Nucleus-Targeted Organoiridium-Albumin Conjugate for Photodynamic Cancer Therapy.

Angewandte A Journal of the Gesellschaft Deutscher Chemiker Chemie International Edition www.angewandte.org Accepted Article Title:Nucleus-targeted organoiridium-albumin conjugate for photoactivated cancer therapy Authors:Pingyu Zhang, Huaiyi Huang, Samya Banerjee, Guy Clarkson, Chen Ge, Cinzia Imberti, and Peter J. Sadler This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article. To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201813002 Angew. Chem. 10.1002/ange.201813002 Link to VoR: http://dx.doi.org/10.1002/anie.201813002 http://dx.doi.org/10.1002/ange.201813002 10.1002/anie.201813002 COMMUNICATION Nucleus-targeted organoiridium-albumin conjugate for photodynamic cancer therapy Pingyu Zhang,[a,c] Huaiyi Huang,*[b,c] Samya Banerjee,[c] Guy J. Clarkson,[c] Chen Ge,[a] Cinzia Imberti,[c] Peter J. Sadler*[c] Abstract: A novel organoiridium-albumin bioconjugate (Ir1-HSA) was functionalized metal complexes (e.g. Pt, Ru, Os) have been synthesized via reaction of a pendant maleimide ligand with human developed for cancer therapy, illustrating that HSA plays a key serum albumin. The phosphorescence of Ir1-HSA was enhanced role in augmenting anticancer activity.[10] significantly compared to parent complex Ir1. The long Here we have conjugated a maleimide-functionalized phosphorescence lifetime and high 1O quantum yield of Ir1-HSA are octahedral organo-iridium(III) complex (Ir1, Figure 1a) to HSA, 2 highly favourable properties for photodynamic therapy. Ir1-HSA giving rise to a large enhancement in the phosphorescence of Ir1- mainly accumulated in the nucleus of living cancer cells and showed HSA compared to Ir1. Ir1-HSA is notably nontoxic in the dark, but remarkable photocytotoxicity against a range of cancer cell lines and exhibits potent photo-cytotoxicity with significant selectivity for tumor spheroids (light IC ; 0.8-5 M, photo-cytotoxicity index PI = 40- cancer cells and cancer cell spheroids over normal cells. Ir1-HSA 50 60) while remaining non-toxic to normal cells and normal cell appears to be the first example of an HSA-functionalized iridium spheroids, even after photo-irradiation. This nucleus-targeting conjugate which allows targeting of cell nuclei, as well as being organoiridium-albumin is a strong candidate photosensitizer for an efficient photosensitizer for PDT. anticancer photodynamic therapy. The octahedral organo-iridium(III) complex Ir1, containing two chelated phenylpyridine ligands and two monodentate pyridines functionalized with a maleimide substituent, was synthesized and Photodynamic therapy (PDT) is a non-invasive cancer therapy,[1] fully characterized as described in the Supporting Information. It which uses photosensitizers and light to convert cellular triplet was highly stable in phosphate-buffered saline (PBS) solution for oxygen (3O ) to highly reactive and cell-damaging singlet oxygen 2 12 h in the dark and photostable after 1 h irradiation with blue light (1O).[2] Examples of clinical photosensitizers include 2 (465 nm; Figure S1). hematoporphyrin derivatives (Photofrin) and aminolevulinic acid To investigate the reactivity of the C=C bond of the pendant (ALA, a porphyrin precursor).[3] In recent years, metal complexes maleimides of Ir1, the complex was reacted with cysteine (Cys) in with high luminescence have emerged as promising photo- a molar ratio of 2[Cys]: 1[Ir1] in DMSO-d /D O (2/1 v/v) at 298 K 6 2 theranostic candidates due to their superior photochemical and for 30 min. 1H NMR peaks for the vinyl protons of the maleimide photophysical properties.