A Smart EuropiumâRuthenium Complex as Anticancer Prodrug

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A Smart Europium-Ruthenium Complex as Anticancer Prodrug: Controllable Drug Release and Real-Time Monitoring under Different Light Excitations Hongguang Li, Chen Xie, Rongfeng Lan, Shuai Zha, Chi-Fai Chan, WingYan Wong, Ka-Lok Ho, Brandon Dow Chan, Yuxia Luo, Jing-Xiang Zhang, GaLai Law, William C. S. Tai, Jean-Claude Georges Bunzli, and Ka-Leung Wong J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01162 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

f

School of Chemistry and Environment Engineering, Hanshan Normal University, Chaozhou,

521041, Guangdong, China. KEYWORDS: Prodrug, drug delivery, photoactivation, real-time monitoring, quantitative analysis

ABSTRACT: A unique, dual-function, photo-activatable anticancer prodrug, RuEuL, has been tailored that features a ruthenium(II) complex linked to a cyclen-europium chelate via a πconjugated bridge. Under irradiation at 488 nm, the dark-inactive prodrug undergoes photodissociation, releasing the DNA-damaging ruthenium species. Under evaluation-window irradiation (λirr = one-photon 350 nm or two-photon 700 nm), the drug delivery process can be quantitatively monitored in real-time thanks to the long-lived red europium emission. Linear relationships between released drug concentration and ES-MS or luminescence responses are established. Finally, the efficiency of the new prodrug is demonstrated both in vitro on HeLa cells and in vivo by inhibiting tumours in BALB/c nude mice carrying SW480 xenografts. The results presented in this article also reveal the advantages of the dual-functional RuEuL anticancer prodrug over some existing ones and open the way for decisive improvements in multi-purpose prodrugs.

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INTRODUCTION Development of prodrugs is an increasingly important area in pharmacology. A prodrug is a medication which remains inactive before metabolism but becomes pharmacologically active after biotransformation in the body.1 Because a prodrug contains an active core linked to non-toxic protective groups, adverse effects from parent molecules can be reduced.2 Applications of prodrugs are important and extensive in treatments inducing severe side effects, like platinum(II) complexes in cancer therapy.3-4 Control of drug release by prodrugs is commonly achieved via encapsulation in micelles5 or nanomaterials6 followed by photo-dissociation.7 Photo-activation is frequently accomplished by upconverting nanoparticles8-9 but owing to their low quantum efficiency, better alternative strategies are welcome. For instance, a photoactivatable cisplatinbased prodrug has been developed: the inactive form of the Pt(II) complex is excited by ultraviolet light or infrared irradiation through two-photon absorption and then becomes toxic for tumour cells. Dissociation of the ligand and release of the platinum-based anticancer drug are induced by a π-conjugated ligand absorbing excitation energy followed by internal conversion.10 Another important advantage of prodrugs is their traceability because direct instant monitoring of drug location provides real-time information on their molecular action, which is the basis for minimizing unwanted side effects in therapy.11 Luminescence spectroscopy is a powerful tool for investigating the evolution of a therapy. Recently, a Eu-Pt complex has been reported to function as a prodrug, releasing a Pt(II) drug in a controlled manner and, in addition, generating in vitro luminescence.12 Such prodrugs that are cytotoxic in their active form while simultaneously displaying imaging capabilities are presently promising theranostic agents in medication.13-15 In view of the high cytotoxicity of common platinum(II) drugs,16-17 ruthenium(II) complexes are considered as welcome alternative anticancer agents showing relatively low cytotoxicity before


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conversion into their active form.18-20 For instance, Glazer et al. reported a Ru(II) polypyridyl complex that remains unreactive in the dark but promotes light-induced ligand dissociation under excitation of visible light. When the complex is photoactivated, it causes covalent modification of DNA with a similar pattern as cisplatin.21 These light-activatable Ru(II)-based prodrugs with low dark cytotoxicity provide higher potency in anticancer treatment than traditional cisplatin analogues. So far there have been few studies on prodrug models that simultaneously show light-induced drug release, quantitative traceability, and low dark cytotoxicity. To bridge this knowledge gap, we have previously reported PtEuL with photo-dissociation and evaluation of the quantity of released drug using the same excitation wavelength12. Since, in addition, the dark cytotoxicity of PtEuL is too high (IC50 = 23.6 M for HeLa cells), we herein report a new-generation prodrug with similar structure, RuEuL (Figure 1), the properties of which are established by luminescence spectroscopy, high performance liquid chromatography (HPLC), mass spectrometry, DNA binding and cytotoxicity studies. The large π-conjugated linkage group of the cyclen ligand absorbs visible light and transfers most excitation energy to the dissociative states of the Ru(II) complex while poorly sensitizing Eu(III).12 Cleavage of the Ru(II) moiety is revealed by sensitized Eu(III) emission since the energy absorbed by the antenna ligand is now transferred to the Eu(III) center.22 In addition to luminescence measurements, mass spectroscopy is successfully applied to monitor drug release: a strict linear correlation between the intensities of the molecular peaks of the dissociated cis-[Ru(OH)(bpy)2(H2O)]+ complex and the europium emission is demonstrated. In the final stage of the process, the released Ru(II) moiety is an active form of the anticancer Ru(II) species and subsequently crosslinks on targeted DNA and damages it. The prodrug is tested both on living HeLa cells and in vivo, on a mouse model.


