H), 1.72 (s, 12H), 1.62 (s, 6H), 1.23 (d, J = 22.9 Hz, 6H), 1.07 (s, 6H), 0.90 (d, J = 6.9 Hz, 6H). HRMS: M+, calcd for C79H99F2N2O12+, m/z =1305.7161, found: m/z =1305.7164. 13C NMR (125 MHz, CDCl3) δ 205.55, 187.03, 173.84, 173.36, 167.28, 153.59, 150.73, 142.00, 140.60, 129.35, 128.99, 125.40, 124.80, 122.09, 111.10, 103.97, 101.49, 91.14, 71.91, 71.61, 68.31, 53.83, 49.06, 48.68, 48.50, 48.39, 44.41, 44.00, 35.89, 35.68, 34.37, 33.43, 32.45, 31.15, 29.27 , 28.15, 27.48, 27.21, 25.92, 24.48, 23.07, 16.44, 14.84.
2.3. Cellular localization imaging
Cells were cultured in DEME (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum. One day before imaging, cells were seeded in cell-culture dishes.
8
The next day, 10 µL Cy7-Dex, Cy5-Dex and Cy3-Dex (represented as Cyn-Dexs) solution were added to the media at a final concentration of 10 µM respectively, and then the cells were incubated for another 2 h at 37 °C with 5% CO2. The cells were washed three times with PBS before imaging. For each staining test, the fluorescent imaging pictures were obtained with an equal parameter for control. The cells were stained with Cy7, Cy5, Cy3 also the corresponding physical mixture of cyanine and Dex with the ratio of 1:2 (the concentration of Cyn+Dex is calculated according to that of cyanines) using the same procedure. After rinsing with PBS twice, the same sample was further stained with 100 nM Mito-Tracker Green FM for another 15 min and then imaged.
2.4. MTT assay
The cytotoxicity of compounds Cyn-Dexs, cyanines and Dex were evaluated following a reported approach.10 RAW264.7 cells were seeded at a density of 5×104 cells/mL in a 96-well plate with 100 µL of RPMI-1640 medium and grew for 24 h at 37 °C with 5% CO2. Then, these cells were treated with various concentrations of Cyn-Dexs (12.5, 25, 50, 100 and 200 µM) for 24 h. The cytotoxicity of these compounds was then determined using MTT assays. For comparison, the cytotoxicity of cyanines, Dex and Cyn+Dexs were studied using the same procedure. Absorbance at 490 nm was measured using a microplate reader. Cell survival was calculated as a percentage of the inhibition for viable cells.
2.5. Enzyme hydrolysis test The enzyme hydrolysis assay was carried out with reference to a published method.11 Substrate Cyn-Dexs 50 µM dissolved in DMSO were prepared. Incubate Cyn-Dexs with 9
pooled HLM 100 µg/mL, RLM 100 µg/mL, MLM 100 µg/mL respectively. The reaction was performed at 37 °C for 1 h. It was supposed that cyanine was one of the hydrolysates which had characteristic ultraviolet absorption. Here a HPLC method was used to separate the hydrolysates and detect hydrolysis rate of Cyn-Dexs. Also, the retention time of corresponding cyanines were determined acted as the control group. The chromatographic column was a 2.1150 mm Shim-pack VP-ODS column with a diameter of 5 μm. Eluent was a linear gradient containing acetonitrile/H2O (added 0.2% methanoic acid) found to be the most efficient eluents for this separation. For Cy5-Dex and Cy7-Dex, in 0-5 min, the water ratio was 90%, in 5-8 min, the water ratio changed from 90% to 60%, in 8-10 min, the water ratio changed from 60% to 5%, in 10-13 min, the water ratio was 5% and in 13-17 min, the water ratio was 90% with a flow rate of 0.4 mL/min. For Cy3-Dex, in 0-2 min, the water ratio was 80%, in 2-5 min, the water ratio changed from 80% to 60%, in 5-8 min, the water ratio changed from 60% to 5%, in 8-11 min, the water ratio was 5% and in 11-14 min, the water ratio was 80% with a flow rate of 0.4 mL/min. The wavelength for detection of Cy3, Cy5, and Cy7 was 556 nm, 643 nm, and 780 nm respectively for that they were the maximum absorption wavelength of Cy3, Cy5, and Cy7 in PBS buffer.
2.6. Cytokine assay The anti-inflammatory effects were determined with reference to a reported method.12 RAW264.7 cells were distributed into a 24-well plate with a density of 1.0×106 cells/mL. Cells were incubated for 24 h in the absence or presence of lipopolysaccharide (LPS) (1 µg/mL) and Polymyxin B (PMB, 20 µM), Dex (25 µM), Cyn-Dexs (12.5 µM), Cyn+Dexs (12.5 µM), respectively at 37 °C with 5% CO2. After 24 h incubation, supernatants were collected for 10
ELISA analysis. Cytokine IL-6 and TNF-α were determined by ELISA kits according to the instruction recommended by the manufacture. The optical density of the microplate was read at 450 nm and the IL-6 or TNF-α concentration for each sample was calculated from a standard curve.
3. Results and discussion
3.1. Synthesis of Cyn-Dexs Cy7, Cy5 and Cy3 were synthesized by previously published methods. 13 Cyn-Dexs were obtained by esterification reaction. First, cyanines were activated by 4dimethylaminopyridine (DMAP) in dehydrated dichloromethane solution under the protection of nitrogen. Then, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and Dex were added into the solution to produce the final products, Cyn-Dexs (Scheme 1, ESI†). The crude products were purified by Column Chromatography. Cyanine dyes and Cyn-Dexs were confirmed by 1H NMR, HRMS and 13
C NMR (ESI†).
3.2. Spectral properties of Cyn-Dexs
The normalized UV-vis absorption and fluorescence emission spectra of Cyn-Dexs were obtained to provide fundamental information for cell imaging (Fig. 1, Fig. S1). The corresponding data of single cyanines and Cyn+Dexs were obtained using the same procedure (Fig. S1). The case of Cy7 is taken as an example. The spectra of Cy7Dex and the corresponding Cy7 or Cy7+Dex were almost the same on the UV-vis absorption maxima (785 nm) and fluorescence emission maxima (815 nm). The same 11
situation also occurred in the case of Cy5 and Cy3 (Fig. S1). All these demonstrated that the introduction of Dex didn’t affect the spectral characteristic of cyanine dyes.
3.3. Cellular localization
Regardless of cell types (MCF-7, HL-7702, HepG-2), cells incubated with Cy7 alone or physical mixture Cy7+Dex exhibited little fluorescence, whereas cells incubated with Cy7-Dex showed a significant fluorescence signal (Fig. 2, Fig. S2, S4). The different imaging results probably resulted from the targeting effect of Dex. The localization of Cy7-Dex in living cells was tested by costaining experiments by using commercially available Mito-Tracker Green FM. The red fluorescence from the Cy7Dex evidently colocalized well with green fluorescence from Mito-Tracker Green FM. The Pearson’s correlation coefficient was 0.99 for MCF-7 cells (Fig. 2), 0.94 for HL7702 cells (Fig. S3) and 0.96 for HepG-2 cells (Fig. S5). These findings suggest that the targeting position of Cy7-Dex was mitochondria. The targeting effect of Cy7-Dex was in accordance with previous findings that the action position of Dex was mitochondria.14
To explore whether cyanine groups could influence the original drug effect of Dex, different cyanine groups, Cy5 and Cy3, were covalently linked with Dex using the same method as Cy7 to form Cy5-Dex and Cy3-Dex. In the cellular imaging experiments, the same trend can be observed in the case of Cy5 (Fig. S6-S11) and Cy3 (Fig. S12-S17) under the same circumstance. Cy5-Dex and Cy3-Dex could penetrate into cells and accumulated at mitochondria. Whereas, neither Cy5, Cy5+Dex, Cy3, nor Cy3+Dex could be found in cells.