[4] An octahedral tris-N,N-chelated RuII groups at 6.62 ppm disappeared upon the addition of Cys, and complex (TLD1433) recently entered clinical trials for bladder new peaks appeared between 2.9 ppm and 3.9 ppm assignable PDT,[4a] and WST 11 (TOOKAD® Soluble), a square-planar PdII to conjugated Cys (Figure S2). The intensities of the peaks bacteriochlorophyll derivative, has been approved for vascular- indicated that Cys reacted with each of the two pendant targeted PDT.[4b] maleimides of Ir1. The conjugate was further characterised by Human serum albumin (HSA) conjugates can be used to ESI-MS (Figures S3-S4). delivery anticancer drugs. HSA is abundant in blood serum To investigate whether the free Cys34 thiol of HSA can similarly (ca.0.6 mM), rich in histidine, and contains a free thiol residue at react with the C=C in maleimide, Ir1 (30 M) was incubated with cysteine-34.[5] Moreover, HSA functions as a physiological various amounts of HSA (0-120 M) for 1 h and the products were antioxidant,[6] and binds a wide range of biologically and clinically separated by RP-HPLC. The peak for Ir1 gradually disappeared important molecules.[7] The effectiveness of HSA-coupled with increasing amounts of HSA, with complete reaction observed anticancer drugs has been established clinically for doxorubicin at 120 M HSA (Figure 1b). This molar ratio of [HSA]:[Ir1] of 4:1, (INNO206; aldoxorubicin),[8] and HSA-based nanoparticle- is consistent with the free thiol content of the HSA used, encapsulated paclitaxel (Abraxane®).[9] Recently, HSA- determined via reaction with 2,2’-dithiodipyridine (2,2’-DTDP) using a slightly modified literature approach.[11] This gave a thiol content of 0.27  0.1 mol SH per mol HSA (Figure 1c). Hence, the [a] Dr. P. Zhang, C. Ge concentration of free -SH groups from 120 M HSA was 32.4  College of Chemistry and Environmental Engineering 1.2 M, which reacted with 30 M Ir1, suggesting formation of a Shenzhen University Shenzhen, 518060, China. 1:1 Ir1:HSA adduct. As Cys34 is in a crevice, it is likely that the [b] Dr. H. Huang second maleimide group is not accessible to a second HSA. RP- School of Pharmaceutical Science (Shenzhen), Sun Yat-sen HPLC studies on the time-dependent binding showed that the University, Guangzhou, 510275, China reaction was relatively rapid with a half-life of ca. 20 min (Figure E-mail: huanghy87@mail.sysu.edu.cn [c] Dr. H. Huang, Dr. S. Banerjee, Dr. G. J. Clarkson, Dr. C. Imberti, S5). Prof. P. J. Sadler Department of Chemistry, University of Warwick Coventry, CV4 7AL, UK E-mail: P.J.Sadler@warwick.ac.uk Supporting information and the ORCID identification numbers for the authors of this article can be found under:https://doi.org/xxxxxx tpircsunaM detpeccA Angewandte Chemie International Edition This article is protected by copyright. All rights reserved. 10.1002/anie.201813002 COMMUNICATION Figure 2. (a) Emission intensity of HSA (100 M) after reaction with 10 mol equiv cystine (giving disulfide formation at Cys34) in PBS (pH = 7.4) for 24 h at 277 K, followed by treatment with Ir1 for 30 min; Inset: Images of the reaction mixture compared to Ir1-HSA under UVA irradiation showing the decrease in phosphorescence. (b) Emission intensity of Ir1 (4 M) in the presence of various amino acids (100 M) in PBS solution. (c) Emission intensity at 515 nm of Ir1 in the presence (I) vs. the absence (I0) of amino acids ac . c ording to (b). (d) Structure of HSA (PDB:5IJF); Cys34 and His39 are labelled Figure 1. (a) Scheme