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This new design of a dual-function prodrug combining therapeutic effect and imaging not only introduces a Ru(II) complex with lower dark toxicity (IC50 > 200 M) as a substitute for cisplatin but also provides real-time and quantitative traceability of the photo-activation process using a different excitation window for photo-dissociation. RESULTS AND DISCUSSION Synthesis, characterization, and photophysical properties of the prodrug, RuEuL and the proposed dissociation product, EuL EuL was synthesized according to procedures previously reported.12 RuEuL was prepared by reflux of complex EuL with an excess amount of cis-Ru(bpy)2Cl2 (2 eq.) in ethanol to form a monosubstituted complex. The excess of cis-Ru(bpy)2Cl2 can be removed by treatment of the aqueous solution of the crude product with DCM. The two complexes, EuL and RuEuL were characterized by analytical HPLC (Figure S1 and S2) and ESI-MS (Figure S7). The absorption and emission spectra of RuEuL and its dissociation products EuL and [Ru(OH)(bpy)2(H2O)]+ were recorded at room temperature in aqueous solution (Figure 2); EuL and RuEuL both have one similar absorption band located at ca. 330 nm resulting from the π → π* transition of their pyridine-based ligand with molar absorption coefficients of 26,970 M-1cm-1 for RuEuL and 18,860 M-1cm-1 for EuL (Figure 2a). In addition, RuEuL has four other absorption bands assigned to the ruthenium moiety and located at 243 nm (π → π* transition of bpy ligand, ε = 28,340 M1

cm-1) 290 nm (π → π* transition of bpy ligand, ε = 67,380 M-1cm-1), 400 nm (shoulder, MLCT),

and 464 nm (MLCT), ε = 11,200 M-1 cm-1. [Ru(OH)(bpy)2(H2O)]+ shows four absorption bands located at 243 nm (π → π* transition of bpy ligand, ε = 19,862 M-1cm-1) 290 nm (π → π* transition of bpy ligand, ε = 58,192 M-1cm-1), 340 nm (ε = 7,402 M-1 cm-1 ) and 484 nm (MLCT, ε = 8,430


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M-1 cm-1). As for the emission spectra, EuL exhibits strong red emission (corresponding to the 5

D0→7F0-4 transitions of Eu(III)), while RuEuL and [Ru(OH)(bpy)2(H2O)]+ feature only very

weak emission under identical experimental conditions (Figure 2b). The intensely sensitized Eu(III) emission of EuL arises from efficient energy transfer from the antenna chromophore to the excited states of the metal ion.12 Qualitative analysis of photo-dissociation The photo-induced dissociation of RuEuL was performed on the setup shown in Figure S8 and was simultaneously studied by absorption (Figure 3a) and emission (Figure 3b) spectroscopies, and by HPLC (Figure 3c and d) of the RuEuL solution (5 M in H2O) after continuous irradiation with visible light (λirr. = 488 nm, 1 W) during various times (1-8 min). After irradiation, the MLCT band redshifts from 465 to 482 nm while the absorption of the π → π* transition is reduced (Figure 3a). In parallel, turned-on europium emission is observed under the same experimental conditions (Figure 3b). This observation can be explained by the existence of dissociative states on Ru(II), which was proposed by Saddler et al.10 Since our group has reported one- and two-photon induced dissociation of EuL from PtEuL12, which is structurally similar to RuEuL, an analogous energy transfer mechanism can be proposed for RuEuL. In the latter, the excitation energy is transferred by the ligand to the dissociative states of the Ru(II) moiety and little energy to the Eu(III) excited states. Continuous light irradiation leads to complete dissociation of the Ru(II) complex and to the accumulation of EuL with concomitant responsive turned-on europium emission. The photo-dissociation products, EuL and ruthenium (II) complex, [Ru(OH)(bpy)2(H2O)]+, have been further qualitatively identified by HPLC-UV and ESI-MS spectra (Figure 3c and Figure S9). Before light irradiation, only one HPLC peak corresponding to RuEuL (retention time, R = 11.3 min) is observed. After irradiation, this peak disappears, and two peaks of dissociated


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photoproducts appear. Comparing the HPLC trace of the photoproducts with that of the pure complex EuL (R = 12.1 min) and prepared [Ru(OH)(bpy)2(H2O)]+ (R = 4.3 min) prove that the photoproducts are similar to these compounds. The real-time absorption spectra (Figure 3d) of the corresponding peaks in HPLC traces are the same as the absorption spectra of the pure compounds (Figure 2a). These results consistently demonstrate that RuEuL can be photo-activated with visible light to generate EuL and [Ru(OH)(bpy)2(H2O)]+ complexes. 1H 1

NMR analysis of the photo-dissociation

H NMR analysis of the evolution of the photo-dissociation reaction was performed on the

lanthanum(III) analogue (RuLaL) of the prodrug in a mixed solution of deuterium oxide (D2O) and

methanol-d4

(V:V

=

1:1)

with

the

help

of

reference

samples

(LaL

and

[Ru(OH)(bpy)2(H2O)]NO3) (Figure 5). The 50% methanol-d4 in deuterium oxide used in this experiment avoids precipitation of highly concentrated (～ 2 mM) LaL in pure D2O. Assignments of the 1H-NMR spectra are shown in Figure 5, and the spectra confirm the purity of all compounds (LaL, RuLaL and [Ru(OH)(bpy)2(H2O)]NO3)). This is good evidence for the feasibility of methods for the synthesis of the complexes LnL and RuLnL. Blue light (ex = 488 nm, P = 1W) treatment of RuLa induces disappearance of peaks at δ 9.82 and 7.30, assigned to 3a and 3j +3g protons of RuLaL respectively. This is accompanied by an increase in signals at δ 9.38 and 7.14, assigned to 2a and 2g protons of [Ru(OH)(bpy)2(H2O)]NO3, and at δ 8.75 and 8.54, assigned to 1a and 1g protons of LaL. In addition, new peaks at δ 9.71 and 9.64 are observed and assigned to the chlorinated ruthenium(II) bipyridine complexes. The NMR study clearly demonstrates that blue light triggers dissociation of RuLaL to generate LaL and [Ru(OH)(bpy)2(H2O)]+, and serves as a good control experiment for the photo-dissociation of the prodrug, RuEuL. Quantitative analysis of photo-dissociation