12
Results demonstrated that different cyanine groups had little influence on the targeting position of Dex. All these findings further illustrated that the targeting position of Cyn-Dexs was mitochondria, similar to that of Dex. This finding means that Dex could probably be utilized as a mitochondrial targeting functional group. Furthermore, MTT assay was performed. Negligible toxicity can be observed even under a relative higher concentration employed (the concentration of Dex, cyanines, Cyn-Dexs and Cyn+Dexs was 25 μM, 12.5 μM, 12.5 μM and 12.5 μM respectively), thereby proving the viability of the proposed method (Fig. S18).
3.4. Enzyme hydrolysis test
The proposed prodrugs consist of drug moiety Dex and fluorophore component cyanines, which are combined via carboxylic ester bond. In vitro or in vivo, these prodrugs are anticipated to be divided into two parts, namely, the drug Dex and fluorophore cyanines, mainly through enzyme hydrolysis.15 Enzyme hydrolysis experiments were conducted to verify any difference among the hydrolysis rates of different Cyn-Dexs, which might have different influences on the drug effect of Dex.
Two main peaks were observed in the chromatogram of all the three Cyn-Dexs, namely, one for Cyn-Dexs and the other for cyanines, which acted as a contrast (Fig. S19). The case of Cy7 is taken as an example (Fig. 3). No peak that corresponded to Cy7 appeared in the reaction system of Cy7-Dex with any kind of microsome, thereby showing that Cy7-Dex could hardly be hydrolyzed in these microsomes. In comparison, a low peak appeared in the Cy5-Dex solution added with microsomes. After separation and collection, the hydrolysis rate was calculated as 0.26% for HLM, 13
0.83% for RLM, and 2.92% for MLM. A much higher peak appeared in the Cy3-Dex solution added with microsomes. After collection, the hydrolysis rate was calculated as 0.16% for HLM, 6.65% for RLM, and 13.00% for MLM.
From Cy7-Dex to Cy3-Dex, the hydrolysis rates gradually rose, thereby suggesting an easier tendency to be hydrolyzied. The difference in hydrolysis rates was speculated to have resulted from the different affinity of Cyn-Dexs to enzymes and the smaller size accelerated the interaction of Cyn-Dexs and enzymes. Results showed that different fluorophores probably had different influences on the fluorophore-conjugated drug, which potentially had diverse impacts on drug effect. Cy3-Dex was preferred for hydrolysis in the presence of microsomes. Thus, Cy3-Dex might have the best drug release effect and furthermore the minor influence on the therapeutic effect of Dex. By contrast, Cy7-Dex might be the better choice for simple drug monitoring in that it was stable under hydrolytic enzyme systems.
3.5. Anti-inflammatory effects of Cyn-Dexs
Interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) are key inflammatory cytokines in many infectious and inflammatory diseases.12, 16 The concentration of IL-6 and TNF-α can reflect the inflammatory level. LPS can induce marked increase in IL-6 and TNF-α production. Whereas, Dex can decrease the production and release of IL-6 and TNF-α.17 Cytokine assay was performed to examine whether the modification of Dex by cyanines could influence the drug effect of Dex, and to verify if a difference in the drug effects of Cy7-Dex, Cy5-Dex, and Cy3-Dex existed. RAW264.7 cells were incubated in the absence or presence of LPS and
14
PMB, Dex, Cyn-Dexs, Cyn+Dexs, respectively. Cytokine IL-6 and TNF-α were determined. PMB, a kind of antibiotic, acted as a positive control. Cyn+Dexs also acted as a control group to verify if cyanines could cause influence on the anti-inflammatory effect of Dex.
As shown in Fig. 4a and Fig. 4b, RAW264.7 cells with no LPS stimulation secreted a basal level of TNF-α and IL-6. However, with the addition of LPS, the TNF-α and IL-6 protein level increased by approximately four folds, and the additions of Dex, Cyn-Dexs, and Cyn+Dexs was able to inhibit the production of TNF-α and IL-6. After the cells were treated by Dex, the TNF-α production was reduced to approximately 50% and IL-6 production was reduced to 25%; these values for TNF-α and IL-6 were 36% and 33% in Cy7-Dex, 50% and 30% in Cy5Dex, 60% and 36% in Cy3-Dex, respectively. Cy5-Dex showed a similar inhibition ability with Dex in TNF-α and IL-6 secretion, but Cy3-Dex and Cy7-Dex exhibited lower or higher inhibition ability than Dex, respectively. Cy7+Dex, Cy5+Dex and Cy3+Dex, exhibited a similar inhibition ability toward Dex in TNF-α and IL-6 secretion, which indicated cyanines exerted little influence on the anti-inflammatory effect of Dex. Generally, different cyanines, Cy7, Cy5, Cy3 modified Dex exhibited different abilities to inhibit cytokine secretion. The inhibition effect of Cy3-Dex, Cy5-Dex, and Cy7-Dex on TNF-α production improved. No evident trend among the three Cyn-Dexs was observed in IL-6 production. b
4. Conclusion
15
In summary, Dex that is, covalently connected with Cy7, facilitated the penetration of Cy7 into cells and targeted mitochondria unlike single Cy7 and Cy7+Dex. The same trend occurred in the case of Cy5 and Cy3. Results have revealed a new mitochondrial targeting probe based on a commonly used anti-inflammatory drug, Dex, which can target mitochondria and achieve an anti-inflammatory effect. In addition, different cyanine groups had different influences on the hydrolysis rates of Cyn-Dexs and anti-inflammatory effect of Dex. In drug labeling and monitoring, the appropriate fluorophore should be selected to avoid possible false conclusions.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (Nos. 21272030, 21472016, 21306019, 21576042, 21421005).
References
1.
(a) F. G. De Felice and S. T. Ferreira, Diabetes, 2014, 63, 2262; (b) L. Galluzzi, N. Larochette, N.
Zamzami and G. Kroemer, Oncogene, 2006, 25, 4812; (c) S. Bonnet, S. L. Archer, J. Allalunis-Turner, A. Haromy, C. Beaulieu, R. Thompson, C. T. Lee, G. D. Lopaschuk, L. Puttagunta, S. Bonnet, G. Harry, K. Hashimoto, C. J. Porter, M. A. Andrade, B. Thebaud and E. D. Michelakis, Cancer Cell, 2007, 11, 37; (d) S. Fulda, L. Galluzzi and G. Kroemer, Nat Rev Drug Discov, 2010, 9, 447. 2.
(a) X. Xiong, F. Song, J. Wang, Y. Zhang, Y. Xue, L. Sun, N. Jiang, P. Gao, L. Tian and X. Peng, J Am
Chem Soc, 2014, 136, 9590; (b) M. Abo, Y. Urano, K. Hanaoka, T. Terai, T. Komatsu and T. Nagano, J Am Chem Soc, 2011, 133, 10629; (c) T. T. Chen, X. Tian, C. L. Liu, J. Ge, X. Chu and Y. Li, J Am Chem Soc, 2015, 137, 982. 16
3.
(a) C. W. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Lam and B. Z. Tang, J Am Chem Soc, 2013, 135,
62; (b) S. Wu, Q. Cao, X. Wang, K. Cheng and Z. Cheng, Chem Commun (Camb), 2014, 50, 8919; (c) A. R. Sarkar, C. H. Heo, H. W. Lee, K. H. Park, Y. H. Suh and H. M. Kim, Anal Chem, 2014, 86, 5638. 4.
Y. Li, Z. Lü, M. Liu and G. Xing, Chinese J Org Chem, 2016, 36, 962.
5.
(a) B. Guo, W. Zhang, S. Xu, J. Lou, S. Wang and X. Men, Life Sci, 2016, 144, 1; (b) P. Londono, A.
Komura, N. Hara and D. Zipris, Clin Immunol, 2010, 135, 401; (c) S. Benedetti, B. Pirola, P. L. Poliani, L. Cajola, B. Pollo, R. Bagnati, L. Magrassi, P. Tunici and G. Finocchiaro, Gene Ther, 2003, 10, 188; (d) S. Tsurufuji, K. Sugio and F. Takemasa, 1979; (e) R. Mohammadi, M. Azad-Tirgan and K. Amini, Injury, 2013, 44, 565. 6.