for the conjugation of Ir1 to HSA. (b) Reaction of Ir1 (30 M) with various concentrations of HSA (0, 60, 90 and 120 M, concentrations of Ir1 with histidine resulted in an emission enhancement of ca. of Cys34 free thiol group were 0, 16.2, 24.3, 32.4 M, respectively) studied by 37-fold. In contrast, no significant luminescence enhancement RP-HPLC (UV detection at 280 nm). (c) Variation of absorbance at 340 nm at various HSA:2,2’-DTDP ratios ([HSA] = 80 M; [DTDP] = 0-110 M) and was observed with the other amino acids, including Cys (Figure determination of the thiol content of HSA. (d) Emission spectra of Ir1 (4 M) in 2b,c). Although Ir1 binds to Cys34, other factors appear to be the presence of increasing concentrations of HSA (0-30 M, [Cys34 free thiol] responsible for the enhancement of phosphorescence of Ir1- 0-8.1 M), in PBS (pH = 7.4), reaction time: 20 min at each concentration, ex = HSA. These include interactions with His residues, a strong 405 nm; Inset: photos of Ir1 (1) and Ir1-HSA (2) under UVA irradiation. (e) Dependence of the phosphorescence intensity of Ir1 at = 515 nm on the candidate being nearby His39 (Figure 2d). concentration of free thiol of HSA, as ratio [Ir1]/[free thiol]. Ir(III) complexes with weakly bound ligands are known to bind strongly to amino acids/proteins through ligand substitution reactions, especially to histidine/histidine rich proteins, and their Ir1 (4 M) itself was only weakly emissive in aqueous solution use in protein staining has been reported.[12] Here it is evident that ( ex = 405 nm), whereas Ir1-HSA showed strong histidine can switch on the phosphorescence of Ir1. We recorded phosphorescence under the same conditions (inset graph Figure the ESI-MS of a mixture of Ir1 and His; two peaks at m/z 656.2 1d). The phosphorescence of Ir1 gradually increased with and 903.2 assignable to His-bound Ir(III) species were detected increasing concentrations of HSA (Figure 1d), plateauing at a mol (Figure S6). These results suggest that one sterically-hindered ratio of ca. 1.0 Ir1:HSA(free-SH); with a phosphorescence monodentate maleimide ligand is released after binding of Ir1 to enhancement of ca. 53-fold (Figure 1e). Cys34, being displaced by His39. To remove free thiol groups from HSA (by oxidation to a As Ir1-HSA emitted strong phosphorescence, we evaluated disulphide), a 10-fold molar excess of cystine was added to a 100 Ir1-HSA as a potential photosensitizer for PDT. Firstly, Ir1 (0.4 M HSA solution for 24 h at 277 K. Then this HSA-Cys34-S-S- mM) was dissolved in 20 mL MeOH:H O (1:2 v/v), and HSA (0.4 2 Cys product was reacted with Ir1 for 30 min. The observed mM) was added. The reaction mixture was stirred for 1 h, followed phosphorescence was much weaker compared to the conjugate by Ir1-HSA purification by dialysis (10 kDa cut-off filter) to remove formed by reaction of HSA-Cys34 and Ir1 (Figure 2a), clearly unbound Ir1. The Ir1-HSA conjugate showed a new IR band at suggesting that the free thiol of Cys34 is the binding site of Ir1. 1043 cm-1, characteristic of a new C-S stretch (Figure S7). HSA is a large protein (66.5 kDa) with a single-chain of 585 Next the localization of Ir1-HSA in living A549 lung cancer cells amino acid residues.[5-7] To provide an indication on which was investigated using confocal microscopy. The cells were residues of HSA might be involved in the luminescence incubated with Ir1-HSA ([Ir] = 5 M) for various times. Real-time enhancement of Ir1, the interaction of Ir1 with various amino acids imaging showed that Ir1-HSA mainly accumulated in the was studied by luminescence analysis (Figure 2b,c). Interaction cytoplasm within the first 30 min and then migrated to the nucleus tpircsunaM detpeccA Angewandte Chemie International Edition This article is protected by copyright. All rights reserved. 