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Cancer-Targeting Functionalization of Selenium-Containing Ruthenium Conjugate with Tumor Microenvironment-Responsive Property to Enhance Theranostic Effects.
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Accepted Article
Title: Cancer-targeting Functionalization of Selenium-Containing
Ruthenium Conjugate with Tumor Microenvironment-Responsive
Property to Enhance Theranostic Effects
Authors: Zhennan Zhao, Pan Gao, Yuanyuan You, and Tianfeng Chen
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To be cited as: Chem. Eur. J. 10.1002/chem.201705561
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Cancer-targeting Functionalization of Selenium-Containing
Ruthenium Conjugate with Tumor Microenvironment-Responsive
Property to Enhance Theranostic Effects
Abstract: A mutifunctional ruthenium (Ru)-based conjugate Ru-BSe
was designed and sythesized. The Ru complex with favorable
bioimaging function was covalently linked with a cancer-targeted
molecule that could be effectively internalized by the tumor to realize
enhanced theranostic effects. The pH-response of the Ru conjugate
in tumor acidic microenvironment causes ligand substitution and
release of therapeutic complex. This activated complex remains inert
to the reducing biomolecule-glutathione and terminally locates in
mitochondria, where it triggers oxidative stress, and activates
intrinsic apoptosis. Real-time monitoring reveals that this Ru
conjugate could selectively accumulate in tumor tissue in vivo, which
significantly suppress tumor progression and alleviate the damage to
normal organs, realizing the precise cancer theranosis.
Introduction
Cancer theranosis offers an appealing strategy in cancer
treatment by combination of chemotherapy with early and timely
diagnosis of tumor carcinogenesis.[1] Typically, in vivo
applications of chemotherapeutic agents are hindered by the
drawbacks of low bioavailability, lack of selectivity toward tumor
and intrinsically non-fluorescence etc. [2] Taking advantages of
targeted drug delivery system (DDS), small molecules could be
developed as theranostic prodrug that being real-time monitored
and selectively delivered to tumors. [3] The prodrug can be
activated by intracellular thiols and changes in pH to realize
increased drug bioavailability in the tumor.[4]
Considering that some transition-metal complexes display
favorable phosphorescent properties, it is accessible that metal
complexes could be developed as theranostic agents. [5] Among
anticancer metallodrugs, Ruthenium (Ru) complexes are
potential alternatives for platinum-based cancer drugs. Currently,
the sodium analog of KP1019, i.e., sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
{KP-1339/IT139,
Na
trans[RuCl4(Hind)2]} was selected for clinical trials, while Ru(II)
polypyridyl complexes, TLD-1433, entered phase IB clinical trials
as a photodynamic therapy agent for with bladder cancer.[6]
Meanwhile,
phosphorescent
metal
complexes
with
advantageous photophysical features are desirable for biological
imaging application. Therefore, by tuning auxiliary ligands, the
properties of complexes can be modified to achieve both therapy
and diagnosis functions within a single molecule.[7] Another
effective strategy that uses metal complexes containing
[a]
Z. Zhao, P. Gao, Y. You, Prof. T Chen
Department of Chemistry
Jinan University, Guangzhou 510632 (P. R. China)
E-mail: tchentf@jnu.edu.cn
anticancer ligands with clear action mechanisms has kindled
great interest of chemists. Recently, studies found that
complexes, with conjugation of chemotherapeutic agents,
display potent inhibitive effects on proliferation of cancer cells. [8]
The anticancer potency of selenium (Se)-containing
compounds has been well-documented in previous studies.[9]
Among these agents, organic selenadiazole derivatives exhibit
outstanding anticancer activities,[10] but their drawbacks, like
poor solubility and unsatisfied luminescent properties, limit their
theranostic applications in vivo. Therefore, studies have been
conducted to solve this problem employing metal complexes.
Remarkably, Chao et al. have developed phosphorescent Secontaining iridium(III) complexes that are enable for tracking of
mitochondrial morphological changes in cells.[11] Our previous
studies showed that the introduction of Se-containing ligand into
Ru complexes effectively enhanced the apoptosis-inducing
activity against cancer cells, and targeted DDS was capable to
enhance the selectivity of metal complexes towards cancer
cells.[12] Therefore, the introduction of Se into luminescent metal
complexes and the further cancer targeting design may be a
potent strategy for discovery of theranostic agents for precise
cancer treatment.
Bearing these facts in mind, in the study, we have designed
and synthesized a Se-containing Ru conjugate covalently linked
with a cancer-targeted molecule (Ru-BSe, Scheme 1a) that
could selectively accumulate in cancer cells during circulation in
vivo to realize enhanced theranostic effects and alleviate the
systemic toxicity of metal complexes. The phosphorescent
emission property of Ru(II) conjugates allows the real-time
tracking and imaging of the drug inside the biological systems.
By utilizing the cancer targeting design, biotinylated Ru-BSe can
be selectively internalized by tumor cells, thus minimizing side
effects towards normal organs in tumor-bearing xenograft mice.
Results and Discussion
Rational design and tumor microenvironment responsive
property of Ru-BSe
In this study, a series of Ru complexes with various structures
were designed and synthesized to examine the effects of Se
substitution and targeted modification on the activity of complex
(Scheme 1). The synthetic procedures of Ru(II) complexes were
illustrated in Figure S1 and the chemical structures were
characterized by ESI-TOFMS analysis, CHN elemental analysis,
1
H and 13C NMR spectroscopy (Figure S2-S16). The
photophysical data (Table S1, Figure S17) show that Ru(II)
complexes possess red phosphorescence (λem≈ 600 nm)
Supporting information for this article is given via a link at the end of
the document.
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Zhennan Zhao, Pan Gao, Yuanyuan You and Tianfeng Chen*
10.1002/chem.201705561
Chemistry - A European Journal
Scheme 1. Rational design and theranostic function of Ru(II) conjugate.
(a) Chemical structures of Ru(II) compounds in this work. (b) The cancer
targeted selenium-containing conjugate Ru-BSe is capable for tumor
diagnosis and therapy.
with long emission lifetime (τem=0.42~0.64 μs), which allows the
real-time tracking and imaging of drug in the biological systems.