(a) G. Kroemer and J. C. Reed, Nat Med, 2000, 6; (b) J. D. Ly, D. R. Grubb and A. Lawen,
Apoptosis, 2003, 8; (c) N. Zamzami, P. Marchetti, M. Castedo, C. Zanin, J. L. Vayssiere, P. X. Petit and G. Kroemer, J Exp Med, 1995, 181. 7.
(a) E. J. Kim, S. Bhuniya, H. Lee, H. M. Kim, C. Cheong, S. Maiti, K. S. Hong and J. S. Kim, J Am
Chem Soc, 2014, 136, 13888; (b) C. Lechner, V. Reichel, U. Moenning, A. Reichel and G. Fricker, Eur J Pharm Biopharm, 2010, 75, 284; (c) J. B. Wu, T. P. Lin, J. D. Gallagher, S. Kushal, L. W. Chung, H. E. Zhau, B. Z. Olenyuk and J. C. Shih, J Am Chem Soc, 2015, 137, 2366; (d) T. Xing, C. Mao, B. Lai and L. Yan, ACS Appl Mater Interfaces, 2012, 4, 5662. 8.
(a) H. Zhang, J. Fan, J. Wang, B. Dou, F. Zhou, J. Cao, J. Qu, Z. Cao, W. Zhao and X. Peng, J Am
Chem Soc, 2013, 135, 17469; (b) H. Zhang, J. Fan, J. Wang, S. Zhang, B. Dou and X. Peng, J Am Chem Soc, 2013, 135, 11663. 9.
(a) J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J. H. Ryu and J. Yoon, J Am Chem Soc, 2014, 136,
5351; (b) Y. Li, Y. Sun, J. Li, Q. Su, W. Yuan, Y. Dai, C. Han, Q. Wang, W. Feng and F. Li, J Am Chem Soc, 17
2015, 137, 6407. 10.
X. Jia, Q. Chen, Y. Yang, Y. Tang, R. Wang, Y. Xu, W. Zhu and X. Qian, J Am Chem Soc, 2016, 138,
10778. 11.
(a) S. C. Liang, G. B. Ge, H. X. Liu, H. T. Shang, H. Wei, Z. Z. Fang, L. L. Zhu, Y. X. Mao and L. Yang,
J Pharm Biomed Anal, 2011, 54, 236; (b) K. Abass, P. Reponen, J. Jalonen and O. Pelkonen, Pestic Biochem Physiol, 2007, 87, 238. 12.
S. Mianji, Y. Hamasaki, S. Yamamoto and S. Miyazaki, Int J Immunopharmacol, 1996, 18, 339.
13.
(a) X. Chen, X. Peng, A. Cui, B. Wang, L. Wang and R. Zhang, J Photochem Photobiol A, 2006,
181, 79; (b) H. Wu, S. C. Alexander, S. Jin and N. K. Devaraj, J Am Chem Soc, 2016, 138, 11429; (c) Y. Guan, Y. Zhang, L. Xiao, J. Li, J. P. Wang, M. D. Chordia, Z. Q. Liu, L. W. Chung, W. Yue and D. Pan, Mol Pharm, 2017, 14, 1. 14.
(a) C. V. Demonacos, N. Karayanni, E. Hatzoglou, C. Tsiriyiotis, D. A. Spandidos and C. E. Sekeris,
Steroids, 1996, 61; (b) C. Tsiriyotis, D. A. Spandidos and C. E. Sekeris, Biochem Bioph Res Co, 1997, 235, 349; (c) C. Demonacos, R. Djordjevic-Markovic, N. Tsawdaroglou and C. E. Sekeris, J Steroid Biochem Mol Biol, 1995, 55, 43. 15.
C. Branneby, P. Carlqvist, A. Magnusson, K. Hult, T. Brinck and P. Berglund, J Am Chem Soc, 2003,
125, 874. 16.
N. Yingkun, W. Zhenyu, L. Jing, L. Xiuyun and Y. Huimin, Inflammation, 2013, 36, 242.
17.
C. M. Williams and J. W. Coleman, Immunology, 1995, 86, 244.
Biographies
Siyuan Liu, received his B.S. degree in 2015 from Yanshan University, China. He is pursuing his master’s degree under the guidance of Prof. Shiguo Sun in State Key Laboratory of Fine 18
Chemicals, School of Chemistry, Dalian University of Technology. His research interest focuses on organic probe for molecular detection.
Guangbo Ge received his B.S. degree in applied chemistry in 2003 from Dalian University of Technology, Ph.D degree in biochemical engineering in 2009 from Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS). Currently he is a full professor at Dalian Institute of Chemical Physics, CAS. His current studies are focused on the development of new techniques or bioanalytical methods for key enzymes participating in endobiotic and xenobiotic metabolism, etc.
Weibing Dong received his Ph.D. degree in applied chemistry in 2008 from Dalian University of Technology. He is currently an associate professor in Liaoning Normal University. His research interests include drug discovery, enzyme inhibitors and so on.
Xiaojun Peng received his Ph.D. degree in applied chemistry in 1989 from Dalian University of Technology. He is currently a professor in State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, China and the director of State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology. His current studies are focused on functional dye and application, fluorescent probe, fine chemical cleaning preparation technique.
Fengyu Liu received her Ph.D. degree in 2006 from Dalian University of Technology. She is currently an associate professor in State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, China. Her research interests include
19
electrochemiluminescence, luminescent/fluorescent sensors, DNA-targeting molecules designing, synthesizing and application.
Su-Shing Chen received his BS in Mathematics from the National Taiwan University, and PhD in Mathematics from the University of Maryland, College Park. His research consists of three stages: Mathematics, Artificial Intelligence, and Computational Biology. He has published hundreds of journal and conference papers on Bioinformatics, Information Technology and Digital Libraries, Neural Networks, Computer Vision and Image Processing, Intelligent Information Systems, Advanced Manufacturing Systems, Dynamical Systems, Manifold Theory etc.
Shiguo Sun received his Ph.D. degree in 2003 from Dalian University of Technology and is currently a professor and doctoral supervisor in the School of Chemistry&Pharmacy, Northwest A&F University, China. His research interests include functional molecules with special light or electrochemistry properties, self-assembly chemistry of carbon nanotube/graphene material for DNA and protein sensing, drug delivery and fluorescence tracing, and electrochemiluminescence and luminescence sensors.
1.2
UV-vis
F
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Normalized A
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Fig. 1 Normalized UV−vis absorption and fluorescence mission spectrum of Cy7-Dex (4 μM) in PBS (pH 7.4).
a
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h
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Fig. 2 Fluorescence imaging of MCF-7 cells stained with Cy7 (10 μM), Cy7+Dex (10 μM), Cy7-Dex (10 μM) respectively (Ex, 635 nm, Em, 651-751 nm), and Mito-Tracker Green FM (100 nM, Ex, 490 nm, Em, 500-520 nm). (a-c) Bright field images of MCF-7 cells stained with Cy7, Cy7+Dex, Cy7-Dex respectively, (d-f) fluorescence images of MCF-7 cells stained with Cy7, Cy7+Dex, Cy7-Dex respectively, (g) bright field image of MCF-7 cells stained with Cy7-Dex, (h) fluorescence image of MCF-7 cells stained with Mito-Tracker Green FM, (i) 21
fluorescence image of MCF-7 cells stained with Cy7-Dex, (j) merged image of h and I, (k) correlation plot of the intensities of Mito-Tracker Green FM and Cy7-Dex (Rr, 0.99), (l) normalized intensity profile of regions of interest across MCF-7 cells.
uV(x100,000) 4.0 3.0 2.0 1.0 0.0 0.0
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Fig. 3 The enzyme hydrolysis test of Cy7-Dex. From top to bottom correspond to product (Cy7), substrate (Cy7Dex), HLM+substrate, MLM+substrate, RLM+substrate.
a
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LP S
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Cy B 3De Cy x 5De Cy x 7D Cy ex 3+ D Cy ex 5+ D Cy ex 7+ De x
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Cy B 3De Cy x 5De Cy x 7D Cy ex 3+ D Cy ex 5+ D Cy ex 7+ De x
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IL-6 (pg/mL)
TNF- (pg/mL)
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E
Fig. 4 Inhibition effects of Dex, Cyn-Dexs and Cyn+Dexs on the LPS-induced increase in inflammatory cytokines production. Cells were incubated for 24 h in the absence or presence of LPS (1 µg/mL) and PMB (20 µM), Dex (25
22
µM), Cyn-Dexs (12.5 µM), Cyn+Dexs (12.5 µM), respectively at 37 °C with 5% CO2. (a) Inhibition effects on TNF-α production, (b) inhibition effects on IL-6 production. Data are means ± S.E.M. (n = 3), *P<0.05 vs LPS alone.