10.1002/anie.201813002 COMMUNICATION Figure 3. Confocal microscopy images of living A549 cells incubated with Ir1-HSA ([Ir] = 5 M) in real time (5, 15, 40, 60, 120 min); ex = 405 nm, em = 550  20 nm. upon further incubation (60 min-120 min) (Figure 3). In contrast, Figure 4. Immunofluorescence staining of HSA in cells exposed to Ir1, HSA, the green luminescence of Ir1 alone was observed throughout the Ir1-HSA ([Ir] = 5 M, 2 h), respectively. ex = 563 nm; em = 580-630 nm. cells, both in cytoplasm and nucleus (Figure S8). This subcellular localization of Ir1 and Ir1-HSA in A549 cells was also confirmed by ICP-MS (Figure S9), demonstrating that iridium localized both in the cytoplasm and nucleus of cells treated with Ir1, whereas it mainly accumulated in the nucleus of Ir1-HSA treated cells. We further confirmed that the luminescence of Ir1-HSA was perfectly co-localized with the blue fluorescence of Hoechst 33258 (a nucleus dye), with a co-localization efficiency of 0.82 in live A549 cells (Figure S10). To address the question as to whether HSA co-migrated with Ir, we used an immunofluorescence approach to determine the intracellular localization of HSA. As expected, HSA was not detected in Ir1 treated A549 cells (Figure 4). However, Ir1-HSA exposure led to the accumulation of HSA in the cytoplasm and the nuclear membrane. When the cells were treated with HSA, the red luminescence was observed as well. This suggests that, although HSA facilitates delivery of the iridium complex to the nucleus, it does not itself penetrate past the nuclear membrane, and is probably released from Ir1-HSA before the Ir(III) complex migrates into the nucleus. The phosphorescence quantum yield of Ir1 was very low (0.001) and its phosphorescence lifetime only 182.7 ns in methanol:PBS (1:1, v/v; Figure 5a; Table S1) at 298 K. Compared to Ir1, the quantum yield for Ir1-HSA increased 36x (to 0.036) and Figure 5. (a) Phosphorescence lifetimes of Ir1 and Ir1-HSA in methanol:PBS its emission lifetime to 871.8 ns. The long phosphorescence (1:1 v/v) at 298 K; (b) EPR spectra of Ir1 and Ir1-HSA with TEMP after 20 min lifetime of Ir1-HSA makes it ideal for 1O generation. We used 2 light irradiation (465 nm, 5.76 J/cm2). (c) Fluorescence intensity of ROS in A549 electron paramagnetic resonance (EPR) spectroscopy with 2,2,6,6-tetramethylpiperidine (TEMP) as a spin trap to detect 1O cells treated with Ir1 or Ir1-HSA ([Ir] = 5 M) and ROS probe in the dark or upon 2 generation by Ir1 and Ir1-HSA under 465 nm irradiation. As light irradiation (5, 10, 20 min). The excitation wavelength of the ROS probe was illustrated in Figure 5b, a characteristic 1:1:1 triplet assignable to 520 nm and the fluorescence was measured at 590-625 nm. 2,2,6,6-tetramethylpiperidine-1-oxyl was observed upon irradiation (20 min). Notably the intensity of the EPR signal generated by Ir1-HSA was significantly stronger than for Ir1. The hepatoma: Hep-G2; cisplatin resistant lung: A549R) and normal 1O quantum yield[13] ((1O )) of Ir1-HSA was 0.83, much higher human cells (lung: MRC-5; liver: LO2). Cells in the ‘light’ plate 2 2 than that for Ir1 (0.06) upon 465 nm light irradiation (Table S1). were incubated with Ir1 or Ir1-HSA for 2 h in the dark, washed The long excited-state lifetime and very high 1O generation with PBS, followed by 