The protonation/deprotonation processes can perturb the
electronic properties of the molecules, especially metal
complexes, [5b, 13] which can result in the change on coordination
ability of the ligand. Therefore, we examined the effect of the
protonation/deprotonation of the imidazole ring within the Bioben
ligand on the stability of Ru(II) conjugate in Na2HPO4/citric acid
buffer.[14] When the pH was changed from weakly basic (8.5) to
acidic (3.1), Ru-BSe experienced spectral changes, including
increased ligand absorption band (290-350 nm), declining
metal–to–ligand charge transfer (1MLCT) absorption band (400530 nm) and decreased emission (3MLCT excited state) (Figure
S18). Additionally, we also found time-dependent changes in the
spectrum of Ru-BSe after incubation in aqueous solution at pH=
6.86 (Figure 1a-b). Such a weakly acidic condition simulates the
environments of solid tumors and hypoxia tissues, which
indicates the tumor microenvironment-responsive property of the
Ru conjugate.[15] This hypothesis was further verified by ESITOFMS analysis, demonstrating these changes were attributed
to the descomposition of Ru-BSe in aqueous solution.
Specifically, the peak of [Ru(phenSe)2(H2O)2]+ was detected
(Figure 1c), suggesting ligand Bioben could be released from
Ru-BSe (Figure S19). Additionally, unlike Pt or Au complexes
[16]
, the aqueous product [Ru(phenSe)2(H2O)2]+ remained stable
in the presence of glutathione (GSH), as no distinct peak
ascribed for the adduct of Ru-BSe and GSH was detected after
6 and 72-h incubation (Figure 2a). GSH is a major antioxidant
with detoxifying properties inside cancer cells[17], preventing cell
damage from the exposure of heavy metals. Further
examination showed that the pH-responsive release of Ru-BSe
Figure 1. Biological response of Ru(II) conjugate in weakly acidic
environment. (a) UV/Vis spectrum and (b) the emission spectrum of Ru-BSe
in PBS solution (pH=6.86, containing 5% DMSO) after incubation at different
time point. Inset: Plot of relative absorption (A/A0, at 340 nm)/emission
intensity (I/I0, at 600 nm) versus the incubation time. A0 and I0 represent the
absorption and emission intensity at 0 h, respectively. (c) The decomposition
of Ru-BSe in MeOH/Milli-Q H2O solution (3:7, v:v ,containing 10 mM
NH4HCO3, pH=6.86) after 24-h incubation before recording by ESI-TOFMS.
was temperature dependent (Figure S20). On the other hand,
Ru-BSe kept stable in DMSO and human plasma solution
(Figure 2b), implying the significance of weakly environment on
the activation of Ru-BSe conjugate. Taken together, these
results indicate the significance of weakly acidic environment on
the decomposition of the conjugate, which could promotes the
release of therapeutic complex, thus minimize the effect of
covalent cancer-targeted unit on the anticancer activity of the
prodrug.
Selective recognition of cancer cells by Ru-BSe to realize
tumor diagnosis in vivo
As expected, the formation of Se-containing Ru(II)
complexes enhances the solubility of selenadiazole derivatives,
resulting in significant influence of cellular uptake (Figure 3a).To
verify our hypothesis of the cancer-targeted potency of Ru-BSe,
biotin receptor-positive cancer cells (HeLa) and biotin receptornegative normal cells (L02)[18] were incubated with Ru(II)
conjugate. We found targeted Ru-BSe was preferentially
uptaken by HeLa cancer cells (Figure 3a and S21). Moreover,
the pretreatment of cells with excess biotin partially blocked the
uptake of biotinylated Ru-BSe. Furthermore, we employed a coculture model of HeLa and L02 cells to investigate the selective
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Figure 3. Selective uptake of targeted Ru(II) conjugate by tumor cells. (a)
Cellular uptake of ligand PhenSe (40 μM) and Se-containing complexes (20
μM) in HeLa and L02 cells. Selective induction of apoptosis by the treatment of
Ru-BSe (20 μM) for 24 h in HeLa-L02 co-culture model was examined by
TUNEL-Cell tracker blue co-staining. DNA fragmentation in cells were (b)
observed by fluorescent microscopy and (c) quantified by flow cytometric
analysis.
Figure 2. The decomposition process of Ru-BSe conjugate. (a) After the
incubation for different period of time, the reaction between Ru-BSe (10 μM)
and GSH (50 μM) was monitored by ESI-TOFMS. The mixture was incubated
in the MeOH/Milli-Q H2O solution (3:7, v:v ,containing 10 mM NH4HCO3,
pH=6.86) at 37℃ for 24 h. (b) Stability of Ru(II) conjugate in various condition.
Ru-BSe was incubated in different conditions: (a) in DMSO for 0 h at 25℃; (b)
in DMSO for 72 h at 25℃; (c) in human blood plasma for 72 h at 37℃
(diazepan used as internal standard); (d) diazepan alone in human blood
plasma for 72 h at 37℃. These samples were analyzed by high-performance
liquid chromatography (HPLC) system (monitor at 300 nm).
induction of apoptosis by Ru-BSe in cancer cells using TUNEL
assay. As shown in Figure 3b, formation of DNA fragmentation
was detected in most HeLa cells (24 h), which was hardly found
in L02 cells. The ratio of apoptotic HeLa cells in the co-culture
population was increased to 20.8% after the treatment of RuBSe, which was higher than that of L02 cells (at 2.1%) (Figure
3c). Moreover, the addition of endocytosis inhibitor influenced
the cellular uptake of the complexes in different degree (Figure
S22), indicating the important contribution of receptor-mediated
endocytotic (energy-dependent) pathway to the uptake of RuBSe.
In vivo examination was also performed in HeLa-inoculated
xenograft mice to investigate the cancer targeting ability of Ru(II)
complexes. As reflected by the fluorescent signals of Ru(II)
complexes (Figure 4a), Ru-BSe exhibited much higher
accumulating efficacy in tumor site within 72-h intravenous
injection, than that of Ru-Se. Ex vivo imaging can clearly display
the biodistribution of Ru(II) complexes in the main organs. The
results showed that Ru-BSe was selectively internalized by
tumors rather than other organs, while Ru-Se preferentially
accumulated in liver and spleen (Figure 4b). The biodistribution
of Ru(II) complexes was further verified by determining Ru
content in organs after 30 days treatment. The Ru content in RuBSe-treated tumor was significantly higher than that of normal
tissues, and approximately 3 times of Ru-Se group (Figure 4c).