23
S0925-4005(17)31384-9 http://dx.doi.org/doi:10.1016/j.snb.2017.07.170 SNB 22829
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
19-6-2017 20-7-2017 24-7-2017
Please cite this article as: Siyuan Liu, Guangbo Ge, Weibing Dong, Xiaojun Peng, Fengyu Liu, Su-Shing Chen, Shiguo Sun, A mitochondrial targeting fluorescent probe based on the covalently linked anti-inflammatory drug dexamethasone and Cy7, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.07.170 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A mitochondrial targeting fluorescent probe based on the covalently linked antiinflammatory drug dexamethasone and Cy7
Siyuan Liu,a Guangbo Ge,b Weibing Dong,c Xiaojun Peng,a Fengyu Liu,a* Su-Shing Chen,d* Shiguo Sune,a*
a
State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Ling gong Road, Dalian 116024, P.R.
China. E-mail: [email protected] b
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
c
School of Life Science, Liaoning Normal University, Dalian 116081, China
d Systems Biology Laboratory,
Department of Computer Information Science and Engineering, University of Florida,
Gainesville, Florida, United States of America e Key
Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education, School of Pharmacy, Shihezi
University, Shihezi 832000, China E-mail: [email protected]
Graphical Abstract
Dex was proposed as a mitochondrial targeting group and covalently linked with Cy7 to facilitate the penetration of Cy7 into cells thus achieving mitochondrial imaging. Highlights
Dex was proposed as a mitochondrial targeting group and covalently linked with Cy7 to form Cy7-Dex, which facilitated the penetration of Cy7 into cells thus achieving mitochondrial imaging. 1
The probe, Cy7-Dex, could not only target mitochondria, but also exhibit the antiinflammatory effect.
Different cyanine groups had different influences on the hydrolysis rates of Cyn-Dexs and anti-inflammatory effect of Dex, which implied, in drug labeling and monitoring, the appropriate fluorophore should be selected to avoid possible false conclusions.
Abstract:
As one of the most important organelles, mitochondria not only produces energy but is also involved in cell metabolism and many pathological processes. Thus, targeting and monitoring mitochondria is of great importance. In this study, a commonly utilized antiinflammatory drug, dexamethasone (Dex), whose action position is mitochondria, was employed as a mitochondrial targeting functional group, and a near-infrared fluorescent dye, Cy7, was covalently linked with Dex to form Cy7-Dex, for fluorescent visualization of mitochondria. The corresponding Cy5-Dex and Cy3-Dex were synthesized in control experiments to explore the effects of different cyanine groups on the parent drug of Dex. Although all these Dex derivatives can fluorescently visualize mitochondria, different enzyme hydrolysis results and anti-inflammatory effects can be observed among them, thereby illustrating that different fluorophores might induce different effects on the properties of the same parent drug. Thus, only the appropriate fluorophore can be utilized to modify the drug to avoid false conclusions.
Keywords: mitochondrial targeting; fluorescent visualization; dexamethasone; cyanine dyes; drug labeling
1. Introduction
2
As one of the most important organelles, mitochondria plays the key role of producing energy, which is involved in cell metabolism and many pathological processes, from Alzheimer’s disease to cancer.1 Thus, targeting and monitoring mitochondria is of great importance. Fluorometry is highly sensitive, selective, nonradiative, rapidly performed, and can provide real-time information on the localization and quantity of the targets of interest.2 To provide such information, triphenylphosphonium salt or quaternary ammonium salt are commonly utilized to target the mitochondria and the corresponding fluorescent dye for visualization.3 Unfortunately, triphenylphosphonium is quite hydrophobic, and quaternary ammonium salt, such as pyridinium salt, tends to interact with negatively charged substances.4
Dexamethasone (Dex), a synthetic glucocorticoid (GC), is widely used to treat inflammatory and autoimmune diseases, including asthma, rheumatoid arthritis, inflammatory bowel disease and other systemic diseases. 5 The mitochondria is the target of Dex, which combines with the GC receptor (GR) in mitochondria.6 The fluorescent technique provides a comprehensive tool for investigating drug delivery in single cells and whole tissue. Many prodrugs have been developed to comprise drug portion and fluorescence group such as heptamethine cyanine, coumarin, and rhodamine dyes, to achieve drug delivery and monitoring. 7 Drug moiety accounts for delivery and the fluorescence group is in charge of monitoring. In previous studies, an anti-inflammatory drug, indomethacin, has been utilized as a targeting group that connects with fluorophores for cyclooxygenase-2 marking because indomethacin is the substrate of cyclooxygenase-2.8 The above findings prompted us to consider that 3
Dex might be employed as a functional group for mitochondrial targeting, which could carry fluorescent dye for mitochondrial visualizing and monitoring. However, whether the prodrug would work exactly like the original one, such as taking the same route, reaching the same target, and achieving the same therapeutic effect, or whether some influence exists after covalently linking with different fluorescent groups on the same parent drug needs to be determined.
Scheme 1 Structure of the proposed Cy7-Dex
Considering cyanine fluorescent probes have received broad attention for their tissue permeability, compatibility with biological systems and strong fluorescence intensity,9 a near-infrared cyanine dye Cy7 was covalently linked with Dex through carboxylic ester bond to form Cy7-Dex (Scheme 1). Results demonstrated that Cy7Dex located at the mitochondria in the cellular localization experiment. However, in the control experiments, neither Cy7 alone nor the mixture of Cy7 and Dex (the physical mixture of cyanines and Dex was represented as Cyn+Dexs) could penetrate into cells thereby showing the acceleration ability of Dex to facilitate cyanine into cells. Furthermore, the influence of different linked cyanine groups, Cy7, Cy5, and Cy3, on Dex was determined by investigating the difference on cellular localization, 4
enzyme hydrolysis and anti-inflammatory effects, respectively. Although these Dex derivatives can fluorescently visualize mitochondria, different enzyme hydrolysis results and anti-inflammatory effects can be observed among them.
2. Experimental section
2.1. Materials and methods
Dex was purchased from State Food and Drug Administration, P. R. China. MitoTracker Green FM was purchased from Invitrogen. Human liver microsome (HLM), rat liver microsome (RLM) and mouse liver microsome (MLM) were obtained from Research Institute for Liver Diseases (shanghai) CO. Ltd. Other reagents and chemical were of analytical reagent grade and used without further purification. Silica gel 60 (Merck, 0.063-0.2 mm) was used for column chromatography. Analytical TLC was performed using Merck 60 F254 silica gel (precoated sheet, 0.25 mm thick). 1
H NMR spectra and 13C NMR spectra were recorded with a VAIAN DLG-500
spectrometer with chemical shifts reported as ppm. Mass spectra data were obtained on a Q-Tof MS spectrometer (Micromass, Manchester, England). Absorption spectra were measured on a Perkin-Elmer Lambda35 UV-Vis spectrophotometer. Fluorescence measurements were performed on an Aglilent Cary Eclipse fluorescence spectrophotometer. Fluorescence imaging was performed using an OLYMPUS FV1000 inverted fluorescence microscope with a 60× objective lens. High performance liquid chromatogram (HPLC) analyses and purification were performed using an
5
Agilent 1100 HPLC system with a diode array UV detector. The UV spectra of the HPLC peaks were obtained using on-line diode array detection.