20 min irradiation using blue LEDs (465 nm, 2 quantum yield make Ir1-HSA a potential PDT agent. To explore 5.76 J/cm2), while the ‘dark’ plate was kept in the dark. Then all this, the dark- and photo-antiproliferative activity of Ir1 and Ir1- cells were allowed to recover over 46 h. No cell death was HSA was determined against human cancer cells (lung: A549; observed for untreated cells exposed to light (Figure S11). Ir1 was tpircsunaM detpeccA Angewandte Chemie International Edition This article is protected by copyright. All rights reserved. 10.1002/anie.201813002 COMMUNICATION Table 1. IC50 (M) values for Ir1 and Ir1-HSA against 2D and 3D (spheroids) In contrast to other well-studied cyclometalated iridium cancer and normal cell lines. complexes, which mainly located in the cytoplasm, Ir1-HSA appears to be the first reported nucleus-targeting photosensitizer. Cell linesa Ir1 Ir1-HSA There are only a few reports of the transport of albumin to the Dark Light Dark Light nucleus: in response to oxidative stress,[14] and via A549 89.63.7 53.34.5 62.32.6 1.10.3 permeabilization of cells with digitonin.[15] In our work, it appears Hep-G2 83.53.7 54.82.6 85.63.2 2.20.3 that albumin plays an important role in the transport and delivery A549R 75.64.1 56.21.5 84.95.9 2.30.2 of Ir1 to the cell nucleus. MRC-5 90.61.7 76.91.6 96.46.1 78.72.3 Importantly, Ir1-HSA exhibited a long phosphorescence lifetime LO2 89.32.3 76.50.9 88.63.0 66.44.5 and remarkably high 1O generation quantum yield along with high 2 A549 spheroid 100 100 65.65.9 4.80.2 photostability, which were essential for efficient PS. Ir1-HSA MRC-5 spheroid 100 100 100 100 exhibited excellent photocytotoxicity against a range of cancer aCells were incubated with the compounds for 2 h in the dark, washed, fresh cell lines and multicellular spheroids with a high photo-cytotoxicity medium added, followed by incubation in the dark or irradiation at 465 nm (20 index while remaining dormant in normal cells/spheroids, even min, 5.76 J/cm2), and a further 46 h incubation. IC50 values for Ir1 and Ir1-HSA after photo-irradiation. All these properties confirm that Ir1-HSA are based on Ir concentration. Under the same experimental conditions, 5- aminoevulinc acid, a clinical PDT agent and cisplatin gave IC50 > 100 M both could be an efficient photosensitizer with novel nucleus-targeting in the dark and upon light irradiation. The IC50 values (concentrations which property for potential clinical PDT applications. caused 50% of cell death) were determined as duplicates of triplicates in three independent sets of experiments. For each data point, the average and standard deviation are reported. For 3D toxicity assays, 8 spheroids were selected for each condition studied. Acknowledgements relatively nontoxic toward A549 cells both in the dark (89.6 M) We thank the EPSRC (grants EP/G006792 and EP/M027503/1 and light (53.3 M) (Table 1). In contrast, Ir1-HSA was non-toxic for PJS), National Natural Science Foundation of China (NSFC, towards A549 cells in the dark (62.3 M), but became highly 21701113), the Science and Technology Foundation of Shenzhen cytotoxic upon irradiation (1.1 M) with a high photocytotoxicity (JCYJ20170302144346218) and the Natural Science Foundation index (PI, PI = dark IC /light IC ) of 56.6 (Figure S11). Similar of SZU (2018036) for PZ, Newton Fund (NF160307 for HH, 50 50 photodynamic efficiency for Ir1-HSA was also observed for Hep- NF151429 for SB) and the Wellcome Trust (209173/Z/17/Z for CI) G2 and A549R cells. Notably, under the same experimental for