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The photo-dissociation of the ruthenium(II) prodrug is consistently demonstrated by the experimental data reported above. However, in order to obtain the exact amount of the photoreleased ruthenium(II) moiety for biomedical purpose and to evaluate drug release in real time, quantitative analysis is required. It can be achieved by LC/ESI-MS for simultaneous detection of the photo-products EuL and [Ru(OH)(bpy)2(H2O)]+ (See Table S3 for conditions). Moreover, the switchable europium(III) emission can be considered as a real-time signalling technique for the released anticancer Ru(II) complexes and can also be exploited quantitatively. To demonstrate the feasibility

of

quantitative

analysis

of

the

photoproducts,

standard

solutions

of

[Ru(OH)(bpy)2(H2O)]NO3 and EuL with concentrations 0.1, 0.2, 0.5, 1, 2 and 5 μM were prepared for establishing calibration curves. A time-resolved correlation relationship was therefore established between Eu(III) emission, MS response, and the level of ruthenium compounds released (Figure 5a and b). There is a high correlation between Eu(III) emission and the amounts of dissociation products (Figure 5b), meaning that the concentration of the released drug can be simply calculated from the Eu(III) emission intensity (Figure 5c). A pseudo-first order rate constant k was determined from these data to be 0.43 min-1 under the same experimental conditions as for the photophysical studies. In the control experiment, RuEuL is stable under dark at 37 oC for 24 hours and [Ru(OH)(bpy)2(OH)2]+ is photostable under the same experimental condition as for RuEuL photo-release (Figure S10 and S11). DNA binding and cytotoxicity of the prodrug Furthermore, the interaction of photo-activated RuEuL with DNA was examined by agarose gel electrophoresis (Figure 5a). Plasmid DNA (22.5 g/mL in Tris/EDTA (TE) buffer) was incubated


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with the compounds (20 μM), irradiated by 488 nm light (dose 50 J m-2) or not (control) and then subjected to 1% agarose gel electrophoresis. The separated DNA was stained with GelRed Nucleic Acid Stain (BIOTIUM) and imaged using Tanon 1600 Gel Imaging imental lane (with light excitation), the migration of DNA is delayed for RuEuL compared to EuL and TE buffer, whereas in the control lanes (without irradiation), RuEuL left the DNA intact and showed normal migration. EuL does not interact with DNA and shows normal migration. The DNA binding activity contributes most to the cytotoxicity of RuEuL. Though it is possible for Ru(II) bipyridine moieties serving as singlet oxygen photosensitizers and cause cell damage, the actual singlet oxygen quantum yield has been measured to be negligible (Figure S12). The delayed shift suggests the released Ru moiety from RuEuL incorporating into DNA and obviously affecting gel migration. To determine the potential of RuEuL as a photo-controlled prodrug cell cytotoxicity studies were performed on human cervical cancer (HeLa) cells, together with PtEuL12 (structure shown in Figure 1) for comparison and EuL as control (Table 1). Cells were incubated 6 hours in the dark before irradiation (488 nm, 8 min); dark controls were run in parallel. As expected, PtEuL showed similar toxicity under light and dark conditions while control compound EuL exhibited almost no toxicity towards HeLa cells. In contrast, photo-dissociative RuEuL showed significant light-induced cytotoxicity. After irradiation in HeLa, RuEuL was comparably more toxic (light IC50 32.5 μM) as PtEuL (light IC50 22.9 μM), but was nontoxic in the dark, with an IC50 value over 200 μM. The approximate 10-fold difference between light and dark IC50 values is highly suitable for a photo activated prodrug. In vitro imaging of the photo-activation of RuEuL The light-induced cytotoxicity of RuEuL towards HeLa cells was also investigated by confocal microscopy (Figure 7) with the PtEuL complex tested under identical conditions for comparison.


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Preliminary studies were performed in pure water; with laser irradiation at 488 nm (7 mW) for 30min, no obvious europium emission was observed, which suggested little dissociation happened in PtEuL, while the strong emission from the RuEuL solution indicated structural changes in the complex. In cell culture studies, red Eu(III) emission from RuEuL was observed after 15-min irradiation. The emission intensity increased with longer irradiation time because more excitation energy induced the dissociation of more Ru(II) moieties from the parent molecules, and the resulting EuL units generated stronger light emission. In addition, the released Ru(II) moiety caused severe cell damage as cell lysis was detected after 15-min irradiation. In contrast, no red emission or cell death was noticed from the HeLa cells incubated with PtEuL (10 μM). This means there was no obvious cell damage induced by light irradiation because 488 nm cannot trigger the release of cisplatin from PtEuL. It has been reported by our group that PtEuL is photo-activated upon ultraviolet A irradiation (λ = 365 nm)12. However, UVA irradiation could cause severe adverse effects on human cells. It is worth noting that the dosage concentration of PtEuL is 10 μM, lower than that of RuEuL. Because according to Table 1, PtEuL is able to induce severe cell death at a low concentration (IC50 around 20 μM) with or without light, half IC50 was chosen as the concentration of PtEuL so that a clear comparison with RuEuL would be made. In vivo photo-induced tumour growth inhibition and ex vivo photo-activation of RuEuL To examine the in vivo effect of blue-light activation on RuEuL, we carried out bi-weekly intratumour injection of 40 μg RuEuL or pre-photo-activated RuEuL (RuEuL + hv) in BALB/c nude mice carrying SW480 xenografts (two mice in each group). Body weight and tumour size were measured three times a week. Body weights were not significantly affected by both drug treatments (data not shown). After a 21-day treatment period, mice were sacrificed and tumours were excised and weighed. Tumour growth was inhibited by RuEuL treatment, with or without 488-nm light