2.2. Synthesis
2.2.1. Cy7-Dex
Cy7-Dex: Cy7 (200 mg, 0.26 mmol) and N,N-4-dimethylaminopyridine (DMAP, 38 mg, 0.31 mmol) were added into a 25 mL round-bottom flask with 10 mL dehydrated dichloromethane dissolved. The solution was stirred for 1 h under nitrogen. Then Dex (204 mg, 0.52 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 57 mg, 0.31 mmol) were added into the solution above which was stirred 12 h. The solvent was removed by reduced pressure distillation. The solid mixture was divided by column chromatography. The purified product weighed 267 mg, 68% yield. 1H NMR (500 MHz, CDCl3) δ 8.34 (d, J = 13.9 Hz, 2H), 7.47 (d, J = 10.1 Hz, 2H), 7.41 – 7.33 (m, 4H), 7.24 (dd, J = 13.8, 6.3 Hz, 2H), 7.15 (d, J = 8.0 Hz, 2H), 6.27 – 6.23 (m, J = 10.6 Hz, 4H), 6.03 (s, 2H), 5.09 (d, J = 17.3 Hz, 2H), 4.84 (d, J = 17.3 Hz, 2H), 4.40 (d, J = 8.8 Hz, 2H), 4.30 (s, 1H), 4.13 (d, J = 4.5 Hz, 4H), 3.46 (s, 1H), 3.22 (s, 1H), 3.08 – 2.94 (m, 2H), 2.72 (s, 4H), 2.56 (dd, J = 17.3, 12.2 Hz, 2H), 2.43 (d, J = 3.8 Hz, 5H), 2.34 – 2.22 (m, 6H), 2.15 (dd, J = 19.1, 11.1 Hz, 7H), 1.96 (d, J = 4.5 Hz, 2H), 1.90 – 1.80 (m, 4H), 1.75 (dd, J = 15.7, 10.2 Hz, 7H), 1.70 (s, 12H), 1.55 – 1.46 (m, 10H), 1.18 (dd, J = 7.2, 4.2 Hz, 2H), 1.02 (s, 6H), 0.88 (d, J = 7.1 Hz, 6H).. HRMS: M+, calcd for C86H106ClF2N2O12 +, m/z =1431.7397, found: m/z =1431.7395. 13C NMR (125 MHz, CDCl3) δ 205.54, 187.16, 173.28, 172.47, 167.44, 153.99, 150.75, 144.61, 142.24, 141.21, 129.36, 129.01, 127.99, 125.45, 124.84, 122.37, 110.98, 101.70, 100.30, 91.28, 71.50, 71.20, 68.41,
6
49.43, 48.65, 48.60, 44.53, 44.13, 36.11, 35.91, 34.49, 33.58, 32.57, 31.30, 28.32, 27.64, 27.10, 26.71, 26.21, 24.53, 23.25, 16.69, 15.13
2.2.2. Cy5-Dex
Cy5-Dex: Cy5 (172 mg, 0.26 mmol) and N,N-4-dimethylaminopyridine (DMAP, 38 mg, 0.31 mmol) were added into a 25 mL round-bottom flask with 10 mL dehydrated dichloromethane dissolved. The solution was stirred for 1 h under nitrogen. Then Dex (204 mg, 0.52 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (57 mg, 0.31 mmol) were added into the solution above which was stirred 12 h. The solvent was removed by reduced pressure distillation. The solid mixture was divided by column chromatography. The purified product weighed 223 mg, 61% yield. 1H NMR (500 MHz, CDCl3) δ 8.29 (t, J = 16.1 Hz, 2H), 7.60 (d, J = 9.1 Hz, 2H), 7.46 (d, J = 10.1 Hz, 2H), 7.25 (t, J = 8.9 Hz, 2H), 7.11-7.07 (m, 2H), 6.61 (dd, J = 34.1, 19.2 Hz, 1H), 6.26 (d, J = 15.1 Hz, 2H) 5.97 (s, 2H), 5. 26 (t, J =12.6 Hz, 2H), 5.02 (d, J = 17.3 Hz, 2H), 4.80 (d, J = 17.3 Hz, 2H), 4.35 (d, J = 8.8 Hz, 2H), 4.24 (s, 1H), 4.07 (t, J = 9.3 Hz, 4H), 3.40 (s, 1H), 3.18 (s, 1H), 2.97 – 2.94 (m, 2H), 2.72– 2.66 (m, 4H), 2.60 (t, J = 8.3 Hz, 2H), 2.41– 2.10 (m, 17H), 1.90 – 1.85 (m, 2H), 1.64 (s, 12H), 1.51 – 1.42 (m, 10H), 1.34-1.31 (m, 4H), 1.13-1.08 (m, 2H), 0.96 (s, 6H), 0.81 (d, J = 7.1 Hz, 6H). 0.73-0.70 (m, 4H). HRMS: M+, calcd for C81H101ClF2N2O12 +, m/z =1331.7317, found: m/z =1331.6270. 13C NMR (125 MHz, CDCl3) δ 206.12, 187.74, 180.87, 180.06, 168.02, 154.58, 145.20, 142.83, 141.79,138.15,130.11, 129.94, 129.59, 128.57, 126.04, 125.43, 122.95, 111.56, 106.58, 102.17, 100.88, 91.86, 72.09, 71.79, 68.99, 50.02, 49.42, 49.18, 45.11, 44.71, 36.69, 36.49, 34.92, 34.16, 33.15, 31.89, 28.84, 28.23, 26.80, 25.11, 17.28, 15.71. 7
2.2.3. Cy3-Dex
Cy3-Dex: Cy3 (161 mg, 0.26 mmol) and N,N-4-dimethylaminopyridine (DMAP, 38 mg, 0.31 mmol) were added into a 25 mL round-bottom flask with 10 mL dehydrated dichloromethane dissolved. The solution was stirred for 1 h under nitrogen. Then Dex (204 mg, 0.52 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (57 mg, 0.31 mmol) were added into the solution above which was stirred 12 h. The solvent was removed by reduced pressure distillation. The solid mixture was divided by column chromatography. The purified product weighed 202 mg, 57% yield. 1H NMR (500 MHz, CDCl3) δ 8.40 (t, J = 12.8 Hz, 1H), 7.38 (dd, J = 16.9, 7.4 Hz, 8H), 7.13 (d, J = 7.9 Hz, 2H), 6.83 (d, J = 13.0 Hz, 2H), 6.32 (d, J = 10.1 Hz, 2H), 6.08 (s, 2H), 5.30 (s, 2H), 4.97 (dd, J = 42.2, 17.3 Hz, 4H), 4.40 (d, J = 10.0 Hz, 2H), 4.16 (s, 4H), 3.07 (s, 4H), 2.62 (d, J = 9.4 Hz, 4H), 2.47 (d, J = 6.6 Hz, 4H), 2.38 – 2.21 (m, 6H), 1.84 (d, J = 13.0 Hz, 16H), 1.72 (s, 12H), 1.62 (s, 6H), 1.23 (d, J = 22.9 Hz, 6H), 1.07 (s, 6H), 0.90 (d, J = 6.9 Hz, 6H). HRMS: M+, calcd for C79H99F2N2O12+, m/z =1305.7161, found: m/z =1305.7164. 13C NMR (125 MHz, CDCl3) δ 205.55, 187.03, 173.84, 173.36, 167.28, 153.59, 150.73, 142.00, 140.60, 129.35, 128.99, 125.40, 124.80, 122.09, 111.10, 103.97, 101.49, 91.14, 71.91, 71.61, 68.31, 53.83, 49.06, 48.68, 48.50, 48.39, 44.41, 44.00, 35.89, 35.68, 34.37, 33.43, 32.45, 31.15, 29.27 , 28.15, 27.48, 27.21, 25.92, 24.48, 23.07, 16.44, 14.84.
2.3. Cellular localization imaging
Cells were cultured in DEME (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum. One day before imaging, cells were seeded in cell-culture dishes.