Activation of mitochondrial dysfunction by Ru-BSe induced
intrinsic apoptosis
Consistent with our previous studies, Se substitution significantly
enhanced the anticancer efficacy of Ru(II) complexes (Table
1).[12a] Firstly, we demonstrated that, the therapeutic metallodrug
[Ru(phenSe)2Cl2] and its aquation product [Ru(phenSe)2Cl2] (aq)
both exhibit potent anticancer activities,
Figure 4. Precise tumor diagnosis of targeted Ru(II) conjugate in vivo. (a)
Fluorescence imaging monitors the accumulation and distribution of Ru-Se
and Ru-BSe (4 μmol/kg) in HeLa xenografts nude mice at different time points.
Fluorescent filter sets (excitation/emission, 500/650 nm) are used for in vivo
fluorescent imaging. (b) Ex vivo-dissected organs (brain, heart, liver, spleen,
lung, kidney, and tumor tissue) fluorescent images of the Ru-Se and Ru-BSe injected xenograft mice after 72 h tail-vein injection. (c) Biodistribution of Ru in
main organs after 30-day treatment of Ru(II) complexes in HeLa xenografts
nude mice by using ICP-AES.
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Table 1. Cytotoxicity of Ru(II) complexes on cancer and normal cell lines.
IC50 (μM)
HeLa A549 MCF-7
MDAHepG2
MB-231
SI*
L02
NCM460
Ru-IP
67.8 104.3
52.7
50.2
57.8
88.4
95.3
1.30
Ru-Se
21.0
19.4
21.2
18.4
17.8
20.3
25.4
0.97
Ru-Bio
44.7
58.6
87.5
57.4
51.8
126.3
112.6
2.83
Ru-BSe
15.3
17.4
21.0
22.7
34.1
77.6
68.5
4.24
Cisplatin
16.5
16.9
15.7
21.7
13.6
7.3
9.4
0.46
*SI (Safe Index)=IC50 (L02)/IC50 (HeLa), which reflects the side effect of complexes.
Figure 5. Ru-BSe triggers mitochondria dynfunction and ER stress in
HeLa cells. (a) Fluorescent image and (b) emission intensity analysis of RuBSe (20 μM) and Mito-tracker after incubation for 6 h. Cells were visualized in
the green channel for Mito-tracker (λex=488 nm, λem=500–560 nm) and red
channel for Ru complex (λex=500 nm, λem=600–650 nm), respectively. (c)
Change of mitochondrial membrane potential examined by JC-1. (f) Western
blot analysis for expression levels of Bcl-2 family members and ER stress
related proteins that regulated by treatment of Ru conjugates (20 μM) for 72 h.
(e) Induction of ROS generation by Ru(II) complexes (20 μM) in 4 h. (f)
Cytoplasmic calcium ion level in HeLa cells exposed to 20 μM Ru conjugates
(30 min).
but they showed low selectivity between cancer and normal cells
(Table S2). After targeting functionalization, Ru-BSe
demonstrated a broad range of anticancer action towards cancer
cell lines. Especially, Ru-BSe displayed favorable tumor
inhibitory effects toward HeLa cells with IC50 value of 15.3 μM,
which was competitive with that of cisplatin (16.5 μM). Despite
this potency, the prodrug Ru-BSe showed higher selectivity
against cancer cells compared with cisplatin and Ru-Se (without
target unit). Studies have proven that the anticancer actions of
drugs are associated to their intracellular localization of
anticancer drugs.[19] Previously, we found that the cellular
localization of iron(II) polypyridyl complexes determines their
anticancer action mechanisms.[20] Cytosolic Iron(II) complexes
exhibited anticancer and antiangiogenic potencies by targeting
mitochondria to trigger cancer cell apoptosis.[21] Therefore, in
this study, we next set out to elucidate the relationship between
the anticancer action mechanisms and cellular localization of
Ru-BSe. Ru-BSe accumulated in the cytoplasm after incubation
for 6 h. The notable merge of mitochondria (green) and the
conjugate (red) was observed (Figure 5a), with Pearson’s
colocalization coefficient at 0.90. Meanwhile, quantification of
the luminescence intensity further confirmed the overlap of
mitochondria and Ru-BSe (Figure 5b). Therefore, we also
examined the effect of Ru-BSe on the mitochondrial membrane
potential (Δψm) by JC-1 flow cytometric analysis. Ru-BSe
significantly induced dose-dependent disruption of Δψm in HeLa
cells, as reflected by the shift of JC-1 fluorescence from red to
green in cells (Figure 5c). In contrast, only slight change in Δψm
of cells was observed in cells exposed to Ru-Bio. The loss of
Δψm in cells caused by the mitochondrial dysfunction is closely
connected with the regulation by Bcl-2 family proteins.[22] Our
results showed that Ru-BSe dramatically suppressed the
expression of Bcl-2 (anti-apoptotic proteins) and upregulated the
expression of Bax (pro-apoptotic proteins) in HeLa cells (Figure
5d). The mitochondrial respiratory chain is a potential source of
ROS, thus the observation of Ru-BSe-inducted mitochondrial
dysfunction encouraged us to examine intracellular levels of
reactive oxygen species (ROS).[23] Treatments of Se-containing
complexes Ru-Se and Ru-BSe significantly triggered excessive
generation of ROS in cells within 4 h, while the complexes
without Se induced much lower ROS generation (Figure 5e).
The accumulation of excessive ROS generation triggers
endoplasmic reticulum (ER) stress, promoting apoptosis in
cancer cells.[24]. The increased expression level of ER stress
related proteins (p-PERK and CHOP) verified elevated ER
stress level in HeLa cells after the treatment of Se-containing
Figure 6. Se-containing Ru(II) conjugates induced apoptosis in HeLa
cells. (a) Treatment of Ru(II) conjugates activated caspase3/9 activity in HeLa
cells, which was determined by synthetic fluorescent substrates. (b) After
treatment of Ru(II) conjugates for 72 h, apoptotic cell death was examined by
flow cytometric analysis.