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activation. At the end of the experiment, the average tumour volume of mice treated with RuEuL and RuEuL + hv decreased by 26.8% and 77.3% respectively, by comparison with the control groups (Figure 8a). Similarly, the average tumour weight decreased by 25.8% and 56.8% after treatment with RuEuL and RuEuL + hv, respectively (Figure 8c). These data clearly demonstrate that photo-activation increases substantially the tumour inhibitory activity of RuEuL. Ex vivo imaging was performed under 365-nm excitation on tumours subjected to inhibitory experiments (Figure 8d). Without injection of RuEuL or EuL (control), tumours only showed the autofluorescence of tumoral tissues. Tumours injected with 0.5 mg of RuEuL under dark and kept under dark for 10 min before UV excitation for imaging did not display any red emission while those injected with the same amount of EuL displayed bright Eu(III) luminescence. On the other hand, tumours injected with RuEuL and irradiated 30 min with 488-nm light, (P = 100 mW) showed turn-on red emission proving the successful photo-activation of the prodrug. CONCLUSIONS In this work, we have demonstrated that one simple ruthenium-europium-based prodrug (RuEuL) with low dark cytotoxicity (IC50 > 200 M) can achieve two important tasks, photo-activated drug delivery and quantitative monitoring via two different excitation wavelengths (488 nm for drug release and 350 nm/700 nm for imaging). The light cytotoxicity in cells and the amount of drug released can be monitored by real-time europium emission of EuL under two-photon excitation in the NIR. This imaging capability could be easily extended to in vivo experiments with appropriate equipment, femtosecond laser, and animal imaging box. Therefore, the multi-functional RuEuL prodrug described here can be considered as a promising model for quantitative theranostics in anti-cancer therapy. EXPERIMENTAL SECTION


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General remarks. All commercial reagents were purchased from Aldrich, TCI or Alfa Aesar and used as received unless otherwise stated. The purity of all complexes was determined by an Agilent 1290 high-performance liquid chromatography (HPLC) system coupled with a DAD detector. The tested HPLC purity of all biologically evaluated compounds, EuL, RuEuL and PtEuL is over 95%. (Figure S1, S2 and S6, Table S1) Synthesis and characterization. The complex RuEuL was prepared by reflux of complex EuL (HPLC purity > 98% (Figure S1))12 and cis-Ru(bpy)2Cl2 (2 eq.) in the solution of ethanol under the protection of nitrogen gas for 12 hours. The solvent was removed under vacuum and the crude product was dissolved in water and then extracted with dichloromethane for three times to remove the excess of cis-Ru(bpy)2Cl2. The aqueous solution was collected and dried under vacuum to obtain a dark red solid as the product. LaL and RuLaL were prepared with the similar procedures of EuL and RuEuL respectively. [Ru(OH)(bpy)2(H2O)]NO3 was prepared according to the literature23. RuEuL: Yield = 65%. HPLC purity > 95% (Figure S2). ESI-MS: Calc. C54H53EuN11O8Ru for [M – H2O - Cl + OH]+ 1238.2334 found 1238.2287; Calc. C54H52ClEuN11O7Ru 1256.1995 for [M – H2O]+ found 1256.1902 (Figure S7) . Elemental Analysis: (C54H54Cl2EuN11O8Ru) C, H, N: calcd 49.55, 4.16, 11.77; found 49.38, 4.17, 11.80. LaL: Yield = 95%. HPLC purity > 96% (Figure S3). ESI-MS: Calc. C34H36LaN7NaO7 for [M – H2O]+ 816.1637 found 816.1622. Elemental Analysis: (C34H38LaN7O8) C, H, N: calcd 50.32, 4.72, 12.08; found 50.14; 4.73, 12.05. RuLaL, Yield = 72%. HPLC purity > 95% (Figure S4). ESI-MS: Calc. C54H53LaN11O8Ru for [M – H2O - Cl + OH]+ 1224.2186 found 1224.2193; Calc. C54H52ClLaN11O7Ru 1242.1847 for [M