8
The next day, 10 µL Cy7-Dex, Cy5-Dex and Cy3-Dex (represented as Cyn-Dexs) solution were added to the media at a final concentration of 10 µM respectively, and then the cells were incubated for another 2 h at 37 °C with 5% CO2. The cells were washed three times with PBS before imaging. For each staining test, the fluorescent imaging pictures were obtained with an equal parameter for control. The cells were stained with Cy7, Cy5, Cy3 also the corresponding physical mixture of cyanine and Dex with the ratio of 1:2 (the concentration of Cyn+Dex is calculated according to that of cyanines) using the same procedure. After rinsing with PBS twice, the same sample was further stained with 100 nM Mito-Tracker Green FM for another 15 min and then imaged.
2.4. MTT assay
The cytotoxicity of compounds Cyn-Dexs, cyanines and Dex were evaluated following a reported approach.10 RAW264.7 cells were seeded at a density of 5×104 cells/mL in a 96-well plate with 100 µL of RPMI-1640 medium and grew for 24 h at 37 °C with 5% CO2. Then, these cells were treated with various concentrations of Cyn-Dexs (12.5, 25, 50, 100 and 200 µM) for 24 h. The cytotoxicity of these compounds was then determined using MTT assays. For comparison, the cytotoxicity of cyanines, Dex and Cyn+Dexs were studied using the same procedure. Absorbance at 490 nm was measured using a microplate reader. Cell survival was calculated as a percentage of the inhibition for viable cells.
2.5. Enzyme hydrolysis test The enzyme hydrolysis assay was carried out with reference to a published method.11 Substrate Cyn-Dexs 50 µM dissolved in DMSO were prepared. Incubate Cyn-Dexs with 9
pooled HLM 100 µg/mL, RLM 100 µg/mL, MLM 100 µg/mL respectively. The reaction was performed at 37 °C for 1 h. It was supposed that cyanine was one of the hydrolysates which had characteristic ultraviolet absorption. Here a HPLC method was used to separate the hydrolysates and detect hydrolysis rate of Cyn-Dexs. Also, the retention time of corresponding cyanines were determined acted as the control group. The chromatographic column was a 2.1150 mm Shim-pack VP-ODS column with a diameter of 5 μm. Eluent was a linear gradient containing acetonitrile/H2O (added 0.2% methanoic acid) found to be the most efficient eluents for this separation. For Cy5-Dex and Cy7-Dex, in 0-5 min, the water ratio was 90%, in 5-8 min, the water ratio changed from 90% to 60%, in 8-10 min, the water ratio changed from 60% to 5%, in 10-13 min, the water ratio was 5% and in 13-17 min, the water ratio was 90% with a flow rate of 0.4 mL/min. For Cy3-Dex, in 0-2 min, the water ratio was 80%, in 2-5 min, the water ratio changed from 80% to 60%, in 5-8 min, the water ratio changed from 60% to 5%, in 8-11 min, the water ratio was 5% and in 11-14 min, the water ratio was 80% with a flow rate of 0.4 mL/min. The wavelength for detection of Cy3, Cy5, and Cy7 was 556 nm, 643 nm, and 780 nm respectively for that they were the maximum absorption wavelength of Cy3, Cy5, and Cy7 in PBS buffer.
2.6. Cytokine assay The anti-inflammatory effects were determined with reference to a reported method.12 RAW264.7 cells were distributed into a 24-well plate with a density of 1.0×106 cells/mL. Cells were incubated for 24 h in the absence or presence of lipopolysaccharide (LPS) (1 µg/mL) and Polymyxin B (PMB, 20 µM), Dex (25 µM), Cyn-Dexs (12.5 µM), Cyn+Dexs (12.5 µM), respectively at 37 °C with 5% CO2. After 24 h incubation, supernatants were collected for 10
ELISA analysis. Cytokine IL-6 and TNF-α were determined by ELISA kits according to the instruction recommended by the manufacture. The optical density of the microplate was read at 450 nm and the IL-6 or TNF-α concentration for each sample was calculated from a standard curve.
3. Results and discussion
3.1. Synthesis of Cyn-Dexs Cy7, Cy5 and Cy3 were synthesized by previously published methods. 13 Cyn-Dexs were obtained by esterification reaction. First, cyanines were activated by 4dimethylaminopyridine (DMAP) in dehydrated dichloromethane solution under the protection of nitrogen. Then, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and Dex were added into the solution to produce the final products, Cyn-Dexs (Scheme 1, ESI†). The crude products were purified by Column Chromatography. Cyanine dyes and Cyn-Dexs were confirmed by 1H NMR, HRMS and 13
C NMR (ESI†).
3.2. Spectral properties of Cyn-Dexs
The normalized UV-vis absorption and fluorescence emission spectra of Cyn-Dexs were obtained to provide fundamental information for cell imaging (Fig. 1, Fig. S1). The corresponding data of single cyanines and Cyn+Dexs were obtained using the same procedure (Fig. S1). The case of Cy7 is taken as an example. The spectra of Cy7Dex and the corresponding Cy7 or Cy7+Dex were almost the same on the UV-vis absorption maxima (785 nm) and fluorescence emission maxima (815 nm). The same 11
situation also occurred in the case of Cy5 and Cy3 (Fig. S1). All these demonstrated that the introduction of Dex didn’t affect the spectral characteristic of cyanine dyes.
3.3. Cellular localization
Regardless of cell types (MCF-7, HL-7702, HepG-2), cells incubated with Cy7 alone or physical mixture Cy7+Dex exhibited little fluorescence, whereas cells incubated with Cy7-Dex showed a significant fluorescence signal (Fig. 2, Fig. S2, S4). The different imaging results probably resulted from the targeting effect of Dex. The localization of Cy7-Dex in living cells was tested by costaining experiments by using commercially available Mito-Tracker Green FM. The red fluorescence from the Cy7Dex evidently colocalized well with green fluorescence from Mito-Tracker Green FM. The Pearson’s correlation coefficient was 0.99 for MCF-7 cells (Fig. 2), 0.94 for HL7702 cells (Fig. S3) and 0.96 for HepG-2 cells (Fig. S5). These findings suggest that the targeting position of Cy7-Dex was mitochondria. The targeting effect of Cy7-Dex was in accordance with previous findings that the action position of Dex was mitochondria.14
To explore whether cyanine groups could influence the original drug effect of Dex, different cyanine groups, Cy5 and Cy3, were covalently linked with Dex using the same method as Cy7 to form Cy5-Dex and Cy3-Dex. In the cellular imaging experiments, the same trend can be observed in the case of Cy5 (Fig. S6-S11) and Cy3 (Fig. S12-S17) under the same circumstance. Cy5-Dex and Cy3-Dex could penetrate into cells and accumulated at mitochondria. Whereas, neither Cy5, Cy5+Dex, Cy3, nor Cy3+Dex could be found in cells.
12
Results demonstrated that different cyanine groups had little influence on the targeting position of Dex. All these findings further illustrated that the targeting position of Cyn-Dexs was mitochondria, similar to that of Dex. This finding means that Dex could probably be utilized as a mitochondrial targeting functional group. Furthermore, MTT assay was performed. Negligible toxicity can be observed even under a relative higher concentration employed (the concentration of Dex, cyanines, Cyn-Dexs and Cyn+Dexs was 25 μM, 12.5 μM, 12.5 μM and 12.5 μM respectively), thereby proving the viability of the proposed method (Fig. S18).
3.4. Enzyme hydrolysis test
The proposed prodrugs consist of drug moiety Dex and fluorophore component cyanines, which are combined via carboxylic ester bond. In vitro or in vivo, these prodrugs are anticipated to be divided into two parts, namely, the drug Dex and fluorophore cyanines, mainly through enzyme hydrolysis.15 Enzyme hydrolysis experiments were conducted to verify any difference among the hydrolysis rates of different Cyn-Dexs, which might have different influences on the drug effect of Dex.