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Complex
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complex. The release of stored calcium in ER is a sensitive
indicator of ER stress,[25] so we tested the calcium ion levels in
cytoplasm by using the calcium fluorescence probe Fluo-3AM. A
large elevation in the cytoplasmic Ca2+ was detectable in cells
exposed to 20 μM Ru-BSe for 30 min, indicating the ROSmediated ER stress was activated (Figure 5f). A rise in
cytoplasmic Ca2+ contributed to the rapid increase of cation in
mitochondria, which would further promote the bioenergetics
failure of the organelle, leading the activation of intrinsic
apoptosis pathways and caspases proteins. Correspondingly,
we also found the treatment of Se-containing Ru(II) complex
contributed to the activation of caspase-3 and 9 (Figure 6a),
which are well-known as the important mediators of intrinsic
apoptosis. Finally, propidium iodide (PI)-flow cytometric analysis
was performed to examine the induction of apoptosis in cancer
by Ru(II) complexes. As reflected by the Sub-G1cell population
(Figure 6b), exposure of HeLa cells to Ru-BSe (40 μM) for 72 h
resulted in an increased percentage of apoptotic cells from 1.1%
to 62.9%. Taken together, the introduction of Se(IV) species into
Ru(II) complexes effectively triggered mitochondrial dysfunction
by regulation of Bcl-2 family proteins, thus leading activation of
intrinsic apoptosis via ROS-mediated ER stress signal pathway.
Targeted delivery of Ru-BSe enhanced anti-tumor efficacy
and alleviated systemic toxicity
Figure 7. Targeted Ru-BSe conjugate nullifies systemic toxicity in HeLa
xenograft mice in vivo. (a, b) The change in tumor volume and body weight
(dose, 4 μmol/kg, every 2 days). (d) TUNEL staining analysis of tumor cell
apoptosis. (d) The toxicity of Ru(II) complexes on major organs after 30-day
treatment (a dose of 4 μmol/kg every 2 days). The arrows highlight the site
with pathological change.
Finally, the in vivo antitumor activity and systemic toxicity of RuBSe were evaluated by using HeLa-inoculated xenograft mice.
After the treatment with the complexes for 30 days, the tumor
inhibition rates were 44.3% for Ru-Se and 64.2% for Ru- BSe,
respectively (Figure 7a). Additionally, no death or obvious
change in body weight of mice was observed at this dosage
(Figure 7b). Moreover, the results of TUNEL staining assay
illustrated that Se-containing complexes induced tumor cells
Figure 8. Hematological analysis of healthy and tumor-bearing nude mice, and those treated with Ru-Se or Ru-BSe (4 μmol/kg) for 30 days. The nude
mice in healthy and tumor-bearing groups were treated with saline. The tested biochemical indexes included uric acid (UA), blood urea nitrogen (BUN), creatinine
(CREA), triglyceride (TG), aminotransferase (AST), total protein (TP), albumin (ALB) and creatine kinase (CK).
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Conclusions
We have demonstrated the rational desing of Se-containing
conjugate Ru-BSe and its application as a potential theranostic
agent for precies tumor diagnosis and therapy. The protonation
process accelerates the decomposition of Ru-BSe, which
promotes release of therapeutic complex from the DDS. The
activated product remains inert to GSH and possesses high
specificity to mitochondria, where it actives the overproduction of
ROS, resulting in intrinsic apoptosis in cancer cells through the
induction of endoplasmic reticulum stress signal pathway. The in
vivo xenograft mice model demonstrates that Ru-BSe
possesses enhanced theranostic effects for cancer treatment
and reduced systemic toxicity. Such all-in-one theranostic
strategy provides a new approach for the rational design of
phosphorescent metal complexes that are competent for precise
tumor diagnosis and therapy.
Experimental Section
Materials and general instruments: Ruthenium chloride
hydrate, cisplatin, metabolic inhibitors, endocytosis inhibitors,
2’,7’-dichlorofluorescin diacetate (H2DCF-DA) and Fluo3-AM
solution (1 mM in DMSO, ≥99.0%) were obtained from SigmaAldrich. The TUNEL assay kit was purchased from Roche
Applied Science. Other chemical agents from commercial
sources were used as received without further purification,
including
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
(EDCI),
N-Hydroxysuccinimide
(NHS),
dichloromethane (DCM), and N,N-Dimethylformamide (DMF).
Stock solutions of cisplatin (3 mM) were prepared in saline,
while Ru complexes (5 mM) were dissolved in DMSO. All stock
solutions were stored at -20°C, thawed and diluted with culture
medium prior to each experiment. The 1H and 13C NMR
spectrum of samples in DMSO-d6 solution were recorded on
Bruker AVANCE AV 500 NMR spectrometer, with TMS used as
an internal reference. A HORIBA Fluorolog system was
employed for emission lifetime measurements. Luminescent
quantum yields of complexes were measured with degassed
[Ru(bpy)3](ClO4)2 in acetonitrile (фr=0.062) as reference.
Fluorescent images of cells were recorded on EVOS FL auto
microscope (Life Technologies). All animal experiments were
performed under the supervision of the Animal Experimentation
Ethics Committee of Jinan University.
Synthesis of L1: The ligand L1 was prepared as previously
described by Schiffmann et al.[26]
Synthesis of L1a: 3,4-diaminobenzoic acid (4.56 g, 30 mmol)
and copper acetate (5.50 g, 30 mmol) were mixed in 100 mL
ethanol/water (1:1, v:v) solution, and then pyridine-2carbaldehyde (2.68 g, 25 mmol) was added in the solution drop
by drop. A dark brown precipitate was formed and continually
heated at 80℃ for 2 h, then filtered off and suspended in 100 mL
of ethanol. To decompose the complex, Na2S·9H2O (7.20 g, 30
mmol) was added in the mixture and black precipitate of the
formed copper complex was filtered off. The filtrates were
concentrated and acidified with HCl in order to help H2S
removing from the solution by heating on the water bath. The
raw produce was purified by alumina column chromatography
with ethyl acetate/methanol (1:1) solution as eluents. Yield:
79.1%, 1H NMR (DMSO-d6, δ ppm): 13.1 (N-H, s, 1H), 8.73 (d,
1H), 8.34 (d, 1H), 8.19 (s, 1H), 7.54 (t, 1H), 7.87 (d, 1H), 7.52 (d,
2H).
Synthesis of compound 2: Compound 2 was synthesized
according to previous methods. [27]
Synthesis of Bioben: Compound L1a (0.49 g, 2.0 mmol) was
dissolved in 20 mL dry DMF in 50 mL round-bottom flask under
argon atmosphere. EDCI (0.48 g, 2.5 mmol) and NHS (0.27 g,
2.3 mmol) were added in the solution and the mixture was
stirred at room temperature for 2 h. Then compound 2 (0.68 g,
2.0 mmol) and 1 mL triethylamine (TEA) were added in the
solution and stirred it for another 2 h. After the reaction was
completed, a dark yellow precipitate was obtained after the
solution was poured into the ice water. The product was filtered
off and dried, then purified by silica gel column chromatography
using MeOH/DCM (1:1) as eluents to afford compound Bioben
as yellow solid. Yield: 42.9%. Anal. Calcd for C29H37N7O3S(%): C,
61.79; H, 6.62; N, 17.39. Found (%): C, 61.80; H,6.59; N, 17.30.