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– H2O]+ found 1242.1852. Elemental Analysis: (C54H54Cl2LaN11O8Ru) C, H, N: calcd 50.05, 4.20, 11.89; found 49.86, 4.21, 11.86. [Ru(OH)(bpy)2(H2O)]NO3: Yield = 92%. HPLC purity > 97% (Figure S5). ESI-MS: Calc. C20H19N4O2Ru for [M -NO3]+ 449.0554 found 449.0550. Elemental Analysis: (C20H19N5O5Ru) C, H, N: calcd 47.06, 3.75, 13.72; found 46.93, 3.76, 13.76. PtEuL (reported)12: HPLC purity > 96% (Figure S6). Photophysical properties measurements and photochemical studies. UV-Visible absorption spectra in the spectral range 200 to 1100 nm were recorded by an HP Agilent UV-8453 spectrophotometer. The emission spectra of RuEuL and EuL were measured with a Horiba Fluorolog-3 spectrophotometer equipped with a 450W continuous xenon lamp for steady-state emission measurement. For two-photon experiments, the 800 nm pump source was from the fundamental of a femtosecond mode-locked Ti:Sapphire laser system (output beam ~ 150 fs duration and 1 kHz repetition rate). The lasers were focused to spot size ~ 50 µm via an f = 10 cm lens onto the sample. The emitting light was collected with a backscattering configuration into a 0.5 m spectrograph and detected by a liquid nitrogen-cooled CCD detector. To study the photochemical properties of RuEuL, 5 M of RuEuL aqueous solution was irradiated with 488-nm blue laser (P = 1 W) by using the setup shown in Figure S8. The absorption and emission spectra (Figure 3a and b) were measured at a different irradiation time point. And at each irradiation time point, 200 L of solution were used for UPLC-MS/MS quantitative analysis of the dissociation product. The data were processed with OriginLab Origin 8.0. HPLC characterization and qualitative analysis. The HPLC characterization of the complexes (Figure S1-S6) and qualitative analysis of the photodissociation of RuEuL (Figure 3c


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and d) were performed on Agilent 1290 Infinity Quaternary LC System coupled with a DAD detector. The column used was a Hypersil GOLD Analytical Column (250 × 4.6 mm, 5 μm) analytical column. The LC elution profiles were shown in Table S1. 1H 1

NMR analysis of the photo dissociation of lanthanum (III) analogues of RuEuL, RuLaL

H NMR spectra characterization of LaL, [Ru(OH)(bpy)2(H2O)]+ and RuLaL and qualitative

analysis of the photodissociation of RuEuL’s analog, RuLaL was performed on a 400 (1H: 400 MHz) spectrometer at 25 oC. LC-MS/MS quantitative analysis. The ESI-MS/MS quantitative analysis was performed by Ultra-high Performance Liquid Chromatography coupled with a triple quadrupole mass spectrometer (UPLC-MS/MS). LC separation was done on an Agilent 1290 Infinity Quaternary LC System while mass analysis was performed with an Agilent 6460 Triple Quadrupole mass spectrometer equipped with an Agilent Jet Stream technology electrospray ionization source. The column used was an Agilent EclipsePlus C18 RRHD (2.1 × 50 mm, 1.8 μm) protected with an Agilent SB-C18 guard column (2.1 ×5 mm, 1.8 μm). The LC elution profiles were shown in Table S2 The autosampler and column temperatures were set at 4 and 25 °C respectively. The injection was achieved by 5-second needle wash in Flush Port mode for 3 times with eluent B. 10 μL was injected each time. For the source parameter, drying gas (nitrogen) temperature was set at 300 °C with 5 L/min flow rate. Nebulizer pressure was 45 psi. Sheath gas temperature was set at 250 °C with 11 L/min flow rate. Capillary voltage was set at 3500 V. For mass detection, scheduled multiple reaction monitoring (MRM) was performed. The information on MRM transitions can be found in Table S3.


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The result was calculated using Agilent MassHunter Workstation software. Calibration curves were fitted linearly without any weighing. Stability tests of RuEuL in aqueous solution under dark at 37 oC. Two methods, emission spectroscopy and UPLC-MS/MS analysis, are used to determine the stability of RuEuL in aqueous solution under dark at 37 oC. For the emission spectrum method, 5 M of RuEuL was stored in the dark in an oven at 37 oC and luminescence spectra were recorded after 0, 3, 6, 12, 18, and 24 hours. Before measuring the emission spectra, solutions were cooled down to 25 oC. No emission enhancement was observed for RuEuL under dark in aqueous solution compared to its fully lightdissociated solution (Figure S10a). For the UPLC-MS/MS method (Figure S10b), 5 M of RuEuL at 37oC were injected every half hour. The detected concentration of [Ru(OH)(bpy)2H2O]+ and EuL were below 0.05 M and no significant change within the incubation time from 0 to 24 hours. The two experiments consistently demonstrate that RuEuL is stable at 37 oC for 24 hours. DNA binding assays. The interaction of photo-activated RuEuL with DNA was examined by agarose gel electrophoresis. Plasmid DNA (225 ng for each lane) was incubated with the compounds (20 M) as indicated, irradiated at 488 nm (dose 50 J m-2) or not (controls) and then subjected to 1% agarose gel electrophoresis. The separated DNA was stained with GelRed Nucleic Acid Stain (BIOTIUM) and imaged using Tanon 1600 Gel Imaging System (Tanon Science & Technology Co., Ltd). Cell culture. Human cervical cancer HeLa cells were grown in Dulbecco’s Modiﬁed Eagle Medium (DMEM). Human colon cancer cell lines SW480, were obtained from the cell resource center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences and grown in