Two main peaks were observed in the chromatogram of all the three Cyn-Dexs, namely, one for Cyn-Dexs and the other for cyanines, which acted as a contrast (Fig. S19). The case of Cy7 is taken as an example (Fig. 3). No peak that corresponded to Cy7 appeared in the reaction system of Cy7-Dex with any kind of microsome, thereby showing that Cy7-Dex could hardly be hydrolyzed in these microsomes. In comparison, a low peak appeared in the Cy5-Dex solution added with microsomes. After separation and collection, the hydrolysis rate was calculated as 0.26% for HLM, 13
0.83% for RLM, and 2.92% for MLM. A much higher peak appeared in the Cy3-Dex solution added with microsomes. After collection, the hydrolysis rate was calculated as 0.16% for HLM, 6.65% for RLM, and 13.00% for MLM.
From Cy7-Dex to Cy3-Dex, the hydrolysis rates gradually rose, thereby suggesting an easier tendency to be hydrolyzied. The difference in hydrolysis rates was speculated to have resulted from the different affinity of Cyn-Dexs to enzymes and the smaller size accelerated the interaction of Cyn-Dexs and enzymes. Results showed that different fluorophores probably had different influences on the fluorophore-conjugated drug, which potentially had diverse impacts on drug effect. Cy3-Dex was preferred for hydrolysis in the presence of microsomes. Thus, Cy3-Dex might have the best drug release effect and furthermore the minor influence on the therapeutic effect of Dex. By contrast, Cy7-Dex might be the better choice for simple drug monitoring in that it was stable under hydrolytic enzyme systems.
3.5. Anti-inflammatory effects of Cyn-Dexs
Interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) are key inflammatory cytokines in many infectious and inflammatory diseases.12, 16 The concentration of IL-6 and TNF-α can reflect the inflammatory level. LPS can induce marked increase in IL-6 and TNF-α production. Whereas, Dex can decrease the production and release of IL-6 and TNF-α.17 Cytokine assay was performed to examine whether the modification of Dex by cyanines could influence the drug effect of Dex, and to verify if a difference in the drug effects of Cy7-Dex, Cy5-Dex, and Cy3-Dex existed. RAW264.7 cells were incubated in the absence or presence of LPS and
14
PMB, Dex, Cyn-Dexs, Cyn+Dexs, respectively. Cytokine IL-6 and TNF-α were determined. PMB, a kind of antibiotic, acted as a positive control. Cyn+Dexs also acted as a control group to verify if cyanines could cause influence on the anti-inflammatory effect of Dex.
As shown in Fig. 4a and Fig. 4b, RAW264.7 cells with no LPS stimulation secreted a basal level of TNF-α and IL-6. However, with the addition of LPS, the TNF-α and IL-6 protein level increased by approximately four folds, and the additions of Dex, Cyn-Dexs, and Cyn+Dexs was able to inhibit the production of TNF-α and IL-6. After the cells were treated by Dex, the TNF-α production was reduced to approximately 50% and IL-6 production was reduced to 25%; these values for TNF-α and IL-6 were 36% and 33% in Cy7-Dex, 50% and 30% in Cy5Dex, 60% and 36% in Cy3-Dex, respectively. Cy5-Dex showed a similar inhibition ability with Dex in TNF-α and IL-6 secretion, but Cy3-Dex and Cy7-Dex exhibited lower or higher inhibition ability than Dex, respectively. Cy7+Dex, Cy5+Dex and Cy3+Dex, exhibited a similar inhibition ability toward Dex in TNF-α and IL-6 secretion, which indicated cyanines exerted little influence on the anti-inflammatory effect of Dex. Generally, different cyanines, Cy7, Cy5, Cy3 modified Dex exhibited different abilities to inhibit cytokine secretion. The inhibition effect of Cy3-Dex, Cy5-Dex, and Cy7-Dex on TNF-α production improved. No evident trend among the three Cyn-Dexs was observed in IL-6 production. b
4. Conclusion
15
In summary, Dex that is, covalently connected with Cy7, facilitated the penetration of Cy7 into cells and targeted mitochondria unlike single Cy7 and Cy7+Dex. The same trend occurred in the case of Cy5 and Cy3. Results have revealed a new mitochondrial targeting probe based on a commonly used anti-inflammatory drug, Dex, which can target mitochondria and achieve an anti-inflammatory effect. In addition, different cyanine groups had different influences on the hydrolysis rates of Cyn-Dexs and anti-inflammatory effect of Dex. In drug labeling and monitoring, the appropriate fluorophore should be selected to avoid possible false conclusions.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (Nos. 21272030, 21472016, 21306019, 21576042, 21421005).
References
1.
(a) F. G. De Felice and S. T. Ferreira, Diabetes, 2014, 63, 2262; (b) L. Galluzzi, N. Larochette, N.
Zamzami and G. Kroemer, Oncogene, 2006, 25, 4812; (c) S. Bonnet, S. L. Archer, J. Allalunis-Turner, A. Haromy, C. Beaulieu, R. Thompson, C. T. Lee, G. D. Lopaschuk, L. Puttagunta, S. Bonnet, G. Harry, K. Hashimoto, C. J. Porter, M. A. Andrade, B. Thebaud and E. D. Michelakis, Cancer Cell, 2007, 11, 37; (d) S. Fulda, L. Galluzzi and G. Kroemer, Nat Rev Drug Discov, 2010, 9, 447. 2.
(a) X. Xiong, F. Song, J. Wang, Y. Zhang, Y. Xue, L. Sun, N. Jiang, P. Gao, L. Tian and X. Peng, J Am
Chem Soc, 2014, 136, 9590; (b) M. Abo, Y. Urano, K. Hanaoka, T. Terai, T. Komatsu and T. Nagano, J Am Chem Soc, 2011, 133, 10629; (c) T. T. Chen, X. Tian, C. L. Liu, J. Ge, X. Chu and Y. Li, J Am Chem Soc, 2015, 137, 982. 16
3.
(a) C. W. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Lam and B. Z. Tang, J Am Chem Soc, 2013, 135,
62; (b) S. Wu, Q. Cao, X. Wang, K. Cheng and Z. Cheng, Chem Commun (Camb), 2014, 50, 8919; (c) A. R. Sarkar, C. H. Heo, H. W. Lee, K. H. Park, Y. H. Suh and H. M. Kim, Anal Chem, 2014, 86, 5638. 4.
Y. Li, Z. Lü, M. Liu and G. Xing, Chinese J Org Chem, 2016, 36, 962.
5.
(a) B. Guo, W. Zhang, S. Xu, J. Lou, S. Wang and X. Men, Life Sci, 2016, 144, 1; (b) P. Londono, A.
Komura, N. Hara and D. Zipris, Clin Immunol, 2010, 135, 401; (c) S. Benedetti, B. Pirola, P. L. Poliani, L. Cajola, B. Pollo, R. Bagnati, L. Magrassi, P. Tunici and G. Finocchiaro, Gene Ther, 2003, 10, 188; (d) S. Tsurufuji, K. Sugio and F. Takemasa, 1979; (e) R. Mohammadi, M. Azad-Tirgan and K. Amini, Injury, 2013, 44, 565. 6.
(a) G. Kroemer and J. C. Reed, Nat Med, 2000, 6; (b) J. D. Ly, D. R. Grubb and A. Lawen,
Apoptosis, 2003, 8; (c) N. Zamzami, P. Marchetti, M. Castedo, C. Zanin, J. L. Vayssiere, P. X. Petit and G. Kroemer, J Exp Med, 1995, 181. 7.
(a) E. J. Kim, S. Bhuniya, H. Lee, H. M. Kim, C. Cheong, S. Maiti, K. S. Hong and J. S. Kim, J Am
Chem Soc, 2014, 136, 13888; (b) C. Lechner, V. Reichel, U. Moenning, A. Reichel and G. Fricker, Eur J Pharm Biopharm, 2010, 75, 284; (c) J. B. Wu, T. P. Lin, J. D. Gallagher, S. Kushal, L. W. Chung, H. E. Zhau, B. Z. Olenyuk and J. C. Shih, J Am Chem Soc, 2015, 137, 2366; (d) T. Xing, C. Mao, B. Lai and L. Yan, ACS Appl Mater Interfaces, 2012, 4, 5662. 8.