ESI-TOFMS (CH3OH): m/z 586.6404 [M+Na]+, 1150.2517
[2M+Na]+, 1H NMR: (DMSO-d6, δ ppm): 13.33 (d, 1H), 8.77 (d,
1H), 8.51 (d, 1H), 8.35 (d, 1H), 8.27 (s, 1H), 8.03 (m, 1H), 7.83 (t,
1H), 7.74 (q, 1H), 7.56 (t, 1H), 6.40 (d, 2H), 4.29 (t, 1H), 4.13 (t,
1H), 3.29 (q, 2H), 3.09 (q, 1H), 3.04 (q, 2H), 2.80 (dd, 1H), 2.57
(d, 1H), 2.08 (t, 2H), 1.65-1.40 (m, 9H), 1.40-1.20 (m, 6H).13C
NMR: (DMSO-d6, δ ppm): 172.33, 166.97, 163.19, 149.95,
148.64, 142.99, 138.13, 136.60, 134.11, 133.64, 127.78, 125.51,
122.16, 119.00, 110.82, 61.52, 59.66, 55.91, 38.78, 35.70, 29.67,
29.64, 28.69, 28.52, 26.73, 26.65, 25.82.
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apoptosis in a dose-dependent manner in vivo (Figure 7c). To
evaluate the toxic side effects of the complexes in vivo,we
examined the pathological changes of the tissues by utilizing
H&E staining. The targeted complex was capable to deliver RuBSe to tumor, thus reducing the toxic effects on liver, lung and
kidney (Figure 7d). The hematological analysis was performed
to test the effects of Ru(II) complexes on the liver and kidney
functions of the treated nude mice. These results reveal that,
formation of HeLa xenografts induces damage to the liver and
renal function of nude mice, as reflected by the change of values
of blood biochemical analysis. For instance, treatment of RuBSe (4 μmol/kg) effectively alleviated the blood parameters to
normal levels, including blood urea nitrogen (BUN), albumin
(ALB) uric acid (UA), creatinine (CRE), triglyceride (TG),
aminotransferase (AST), total protein (TP) (Figure 8).
Collectively, all these data demonstrate that, targeted Ru-BSe
could specifically accumulate in the tumor site, thus enhancing
antitumor potency and minimizing the undesirable toxic side
effects.
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Synthesis of PhenSe[12a]: PhenSe( [1,2,5]selenadiazolo[3,4f][1,10]phenanthroline) was prepared based on the previous
method. In brief, a mixture of SeO2 (0.57 g, 0.5 mmol) and 5,6diamino-1,10-phenanthroline (1.05 g, 0.5 mmol) was dissolved
in 200 mL ethanol to reflux for 6 h. Then the solvent was
concentred and washed with 50 mL ethanol. The raw product
was recrystallized by methanol and filtered. The pink solid was
obtained with a yield of 67.8%.
cis-[Ru(II) (L)2Cl2]:The cis-[Ru(II) (L)2Cl2] (L= IP or PhenSe, IP=
1H-imidazo[4,5-f][1,10]phenanthroline)
was
prepared
as
following: RuCl3·3H2O (1 mmol, 0.27 g) and L (2 mmol, 0.44 g
for IP or 0.57 g for PhenSe) were mixed in 10 mL of DMF at
140 ℃ for 6 h under argon atmosphere. After reaction was
completed, the solution was cooled to ambient temperature and
dissolved in 100 mL cold acetone, and then the formed
precipitate was filtered and washed with acetone and diethyl
ether. [28] Yield: 47.6% for cis-[Ru(II) (IP)2Cl2] and 59.3% for cis[Ru(II) (PhenSe)2Cl2].
Synthetic procedure for the complexes: Ligand L1 or Bioben
(1 equiv) and the appropriate cis-[Ru(II)(L)2Cl2] (1 equiv) were
suspended in deoxygenated solution (2-methoxyethanol:
H2O=3:1) and refluxed for 6 h under argon atmosphere in dark.
The precipitate was obtained by addition of a saturated aqueous
NaClO4 solution, then filtered off and dried in vacuo. The raw
products were purified by alumina column chromatography,
gradually changing the eluents (DCM /MeOH) from 40:1 to 10:1.
[Ru(II)(IP)2(L1)](ClO4)2 (Ru-IP): Complex Ru-IP was obtained
as an orange powder. Yield: 53.6%. Anal. Calcd for C 38H25
Cl2N11O8Ru (%): C, 48.78; H, 2.69; N, 16.47. Found (%): C,
48.68; H, 2.66; N, 16.39. ESI-TOFMS (CH3OH): m/z 736.5047
[M−2ClO4−H]+, 368.8029 [M−2ClO4]2+. 1H NMR (DMSO-d6, δ
ppm): 9.03 (d, 2H), 9.01 (d, 2H), 8.94 (d, 1H), 8.75 (s, 2H), 8.94
(d, 2H), 8.75 (s, 2H), 8.73 (s, 1H), 8.71 (s, 1H), 8.06-8.04 (dd,
4H), 7.95 (s, 4H), 7.79-7.77 (q, 4H). 13C NMR (DMSO-d6, δ ppm):
173.35, 159.72, 155.79, 151.17, 150.37, 149.94, 149.51, 149.45,
147.83, 146.85, 146.54, 146.34, 146.08, 145.70, 144.88, 144.65,
137.62, 129.49, 129.37, 129.04, 126.29, 126.18, 126.12, 125.65,
125.02, 124.66, 124.44, 124.17, 124.07, 121.92, 121.11, 120.31,
119.59, and 112.90.