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ATCC-formulated Leibovitz's L-15 Medium, (Catalog No. 30-2008) and supplemented with 10% (v/v) fetal bovine serum, 1% penicillin and streptomycin at 37 oC and 5 % CO2. MTT cell cytotoxicity assays. For the dark cytotoxicity, HeLa cells were treated with testing complexes for 24 hours at 7 concentrations (RuEuL and EuL : 0, 1, 5, 10, 50, 100 and 500 µM; PtEuL : 0, 1, 5, 10, 20, 50 and 100 µM). The cell monolayers were rinsed with phosphate buffer saline (PBS) once and incubated with 0.5 mg/mL MTT (3-(4, 5-dimethylthiazol-2-yl)-2 and 5diphenyltetrazolium bromide) solution. The cellular inhibitory potency of the complexes was examined by the formation of formazan after addition of MTT for 3 hours to allow formazan production during cell metabolism. After that, formazan was fully dissolved in DMSO through oscillation. Finally, the absorbance of the solution was measured with Biotek Power wave xsMicroplate Reader at the wavelengths of 570 nm and 690 nm. For the Photo-cytotoxicity, HeLa cells (3103) were treated with testing complexes for 24 hours at 5 concentrations (RuEuL and PtEuL : 0, 1, 5, 10 and 50 µM; EuL: 0, 10, 50, 100 and 500 µM). Afterward, the cells were exposed to blue light (488 nm, 1 J/cm2). Cell viability was determined by the MTT reduction assay at 24-hour post light irradiation. In vitro photoactivation and two-photon in vitro imaging. The HeLa cells were incubated with testing compound (50 μM of RuEuL or 10 μM of PtEuL (negative control)) for 6 hours. The complexes (cell impermeable and dissolved in the medium) were washed out with PBS buffer. The cells with complexes were placed in a tissue culture chamber (5% CO2, 37 oC) inside the twophoton confocal microscope. In vitro images were captured by using the Leica SP8 (upright configuration) confocal microscope which was equipped with a femto-second laser (wavelength: 700 to 1000 nm) and 40×/60×oil immersion objective. The in vitro images were taken at different


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times of laser excitation (ex = 488 nm, Power = 7 mW). The pictures were acquired with twophoton excitation (ex = 700 nm, Power = 420 mW). In vivo photo-induced tumor growth inhibition and ex vivo imaging of photo activation of the prodrug. For tumor xenograft studies, SW480 cells (5 × 106) were suspended in 100 µL serum-free RPMI medium and mixed with an equal volume of matrigel (Gibco, USA) (1:1 ratio) before being injected into the right flanks of male 8-week-old BALB/c nude mice. After 14-day inoculation, when tumors grew to an average volume of 180 mm3, mice were randomly put into different experimental groups. RuEuL without blue light activation (RuEuL) and that with blue light activation (488-nm blue light (P = 100 mW) irradiation for 30 min) (RuEuL + hv) were diluted to the desired concentrations in 0.9% saline and were injected directly into the tumor by using 29-gauge syringes. Mice receiving an equivalent volume of 0.9% saline alone served as controls. Intratumoral injections were repeated twice weekly for three weeks. Body weight and tumor volume were measured three times per week. Tumor volume was calculated by (length × width2)/2. After the 21-day experimental period, mice were sacrificed and their tumors were harvested and weighed. For the ex vivo imaging of the photo-activation of RuEuL, it was performed on the tumors after the inhibitor experiment. Two tumors were imaged on a 365 nm UV lamp. Then the tumors were injected with 0.5 mg of RuEuL or EuL (control) under dark and incubated for 10 more minutes and then imaged. To investigate the ex vivo photo-induced activation ability of RuEuL, a 488-nm blue light continuous-wave (CW) laser (P = 100 mW) was used to treat the tumor for 30 minutes. After that, the tumors were imaged on the UV lamp.

ASSOCIATED CONTENT


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Supporting Information. Supporting figures and tables are shown in the supporting information which is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: KLW [email protected]; GLL [email protected]; WCST [email protected]; JCGB [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was funded by grants from The Hong Kong Research Grants Council (HKBU 12309516), HKBU (FRG 2/16-17/016), and National Natural Science Foundation of China (21402167 and 21401035), Natural Science Foundation of Guangdong Province (2014A030307027) and Dr Kennedy Y. H. Wong Distinguished Visiting Professorship Scheme 2016/17, Hong Kong Baptist University (J.C.G. B.). ABBREVIATIONS CW, continuous-wave; fs, femtosecond; HPLC, high performance liquid chromatography; MLCT, metal to ligand charge transfer; PDI, photodynamic therapy index. REFERENCES (1) Abet, V.; Filace, F.; Recio, J.; Alvarez-Builla, J.; Burgos, C. Prodrug approach: an overview of recent cases. Eur. J. Med. Chem. 2017, 127, 810-827.


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(2) Wermuth, C.; Ganellin, C.; Lindberg, P.; Mitscher, L. Glossary of terms used in medicinal chemistry. Pure Appl. Chem. 1998, 70, 1129-1143. (3) Xiao, H.; Yan, L.; Zhang, Y.; Qi, R.; Li, W.; Wang, R.; Liu, S.; Huang, Y.; Li, Y.; Jing, X. A dual-targeting hybrid platinum(IV) prodrug for enhancing efficacy. Chem. Commun. 2012, 48, 10730-10732. (4) Yoong, S. L.; Wong, B. S.; Zhou, Q. L.; Chin, C. F.; Li, J.; Venkatesan, T.; Ho, H. K.; Yu, V.; Ang, W. H.; Pastorin, G. Enhanced cytotoxicity to cancer cells by mitochondria-targeting MWCNTs containing platinum(IV) prodrug of cisplatin. Biomaterials 2014, 35, 748-759. (5) Ma, J. P.; Dong, H. Q.; Zhu, H. Y.; Li, C. W.; Li, Y. Y.; Shi, D. L. Deposition of gadolinium onto the shell structure of micelles for integrated magnetic resonance imaging and robust drug delivery systems. J. Mater. Chem. B 2016, 4, 6094-6102. (6) Li, B. B.; Liu, H.; Sun, C. B.; Ahmad, Z.; Ren, Z. H.; Li, X.; Han, G. R. Core-shell SrTiO3:Yb3+,Er3+@mSiO(2) Nanoparticles for controlled and monitored doxorubicin delivery. RSC Adv. 2016, 6, 26280-26287. (7) Zhao, P. F.; Zheng, M. B.; Luo, Z. Y.; Gong, P.; Gao, G. H.; Sheng, Z. H.; Zheng, C. F.; Ma, Y. F.; Cai, L. T. NIR-driven smart theranostic nanomedicine for on-demand drug release and synergistic antitumour therapy. Sci. Rep. 2015, 5, 14258. (8) Dai, Y. L.; Xiao, H. H.; Liu, J. H.; Yuan, Q. H.; Ma, P. A.; Yang, D. M.; Li, C. X.; Cheng, Z. Y.; Hou, Z. Y.; Yang, P. P.; Lin, J. In vivo multimodality imaging and cancer therapy by nearinfrared light-triggered trans-platinum pro-drug-conjugated upconverison nanoparticles. J. Am. Chem. Soc. 2013, 135, 18920-18929.