support. We thank Dr. Lijiang Song for excellent assistance conditions, both Ir1 and Ir1-HSA were non-toxic toward normal with mass spectrometry; Dr. Ben Breeze with EPR experiments; cells (MRC-5 and LO2) (Table 1). and Dr. Ivan Prokes with NMR spectroscopy. We further investigated the photocytoxicity in 3D multicellular spheroids (MCSs) with semidiameters of ∼400 m. The Keywords: Organoiridium, Albumin, Photosensitizer, cytotoxicities of the complexes toward MCSs were determined by Photodynamic therapy measurement of ATP concentrations using the CellTiter-Glo® 3D Cell Viability Assay (Promega). As shown in Table 1, both Ir1 and [1] a) F. Heinemann, J. Karges, G. Gasser, Acc. Chem. Res. 2017, 50, Ir1-HSA were non-toxic towards A549 cancer spheroids and 2727; b) J. D. Knoll, C. Turro, Coord. Chem. Rev. 2015, 282, 110. normal cell spheroids in the dark (IC > 50 M). However, Ir1- [2] a) J. Liu, C. Zhang, T. W. Rees, L. Ke, L. Ji, H. Chao, Coord. Chem. 50 HSA showed strong phototoxic effects on A549 cancer spheroids Rev. 2018, 363, 17; b) H. Chen, J. Tian, W. He, Z. Guo, J. Am. Chem. Soc. 2015, 137, 1539. upon light irradiation, with an IC value of 4.8 M. We also 50 [3] D. E. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev. Cancer 2003, 3, 380. studied the effect of Ir1-HSA on the kinetics of 3D MCSs [4] a) S. Monro, K. L. Colon, H. Yin, J. Roque III, P. Konda, S. Gujar, R. P. regrowth. After treatment with Ir1-HSA ([Ir] = 5 M) in the dark, Thummel, L. Lilge, C. G. Cameron, S. A. McFarland, Chem. Rev. 2018, the diameters of 3D MCSs increased slightly after 48 h. However, DOI: 10.1021/acs.chemrev.8b00211; b) I. S. Gill, A. R. Azzouzi, M. the MCSs treated with Ir1-HSA followed by 20 min light irradiation Emberton, J. A. Coleman, E. Coeytaux, A. Scherz, P. T. Scardino, J. decreased in size over time (Figure S12). Urology 2018, 200, 786; c) P. Zhang, C. K. Chiu, H. Huang, Y. P. Lam, A reactive oxygen species (ROS) detection assay kit was used A. Habtemariam, T. Malcomson, M. J. Paterson, G. J. Clarkson, P. B. to determine whether Ir1-HSA produced ROS within the cells O’Connor, H. Chao, P. J. Sadler, Angew. Chem. Int. Ed. 2017, 56, 14898; d) V. W. Yam, Angew. Chem. Int. Ed. 2015, 54, 8304; e) K. K. 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Vis. 2007, 13, 746. [11] A. J. Stewart, C. A. Blindauer, S. Berezenko, D. Sleep, D. Tooth, P. J. Sadler, FEBS J. 2005, 272, 353. Orchid identification numbers Pingyu Zhang: 0000-0002-2921-9490 Huaiyi Huang: 0000-0002-2091-7954 Samya Banerjee: 0000-0003-4393-4447 Guy Clarkson: 0000-0003-3076-3191 Chen Ge: 0000-0002-4910-6282 Cinzia Imberti: 0000-0003-1187-7951 Peter Sadler: 0000-0001-9160-1941 tpircsunaM detpeccA Angewandte Chemie International Edition This article is protected by copyright. All rights reserved. 10.1002/anie.201813002 COMMUNICATION Entry for the Table of Contents COMMUNICATION Pingyu Zhang, Huaiyi Huang,* Samya Banerjee, Guy J. Clarkson, Chen Ge, Cinzia Imberti, Peter J. Sadler* Page No. – Page No. Nucleus-targeted organoiridium- We report the first example of a nucleus-targeted organo-iridium-HSA conjugate for photodynamic albumin conjugate for photodynamic therapy. Phosphorescence of a weakly emissive maleimide-functionalized Ir complex is greatly cancer therapy enhanced when conjugated to human serum albumin. The iridium-HSA conjugate targets the nucleus and generates 1O for photodynamic therapy upon irradiation with visible light. 2 tpircsunaM detpeccA Angewandte Chemie International Edition This article is protected by copyright. All rights reserved.