[Ru(II)(PhenSe)2(L1)](ClO4)2 (Ru-Se): Complex Ru-Se was
obtained as a red powder. Yield: 43.7%. Anal. Calcd for C36H21
Cl2N11O8RuSe2 (%): C, 40.58; H, 1.99; N, 14.46; Found (%): C,
40.46; H, 2.05; N, 14.27. ESI-TOFMS (CH3OH): m/z 866.4620
[M−2ClO4−H]+, 433.8093 [M−2ClO4]2+. 1H NMR (DMSO-d6, δ
ppm): 9.00 (m, 4H), 8.42 (t, 2H), 8.32 (d, 1H), 8.24 (d, 1H), 8.01
(t, 1H), 7.93 (m, 3H), 7.81 (q, 1H), 7.77 (q, 1H), 7.65 (d, 1H),
7.55 (d, 1H), 7.22 (t, 1H), 7.08 (t, 1H), 6.87 (t, 1H), 6.54 (t, 1H)
13
C NMR (DMSO-d6, δ ppm): 173.39, 159.76, 155.83, 151.20,
150.41, 149.97, 149.55, 149.49, 147.87, 146.88, 146.57, 146.38,
137.66, 129.52, 129.40, 129.07, 126.32, 126.21, 126.16, 125.69,
125.06, 121.96, 121.14, 120.34, 119.63, 112.94, 173.39, 144.92,
144.69, 124.69, 124.48, 124.11, and 124.20.
[Ru(II)(IP)2(Bioben)] (ClO4)2 (Ru-Bio): Conjugate Ru-Bio was
obtained as a bright red powder. Yield: 49.3%. Anal. Calcd for
C55H53Cl2N15O11RuS (%): C, 50.65; H, 4.10; N, 16.11 Found (%):
C, 50.71; H, 4.11; N, 16.13. ESI-TOFMS (CH3OH): m/z
1104.7496 [M−2ClO4−H]+. 1H NMR (DMSO-d6, δ ppm): 13.48 (d,
1H), 9.03 (t, 2H), 8.97 (t, 2H), 8.72 (d, 2H), 8.64 (s, 1H), 8.558.46 (m, 4H), 8.33 (d, 1H), 8.13 (d, 1H), 7.92 (d, 1H), 7.79 (t, 1H),
7.73 (t, 2H), 7.65 (t, 2H), 7.57-7.34 (m, 5H), 6.43 (d, 2H), 4.48 (t,
1H), 4.41 (t, 1H), 3.64 (q, 2H), 3.44 (q, 1H), 3.39 (q, 2H), 3.21 (t,
1H), 2.98 (d, 2H), 2.42 (t, 2H), 1.99-1.79 (m, 6H), 1.73 (t, 3H),
1.63 (m, 6H). 13C NMR (DMSO-d6, δ ppm): 172.33, 166.97,
164.50, 163.34, 163.19, 160.05, 159.45, 157.02, 156.08, 155.54,
154.50, 153.38, 151.64, 150.71, 149.95, 148.64, 145.03, 138.13,
137.12, 135.90, 135.03, 132.25, 131.53, 128.20, 127.67, 127.06,
124.91, 121.30, 117.08, 116.47, 61.52, 59.67, 55.91, 38.78,
35.69, 29.67, 29.63, 28.69, 28.52, 26.74, 26.65, 25.83.
[Ru(II)(PhenSe)2(Bioben)](ClO4)2 (Ru-BSe): Conjugate RuBSe was obtained as a red powder. Yield: 37.1%. Anal. Calcd
for C53H49Cl2N15O11RuSe2S (%): C, 44.39; H, 3.44; N, 14.65;
Found (%): C, 44.41; H, 3.56; N, 14.55. ESI-TOFMS (CH3OH):
m/z 1234.7032 [M−2ClO4−H]+, 628.8790 [M−2ClO4+Na]2+,
618.4017 [M−2ClO4]2+. 1H NMR (DMSO-d6, δ ppm): 13.33 (d,
1H), 8.77 (d, 1H), 8.51 (d, 1H), 8.35 (d, 1H), 8.27 (s, 1H), 8.03
(m, 1H), 7.83 (t, 1H), 7.74 (q, 1H), 7.56 (t, 1H), 6.40 (d, 2H), 4.29
(t, 1H), 4.13 (t, 1H), 3.29 (q, 2H), 3.09 (q, 1H), 3.04 (q, 2H), 2.80
(dd, 1H), 2.57 (d, 1H), 2.08 (t, 2H), 1.65-1.40 (m, 9H), 1.40-1.20
(m, 6H). 13C NMR (DMSO-d6, δ ppm): 177.58, 171.88, 168.16,
165.91, 164.69, 162.88, 161.66, 159.57, 158.14, 157.08, 156.48,
155.08, 154.62, 152.33, 142.41, 140.81, 139.29, 135.94, 134.16,
133.80, 132.97, 131.13, 130.00, 129.60, 126.54, 126.04, 117.68,
63.13, 61.21, 57.34, 36.46, 31.05, 30.22, 30.20, 29.21, 29.03,
27.20, 27.11, 26.26.
Absorption and fluorescence measurements:The spectrum
of compounds were recorded under physiological condition in
the PBS-DMSO solution (PBS:DMSO = 95:5, pH=7.4) at 37℃.
The UV-Vis spectrum of compounds was obtained by using a
Cary 5000 UV-2450 spectrophotometer. A Shimadzu RFPC5301 spectrofluorometer was used for recording the
fluorescence spectrum.
To gain insight into the process of structural decomposition, RuBSe (30 μM) was incubated in 1 mM Na2HPO4/citric acid
solution (pH=6.86) for different period of time, then both of the
absorption and fluorescence spectrum were recorded. In other
experiment, Ru-BSe (30 μM) was incubated in PBS-DMSO
solution at different pH condition (ranging from 3.1 to 8.5) for 0
and 24 h before recording the spectrum.
Stability of Ru(II) conjugate:For ESI-TOFMS analysis, 10 mM
NH4HCO3 solution was adjusted to pH=6.86 by adding 0.1 M
HCl, and then mixed with HPLC grade MeOH at the ratio of 7:3.
The decomposition process of Ru-BSe was performed as
following description: Ru-BSe (10 μM) was incubated in the
HPLC grade MeOH/Milli-Q H2O solution (3:7, v:v ,containing 10
mM NH4HCO3, pH=6.86) for 24 h at 37℃, then 0.1 mL sample
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was diluted in 1 mL HPLC grade MeOH. The diluted sample was
loaded in fixed conditions and analysed by using ESI-TOFMS.