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(9) Min, Y.; Li, J.; Liu, F.; Yeow, E. K.; Xing, B. Near-infrared light-mediated photoactivation of a platinum antitumor prodrug and simultaneous cellular apoptosis imaging by upconversionluminescent nanoparticles. Angew. Chem., Int. Ed. 2014, 53, 1012-1016. (10) Zhao, Y.; Roberts, G. M.; Greenough, S. E.; Farrer, N. J.; Paterson, M. J.; Powell, W. H.; Stavros, V. G.; Sadler, P. J. Two-photon-activated ligand exchange in platinum(II) complexes. Angew. Chem., Int. Edit. 2012, 51, 11263-11266. (11) Lammers, T.; Aime, S.; Hennink, W. E.; Storm, G.; Kiessling, F. Theranostic nanomedicine. Acc. Chem. Res. 2011, 44, 1029-1038. (12) Li, H. G.; Lan, R. F.; Chan, C. F.; Jiang, L. J.; Dai, L. X.; Kwong, D. W. J.; Lam, M. H. W.; Wong, K. L. Real-time in Situ monitoring via europium emission of the photo-release of antitumor cisplatin from a Eu-Pt complex. Chem. Commun. 2015, 51, 14022-14025. (13) Zhou, F.; Feng, B.; Yu, H.; Wang, D.; Wang, T.; Liu, J.; Meng, Q.; Wang, S.; Zhang, P.; Zhang, Z.; Li, Y. Cisplatin prodrug-conjugated gold nanocluster for fluorescence imaging and targeted therapy of the breast cancer. Theranostics 2016, 6, 679-687. (14) Cheng, Y.; Huang, F.; Min, X.; Gao, P.; Zhang, T.; Li, X.; Liu, B.; Hong, Y.; Lou, X.; Xia, F. Protease-responsive prodrug with aggregation-induced emission probe for controlled drug delivery and drug release tracking in living cells. Anal. Chem. 2016, 88, 8913-8919. (15) Jia, X.; Zhao, X.; Tian, K.; Zhou, T.; Li, J.; Zhang, R.; Liu, P. Fluorescent copolymer-based prodrug for pH-triggered intracellular release of DOX. Biomacromolecules 2015, 16. 3624-3631. (16) Waalboer, D. C.; Muns, J. A.; Sijbrandi, N. J.; Schasfoort, R. B.; Haselberg, R.; Somsen, G. W.; Houthoff, H. J.; van Dongen, G. A. Platinum(II) as bifunctional linker in antibody-drug conjugate formation: coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model. ChemMedChem 2015, 10, 797-803.


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(17) Muscella, A.; Vetrugno, C.; Fanizzi, F. P.; Manca, C.; De Pascali, S. A.; Marsigliante, S. A new platinum(II) compound anticancer drug candidate with selective cytotoxicity for breast cancer cells. Cell Death Dis. 2013, 4, e796. (18) Du, J.; Kang, Y.; Zhao, Y.; Zheng, W.; Zhang, Y.; Lin, Y.; Wang, Z.; Wang, Y.; Luo, Q.; Wu, K.; Wang, F. Synthesis, Characterization, and in vitro antitumor activity of ruthenium(II) polypyridyl complexes tethering EGFR-inhibiting 4-anilinoquinazolines. Inorg. Chem. 2016, 55, 4595-4605. (19) Sun, W.; Li, S.; Haupler, B.; Liu, J.; Jin, S.; Steffen, W.; Schubert, U. S.; Butt, H. J.; Liang, X. J.; Wu, S. An amphiphilic ruthenium polymetallodrug for combined photodynamic therapy and photochemotherapy in vivo. Adv. Mater. 2017, 29, 1603702. (20) Sun, W.; Parowatkin, M.; Steffen, W.; Butt, H. J.; Mailander, V.; Wu, S. Rutheniumcontaining Block Copolymer Assemblies: Red-light-responsive metallopolymers with tunable nanostructures for enhanced cellular uptake and anticancer phototherapy. Adv. Healthc. Mater. 2016, 5, 467-473. (21) Howerton, B. S.; Heidary, D. K.; Glazer, E. C. Strained ruthenium complexes are potent light-activated anticancer agents. J. Am. Chem. Soc. 2012, 134, 8324-8327. (22) Beeby, A.; Faulkner, S.; Parker, D.; Williams, J. A. G. Sensitised luminescence from phenanthridine appended lanthanide complexes: analysis of triplet mediated energy transfer processes in terbium, europium and neodymium complexes. J. Chem. Soc., Perkin Trans. 2 2001, 1268-1273. (23) Singh, T. N.; Turro, C., Photoinitiated DNA binding by cis-[Ru(bpy)(2)(NH3)(2)](2+). Inorg. Chem. 2004, 43, 7260-7262.