(a) H. Zhang, J. Fan, J. Wang, B. Dou, F. Zhou, J. Cao, J. Qu, Z. Cao, W. Zhao and X. Peng, J Am
Chem Soc, 2013, 135, 17469; (b) H. Zhang, J. Fan, J. Wang, S. Zhang, B. Dou and X. Peng, J Am Chem Soc, 2013, 135, 11663. 9.
(a) J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J. H. Ryu and J. Yoon, J Am Chem Soc, 2014, 136,
5351; (b) Y. Li, Y. Sun, J. Li, Q. Su, W. Yuan, Y. Dai, C. Han, Q. Wang, W. Feng and F. Li, J Am Chem Soc, 17
2015, 137, 6407. 10.
X. Jia, Q. Chen, Y. Yang, Y. Tang, R. Wang, Y. Xu, W. Zhu and X. Qian, J Am Chem Soc, 2016, 138,
10778. 11.
(a) S. C. Liang, G. B. Ge, H. X. Liu, H. T. Shang, H. Wei, Z. Z. Fang, L. L. Zhu, Y. X. Mao and L. Yang,
J Pharm Biomed Anal, 2011, 54, 236; (b) K. Abass, P. Reponen, J. Jalonen and O. Pelkonen, Pestic Biochem Physiol, 2007, 87, 238. 12.
S. Mianji, Y. Hamasaki, S. Yamamoto and S. Miyazaki, Int J Immunopharmacol, 1996, 18, 339.
13.
(a) X. Chen, X. Peng, A. Cui, B. Wang, L. Wang and R. Zhang, J Photochem Photobiol A, 2006,
181, 79; (b) H. Wu, S. C. Alexander, S. Jin and N. K. Devaraj, J Am Chem Soc, 2016, 138, 11429; (c) Y. Guan, Y. Zhang, L. Xiao, J. Li, J. P. Wang, M. D. Chordia, Z. Q. Liu, L. W. Chung, W. Yue and D. Pan, Mol Pharm, 2017, 14, 1. 14.
(a) C. V. Demonacos, N. Karayanni, E. Hatzoglou, C. Tsiriyiotis, D. A. Spandidos and C. E. Sekeris,
Steroids, 1996, 61; (b) C. Tsiriyotis, D. A. Spandidos and C. E. Sekeris, Biochem Bioph Res Co, 1997, 235, 349; (c) C. Demonacos, R. Djordjevic-Markovic, N. Tsawdaroglou and C. E. Sekeris, J Steroid Biochem Mol Biol, 1995, 55, 43. 15.
C. Branneby, P. Carlqvist, A. Magnusson, K. Hult, T. Brinck and P. Berglund, J Am Chem Soc, 2003,
125, 874. 16.
N. Yingkun, W. Zhenyu, L. Jing, L. Xiuyun and Y. Huimin, Inflammation, 2013, 36, 242.
17.
C. M. Williams and J. W. Coleman, Immunology, 1995, 86, 244.
Biographies
Siyuan Liu, received his B.S. degree in 2015 from Yanshan University, China. He is pursuing his master’s degree under the guidance of Prof. Shiguo Sun in State Key Laboratory of Fine 18
Chemicals, School of Chemistry, Dalian University of Technology. His research interest focuses on organic probe for molecular detection.
Guangbo Ge received his B.S. degree in applied chemistry in 2003 from Dalian University of Technology, Ph.D degree in biochemical engineering in 2009 from Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS). Currently he is a full professor at Dalian Institute of Chemical Physics, CAS. His current studies are focused on the development of new techniques or bioanalytical methods for key enzymes participating in endobiotic and xenobiotic metabolism, etc.
Weibing Dong received his Ph.D. degree in applied chemistry in 2008 from Dalian University of Technology. He is currently an associate professor in Liaoning Normal University. His research interests include drug discovery, enzyme inhibitors and so on.
Xiaojun Peng received his Ph.D. degree in applied chemistry in 1989 from Dalian University of Technology. He is currently a professor in State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, China and the director of State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology. His current studies are focused on functional dye and application, fluorescent probe, fine chemical cleaning preparation technique.
Fengyu Liu received her Ph.D. degree in 2006 from Dalian University of Technology. She is currently an associate professor in State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, China. Her research interests include
19
electrochemiluminescence, luminescent/fluorescent sensors, DNA-targeting molecules designing, synthesizing and application.
Su-Shing Chen received his BS in Mathematics from the National Taiwan University, and PhD in Mathematics from the University of Maryland, College Park. His research consists of three stages: Mathematics, Artificial Intelligence, and Computational Biology. He has published hundreds of journal and conference papers on Bioinformatics, Information Technology and Digital Libraries, Neural Networks, Computer Vision and Image Processing, Intelligent Information Systems, Advanced Manufacturing Systems, Dynamical Systems, Manifold Theory etc.
Shiguo Sun received his Ph.D. degree in 2003 from Dalian University of Technology and is currently a professor and doctoral supervisor in the School of Chemistry&Pharmacy, Northwest A&F University, China. His research interests include functional molecules with special light or electrochemistry properties, self-assembly chemistry of carbon nanotube/graphene material for DNA and protein sensing, drug delivery and fluorescence tracing, and electrochemiluminescence and luminescence sensors.
1.2
UV-vis
F
0.8
Normalized F
Normalized A
1.0
0.6 0.4 0.2 0.0 600
650
700
750
800
Wavelength/nm
20
850
900
Fig. 1 Normalized UV−vis absorption and fluorescence mission spectrum of Cy7-Dex (4 μM) in PBS (pH 7.4).
a
b
c
d
e
f
g
h
i
j
k
l
Fig. 2 Fluorescence imaging of MCF-7 cells stained with Cy7 (10 μM), Cy7+Dex (10 μM), Cy7-Dex (10 μM) respectively (Ex, 635 nm, Em, 651-751 nm), and Mito-Tracker Green FM (100 nM, Ex, 490 nm, Em, 500-520 nm). (a-c) Bright field images of MCF-7 cells stained with Cy7, Cy7+Dex, Cy7-Dex respectively, (d-f) fluorescence images of MCF-7 cells stained with Cy7, Cy7+Dex, Cy7-Dex respectively, (g) bright field image of MCF-7 cells stained with Cy7-Dex, (h) fluorescence image of MCF-7 cells stained with Mito-Tracker Green FM, (i) 21
fluorescence image of MCF-7 cells stained with Cy7-Dex, (j) merged image of h and I, (k) correlation plot of the intensities of Mito-Tracker Green FM and Cy7-Dex (Rr, 0.99), (l) normalized intensity profile of regions of interest across MCF-7 cells.
uV(x100,000) 4.0 3.0 2.0 1.0 0.0 0.0
2.5
5.0
7.5
10.0
12.5
15.0
min
Fig. 3 The enzyme hydrolysis test of Cy7-Dex. From top to bottom correspond to product (Cy7), substrate (Cy7Dex), HLM+substrate, MLM+substrate, RLM+substrate.
a
60
500
20
LP S
PM
Bl an k
De x
Cy B 3De Cy x 5De Cy x 7D Cy ex 3+ D Cy ex 5+ D Cy ex 7+ De x
PM
LP S
0 Bl an k
0
40
De x
Cy B 3De Cy x 5De Cy x 7D Cy ex 3+ D Cy ex 5+ D Cy ex 7+ De x
1000
IL-6 (pg/mL)
TNF- (pg/mL)
1500
E
Fig. 4 Inhibition effects of Dex, Cyn-Dexs and Cyn+Dexs on the LPS-induced increase in inflammatory cytokines production. Cells were incubated for 24 h in the absence or presence of LPS (1 µg/mL) and PMB (20 µM), Dex (25
22
µM), Cyn-Dexs (12.5 µM), Cyn+Dexs (12.5 µM), respectively at 37 °C with 5% CO2. (a) Inhibition effects on TNF-α production, (b) inhibition effects on IL-6 production. Data are means ± S.E.M. (n = 3), *P<0.05 vs LPS alone.
23