To gain more insight of the interaction between Ru-BSe and
glutathione (GSH), the mixture was monitored by using ESITOFMS. The fresh prepared GSH (50 μM) and Ru-BSe (10 μM)
were mixed in MeOH/Milli-Q H2O solution. After the incubation at
37℃ for different period of time, 0.1 mL sample was diluted in 1
mL MeOH and loaded in fixed conditions for the ESI-TOFMS
analysis.
HPLC analysis of stability of Ru-BSe in human plasma and
DMSO: The plasma stability experiment was carried out
according to previous studies.[19a, 29] Diazepam (internal standard)
was purchased from Sigma-Aldrich and dissolved in DMSO at
the concentration of 800 μM. Stock of Ru-BSe and diazepam
and DMSO were diluted in the human plasma solution (975 μL)
to a total volume of 1000 μL, and final concentrations of 20 μM
for Ru-BSe and 10 μM for diazepam. The resulting sample was
incubated for 72 h at 37 °C with continuous and gentle shaking
(ca. 300 rpm). The reaction was stopped by addition of 2 mL
methanol, and the mixture was centrifuged for 45 min at 650 g at
4 °C. The methanolic solution was evaporated, and the residue
was suspended in 200 μL of 1:1 (v/v) acetonitrile/H2O. The
suspension was filtered before the HPLC analysis. To examine
the stability of Ru-BSe in DMSO, the DMSO stock solution of Ru
complex (1 mM) was incubated for 72 h at 25℃ with gentle
shaking (ca. 300 rpm). Afterwards, Ru-BSe (20 μM) was diluted
in 200 μL of 1:1 (v/v) acetonitrile/H2O before the HPLC analysis.
All these samples mentioned above were analyzed by HPLC
system (Agilent Technologies 1260 Infinity). The C18 reverse
phase column was employed with a flow rate of 0.5 mL/min and
UV-absorption was monitored at 300 nm. The runs were
performed with a linear gradient of A (acetonitrile (HPLC-grade))
and B (Milli-Q water containing 0.1% trifluoroacetic acid (TFA)): t
= 0−3 min, 20% A; t = 7 min, 50% A; t = 20 min, 90% A; t = 23
min, 100% A; t = 25 min, 100% A; t = 28 min, 20% A).
Cell culture and in vitro cytotoxicity evaluation: Normal
human colon mucosal epithelial cell line (NCM460) was obtained
from INCELL (San Antonio, TX) and maintained in INCELL’s
enriched M3:10 medium. The normal human liver cell line (L02)
was obtained from Nanjing KeyGEN Biotech (Nanjing, China).
Other human cancer cell lines, including HeLa, HepG2, MCF-7,
MDA-MB-231 and A549 were obtained from American Type
Culture Collection (ATCC). All cell lines were cultured in DMEM
medium (except for NCM460 cells) containing 10% of the fetal
bovine serum, 100 units/mL of the penicillin and 50 units/mL of
the streptomycin at 37℃ in humidified incubator with 5% CO2
atmosphere. Cell viability was determined by MTT assay that
based on the capability of living cells to transform MTT to purple
formazan dye. To value the anticancer potency of released
therapeutic Ru complexes, the aqueous products [Ru(IP)2Cl2]
(aq) and [Ru(phenSe)2Cl2] (aq) were prepared by the preincubation of [Ru(IP)2Cl2] and [Ru(phenSe)2Cl2] in medium at
37℃ for 12 h.
Cellular uptake of selenium compound: Briefly, 6×105 HeLa
cells were seeded in 6 cm dishes and supplemented with 6 mL
cell culture media for 24 h. Cells were incubated with PhenSe(40
μM), Ru-Se (20 μM) or Ru-BSe (20 μM) for 2 h, and washed
with PBS buffer twice. To confirm internalization via biotin
receptor-mediated endocytosis, a competition assay was carried
out via adding an excessive of biotin (100 μM) to the media.
After 1 h-incubation, cells were supplied with fresh media before
the treatment of selenium compounds for another 2 h and
observed in fluorescent microscope. In other experiments, the
intracellular concentration of Se was determined by ICP-MS
analysis. After incubation of selenium compounds, cells were
washed with PBS buffer twice and counted.
To investigate the mechanisms of internalization, the cellular
uptake of selenium compounds was measured under different
conditions.[30] 1×104 HeLa cells/well were seeded into the 96well plates and incubated for 24 h. Cells were pretreated with
sodium azide (NaN3) 10 mM and 2-deoxy-dglucose (DOG) 50
mM, or with 100 μM biotin, or with nystatin 10 μg mL−1, or with
50 mM NH4Cl, for 1 h at 37°C. Cells were washed with PBS
buffer before incubated with 10 μM Ru(II) complexes in PBS
buffer for 2 h. The effect of Ru(II) complexes diffusion into cells
was examined by using hypertonic treatment. Cells were
incubated with a solution of Ru(II) complexes (10 μM) and PBS
solution containing 20% sucrose. After all these treatments
mentioned above, cells were washed with PBS buffer for three
times, followed by the lysis of cells and measure of the emission
intensity at 600 nm (λex = 476 nm) of each well.
Determination of Ru and Se content: The samples (cells or
tissues) were digested with the 3 mL acid solution (VHNO3 : VHClO4
= 3:1) in an infrared rapid digestion system (Gerhardt) at 180℃
for 1.5 h. The digested solution was reconstituted with Milli-Q
H2O. The Ru content in tissues was examined by ICP-AES
analysis, while the intracellular Se content was tested by ICPMS analysis.
Co-culture model for examination of selective induction of
cell apoptosis: The selectivity of Ru-BSe between human
cancer and normal cells was examined by co-culture model
using TUNEL-CellTrakcer Blue co-staining assay. Briefly, 8×104
L02 cells were seeded in 35-mm confocal glass dishes and
allowed to attach for 24 h. Adherent L02 cells were preincubated with 5 μM CellTracker Blue for 1 h. After washed with
PBS solution twice, L02 cells were supplied with fresh medium.
Similar amount of suspended HeLa cells were cultured with
adherent L02 cells for another 24 h. After treated with 20 μM of
Ru-BSe for 24 h, the cells were fixed with 3.7% formaldehyde
and permeabilized with 0.1% Triton X-100 in 10% sodium citrate
solution. The fixed cells were incubated with fresh TUNEL
working solution (500 μL) for 1 h at 37℃, and observed by
fluorescence microscopy.