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Figure 1. Schematic illustration of the photo-induced cleavage of PtEuL and RuEuL in previous work and this work, respectively as photo-responsive anticancer prodrugs generating highly luminescent europium off - on signals and cytotoxic species.


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Figure 2. Absorption (a) and emission (ex = 350 nm) spectra (b) of RuEuL and EuL 5 μM in aqueous solution. Inset of (b): Two-photon induced emission spectrum of EuL (120 M in aqueous solution, ex = 800 nm).


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Figure 3. Qualitative analysis of photo-dissociation of RuEuL by absorption (a) emission (b) spectroscopy and HPLC analysis (c and d). Absorption (a) and emission (ex = 350 nm) (b) spectra of RuEuL 5 μM in aqueous solution after various times of laser irradiation at 488 nm (P = 1 W). Inset of b) Photographic image of europium emission enhancement of RuEuL after 8 min laser irradiation at 488 nm (P = 1 W). (c) HPLC qualitative analysis of the photodissociation of RuEuL in aqueous solution and of reference samples: EuL (red), [Ru(OH)(bpy)2(H2O)]+ (green), RuEuL (blue), and RuEuL after 2 min (cyan) and 8 min (black) of 488 nm laser irradiation (P = 1 W). The concentration of each sample is 5 M in aqueous solution. (d) Real-time absorption spectra of the corresponding peaks in the HPLC trace with retention times = 12.1, 4.3 and 11.3 min, respectively.


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Figure 4. 1H NMR analysis of the photo-dissociation of the analogue of RuEuL, RuLaL, in a mixed solution of deuterium oxide (D2O) and methanol-d4 (1:1, V:V) and of reference samples: LaL (red), [Ru(OH)(bpy)2(H2O)]+ (green), RuLaL (blue), and RuEuL after 15 min (cyan) and 30 min (pink) of 488 nm laser irradiation (P = 1 W). The concentration of each sample is ～ 2 mM.


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Figure 5. Quantification of photodissociation products of RuEuL after laser irradiation at 488 nm (P = 1 W). (a) Calibration curves of the ESI-MS response vs concentration of [Ru(OH)(bpy)2(H2O)]+ or EuL and emission intensity vs concentration of EuL. (b) Quantitative analysis of the photo-dissociation products [Ru(OH)(bpy)2(H2O)]+ and EuL after irradiation of RuEuL 5 M with 488 nm laser light (P = 1 W). (c) Three-dimensional diagram representing the quantitative analysis of the photo-dissociation of RuEuL. The photo-dissociation products, EuL and [Ru(OH)(bpy)2(H2O)]+ are indicated in the diagram by red and blue colours, respectively.


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Figure 6. (a) DNA binding profiles of RuEuL and EuL (control) in TE buffer (10 mM Tris-HCl, 1mM EDTA, pH = 8.0) under dark and after photo-activation. Experimental details are shown in the supporting information. (b) MTT assays of dark and photo-cytotoxicity of RuEuL, PtEuL (control) and EuL (control). PDI is the photodynamic therapy index, equal to the ratio of dark IC50 with respect to light IC50. The IC50 and PDI values are summarized in Table 1.


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Figure 7. (Left) Two-photon excited monitoring of photo-induced dissociation of RuEuL and PtEuL (λex. = 700 nm) in aqueous solution and (Right) in vitro imaging of the photo-induced drug release of RuEuL and PtEuL (control). The HeLa cells were incubated with 50 M of RuEuL or 10 M of PtEuL for 6 hours and then irradiated with 488-nm laser (P = 7 mW); the images were acquired under two-photon 700-nm excitation.


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Figure 8. In vivo photo-induced tumour growth inhibition (a, b and c) and ex vivo photo-activation by RuEuL (d). Effect of RuEuL (with or without blue light activation) on inhibition of tumour growth in SW480 xenografted nude mice. SW480 tumour cells (5 × 106) were injected subcutaneously into the right flanks of BALB/c nude mice. When the tumours grew to an average volume of 180 mm3, mice were randomized into 3 different experimental groups. Mice either received (i) 0.9% saline, (ii) 40 μg RuEuL or (iii) 40 μg 488 nm blue light activated RuEuL (RuEuL+ hv) twice a week via intra-tumour injection. Three times a week, tumour volumes were measured and calculated using the formula (length × width2)/2. On day 21, mice were sacrificed and their tumours were harvested and weighed. (a) Tumour volume, (b) Representative tumour images and (c) Tumour weight. (d) Ex vivo imaging of blue-light activation of RuEuL and EuL (control).


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Table 1. IC50 (μM) values of RuEuL, PtEuL (control) and EuL (control) for HeLa cells under dark and blue-light irradiation. [a] IC50 in HeLa cells [M] Complex

PDI Dark

Light

[b]

RuEuL

277.0 ±7.1

32.5 ±8.2

8.5

PtEuL

23.6 ±0.8

22.9 ±3.2

1.0

EuL

510.6 ±12.9

508.2 ±20.2

1.0

a) Incubation time = 24 hours; b) λirr. = 488 nm, light dosage = 1 J∙cm-2. Raw data shown in Figure S13.


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A smart europium-ruthenium complex as anticancer prodrug: controllable drug release and real-time monitoring under different light excitations


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A Smart EuropiumâRuthenium Complex as Anticancer Prodrug

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