In other experiment, after the treatment with Ru-BSe, the coculture cells mentioned above were harvested before fixing with
3.7% formaldehyde. After permeabilized with 0.1% Triton X-100
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solution, cells were incubated with TUNEL working solution and
analyzed by flow cytometry.
containing 20 μM Ru(II) complexes. After 30-min incubation, the
HeLa cells were observed using a fluorescence microscopy.
Evaluation of mitochondrial membrane potential: After the
treatment of Ru(II) complexes, the suspended HeLa cells were
incubated in 0.5 mL JC-1 working solution at 37 ℃ for 10 min.
Cells were centrifuged and resuspended in PBS solution before
analysed by flow cytometry. The percentage of cells that lost
mitochondrial membrane potential were reflected by the green
fluorescence form JC-1.
Mouse xenograft model: About 1×106 cells were
subcutaneously injected in the left leg of BALB/c nude mice to
construct the HeLa xenograft model. When the tumor volume
reach upon 100 mm3, the tumor-bearing mice were randomly
divided in 5 groups (n=6 each group). To examine the
therapeutic effect of Se-containing complexes, the HeLa
xenograft models were respectively received tail intravenous
injection treatment of 2 μmol/kg or 4 μmol/kg Ru-Se, 2μmol/kg
or 4 μmol/kg Ru-BSe per 2 days for 30 days treatment. The
control group mice were injected with equal amount of saline.
The maximal length (l) and the width (w) of the tumor were
measured for the calculation of tumor volume by following the
equation: Volume (mm3) =l×w2/2. At the end of treatment, the
tumor and the normal organs were obtained for histological
analysis and determination of Ru content. To detect the
apoptotic cells in tumor, the TUNEL staining assay was carried
out.
Cellular localization of Ru(II) complexes: The HeLa cells were
cultured in confocal dishes for 24 h before treatment of 10 μM
Ru(II) complexes for 6 h, and 1 μg mL−1 of DAPI and 100 nM
Mito-Tracker Green for 30 min. After washing with PBS buffer
twice, cell images were obtained by a fluorescence microscopy
(EVOS FL auto, Life Technologies). Cells were visualized in the
blue channel for DAPI (λex=345 nm, λem= 430-480 nm), the
green channel for Mito-tracker (λex=488 nm, λem=500–560 nm)
and red channel for Ru complex (λex=520 nm, λem=600–650 nm),
respectively. To determine the intracellular localization of RuBSe, cell images were quantified by the software Image-Pro
plus 6.0.
Determination of caspases activities: The intracellular
proteins were extracted with cell lysis after the treatment of 20
μM Ru(II) complexes for 72 h. The concentration of total proteins
was tested by BCA assay, and then the Caspase-3/9 activities
were measured by using caspase activity kit (BD Biosciences).
Western blot analysis: The extracted proteins from Ru(II)
complexes-treated HeLa cells were analyzed by Western blot
according to our previous study.[31] After separated proteins were
transferred to nitrocellulose membrane, the membranes were
blocked by 5% non-fat milk and incubated with primary
antibodies at 4℃ overnight. Protein bands were visualized on Xray film via the application of a chemiluminescence working
solution. To confirm equal amount of proteins were loaded in
each lane, β-actin was used as loading control.
Determination of ROS generation: The ROS generation in
cancer cells was revealed by the increase of fluorescence
intensity of DCF. Briefly, HeLa cells were seeded in 96-well
plates at a density of 2×104 cells/well for 24 h. Cells were
washed with PBS buffer twice and stained with DCFH-DA for 30
min, and then maintained in fresh PBS buffer before the
treatment of Ru(II) complexes (40 μM). The fluorescence
intensity of DCF (excitation/emission, 500/530 nm) was
measured with fluorescence microplate reader (Tecan SAFIRE)
at different time point.
Release of calcium: The content of calcium in cytoplasm was
detected by Fluo 3-AM probe. 8×104 HeLa cells were seeded in
2 cm dishes for 24 h and then replaced by HBSS (HBSS,
calcium free, magnesium free, Gibco cat. no. 14170−112). The
cells were incubated with Fluo-3AM working solution in HBSS
for 30 min at 37℃ and then replaced by fresh DMEM medium
In vivo and ex vivo fluorescence imaging: To evaluate the
theranostic effect of Ru(II) complexes, 4 μmol/kg of complexes
in 0.2 mL of 0.9% NaCl solution was injected into the tail-vein of
HeLa xenografts nude mice. Afterwards, the mice were
anesthetized and monitored with fluorescence imaging system
(Night OWL II LB 983) at different time points (0, 24, 48 and 72
h). After 72 h treatment, the brain, heart, liver, spleen, lungs,
kidney and tumor of each group were collected for the
determination of biodistribution of Ru complexes by using
fluorescence imaging technique. Fluorescent filter sets are used
for in vivo and ex vivo fluorescent imaging (excitation/emission,
500/640 nm), which are similar with previous studies. [3]
Hematology analysis of nude mice: After the treatment of
Ru(II) complexes, the blood samples were collected from normal
and xenograft mice. The plasma was obtained by centrifuging
blood samples at 3,000 rpm for 10 min and then subjected to
hematology analysis. The tested biochemical indexes included
uric acid (UA), blood urea nitrogen (BUN), creatinine (CREA),
triglyceride (TG), aminotransferase (AST), total protein (TP),
albumin(ALB) and creatine kinase (CK).
Acknowledgements
Acknowledgements Text. This work was supported by National
High-level Personnel of Special Support Program (2014189),
YangFan Innovative & Entepreneurial Research Team Project
(201312H05), Guangdong Special Support Program and
Fundamental Research Funds for the Central Universities.
Keywords: selenium • theranosis • tumor microenvironmentresponsive • cancer targeting.
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Accepted Manuscript
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10.1002/chem.201705561
Chemistry - A European Journal
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Zhennan Zhao, Pan Gao, Yuanyuan
You and Tianfeng Chen*
M itochondria
T um or M icroenvironm ent
A poptosis
Cancer-targeting Functionalization of
Selenium-containing Ruthenium
Conjugate with Tumor
Microenvironment- Responsive
Property to Enhance Theranostic
Effects
Herein we demonstrate the rational design and synthesis of a selenium-containing
conjugate that could selectively recognize cancer cells to realize enhanced
theranostic effects and nullify the systemic toxicity. The protonation process of
Ru(II) conjugate in tumor acidic microenvironment cause ligand substitution,
resulting in release of activated metallodrug that trigger apoptosis in tumor tissue.
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pH response