LuminescentIridiumComplex-PeptideHybrids(IPHs)for...

Research ArticleLuminescent Iridium Complex-Peptide Hybrids (IPHs) forTherapeutics of Cancer Design and Synthesis of IPHs forDetection of Cancer Cells and Induction ofTheir Necrosis-Type Cell Death

Abdullah-Al Masum1 Yosuke Hisamatsu1 Kenta Yokoi1 and Shin Aoki 12

1Faculty of Pharmaceutical Sciences Tokyo University of Science 2641 Yamazaki Noda Chiba 278-8510 Japan2Imaging Frontier Center Tokyo University of Science 2641 Yamazaki Noda Chiba 278-8510 Japan

Correspondence should be addressed to Shin Aoki shinaokirsnodatusacjp

Received 29 December 2017 Accepted 31 May 2018 Published 1 August 2018

Academic Editor Viktor Brabec

Copyright copy 2018 Abdullah-Al Masum et al is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Death receptors (DR4 and DR5) offer attractive targets for cancer treatment because cancer cell death can be induced byapoptotic signal upon binding of death ligands such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) withdeath receptors Cyclometalated iridium(III) complexes such as fac-Ir(tpy)3 (tpy 2-(4-tolyl)pyridine) possess a C3-sym-metric structure like TRAIL and exhibit excellent luminescence properties erefore cyclometalated Ir complexes func-tionalized with DR-binding peptide motifs would be potent TRAIL mimics to detect cancer cells and induce their cell deathIn this study we report on the design and synthesis of C3-symmetric and luminescent Ir complex-peptide hybrids (IPHs)which possess cyclic peptide that had been reported to bind DR5 e results of 27MHz quartz-crystal microbalance (QCM)measurements of DR5 with IPHs and costaining experiments of IPHs and anti-DR5 antibody suggest that IPHs bind withDR5 and undergo internalization into cytoplasm possibly via endocytosis It was also found that IPHs induce slow cell deathof these cancer cells in a parallel manner to the DR5 expression level ese results indicate that IPHs may offer a promisingtool as artificial luminescent mimics of death ligands to develop a new category of anticancer agents that detect and killcancer cells

1 Introduction

Death receptors (DRs) are often overexpressed on the cellmembrane of cancer cells and bind with death ligands such astumor necrosis factor-related apoptosis-inducing ligand(TRAIL) to send the cell extrinsic apoptotic signal [1] TRAILreceptors comprise five categories plasma membrane-expressed TRAIL-R1 (DR4) TRAIL-R2 (DR5) TRAIL-R3(DcR1) TRAIL-R4 (DcR2) and a soluble receptor osteo-protegerin (OPG) DR4 and DR5 contain death domain (DD)to transduce apoptotic signals and hence named as deathreceptors whereas DcR1 and DcR2 are unable to induce celldeath and regarded as decoy receptors [1ndash7] TRAIL is a C3-symmetric protein consists of three monomeric units andbinds with three DRs [8 9] Upon binding of DR with TRAIL

TRAIL-DR cluster sets up death-inducing signaling complex(DISC) at their cytoplasmic death domain (DD) and recruitsadaptor protein (FADD) via death-effector domain (DED)e signaling activates procaspase-8 to caspase-8 and thencaspase-3 in order to cleave multiple substrates to execute celldeath [10] e nonsignaling DcR1 does not contain cyto-plasmic DD and DcR2 is very similar to death receptors butcontains a truncated form of DD erefore both DcR1 andDcR2 are unable to assemble DISC [4ndash6]

Since death receptors are overexpressed in various typesof cancer cells TRAIL is capable of selectively inducingapoptosis of cancer cells with low cytotoxicity in normal cells[11ndash19] erefore death receptors have been conceived aspromising targets for the treatment and imaging of cancercells To date only limited examples of artificial death

HindawiBioinorganic Chemistry and ApplicationsVolume 2018 Article ID 7578965 18 pageshttpsdoiorg10115520187578965

receptor binders having TRAIL-like functionalities are re-ported Representative examples include DR5 bindingpeptides [20ndash26] zinc-binding site peptide of TRAIL(RNSCWSKD that was screened out from TRAIL (227ndash234))[27] and small molecular TRAIL mimics (bioymi) [28]

Meanwhile cyclometalated iridium (Ir(III)) complexessuch as fac-Ir(tpy)31 (tpy 2-(4-tolyl)pyridine) (Figure 1) drawincreasing attention as one of the imaging tools to study extra-and intracellular events in addition to organic light-emittingdiodes (OLEDs) such as phosphorescent emitters [29ndash33]because of their signicant stability and excellent photophysicalproperties under physiological conditions [31 33ndash39] Suchtypes of Ir complex analogues have been widely applied tooxygen sensors [40 41] chemosensors [42ndash47] and lumi-nescent probes for biological systems [48ndash66] ese advan-tages originate from their high-luminescence quantum yieldsthe long luminescence lifetimes (τsimmicros) that can eliminate theshort-lived autouorescence (τsimns) from biological samples incellular imaging and signicant stokes shift that minimizesself-quenching process [29ndash39] We previously reported someexamples of Ir complexes that can be functionalized asbluesimgreensimred and white color emitters [67ndash71] pH sensors[68ndash70] photosensitizers [68ndash70] and cell death inducer ofcancer cells [68ndash70] Recently we have reported on Ir com-plexes (2a-f and 3a-c) having cationic peptides (typically H2N-KKGG-) (Figure 1) as inducers and detectors of cell death ofJurkat cells (a human T-lymphoma cell line) [72ndash74] eseresults suggest that Ir complexes are potential agents for thediagnosis and treatment of cancer and related diseases and evenfor mechanistic study of cell death processes

Because cyclometalated iridium (Ir(III)) complexes suchas fac-Ir(tpy)3 1 possess a C3-symmetric structure likeTRAIL (the top of Figure 2) it is hypothesized that thesecomplexes could be good scaolds to mimic TRAIL In thismanuscript we report on the design and synthesis of somenew C3-symmetric tris-cyclometalated Ir complexes havingcyclic peptides (Figure 2) [20ndash22] which had been reported

to be able to bind DR5 for selective staining and induction ofcell death of cancer cells e Ir complexes 4ndash6 were syn-thesized by regioselective substitution reactions reported by us[75] and the successive coupling reactions with cyclic peptides[20ndash22] Due to low solubility of 4 in water Ser-Gly-Ser-Gly

MeMe

Me

Me Me

Me

Ir Ir

IrR1

Ir

N

3

3 3

ONH

2a (n = 2)2b (n = 4)2c (n = 6)2d (n = 8)2e (n = 12)2f (n = 16)

3a (R1 = H2N-D-K-D-KGG-)3b (R1 = H2N-KKG-)3c (R1 = H2N-KKKGG-)

H2N-KKGG N

N

NN

nH

N6 HN

N

H

O O O

1 (fac-Ir(tpy)3)

Figure 1 Ir complexes having cationic peptides

TRAIL homotrimer

Receptor binding site

LinkerIr core

Ir complex-peptide homotrimer(structurally similar to TRAIL)

O Me

Ir

N

3

HN

HN

HN

HN

HN

O

O

Inserted for better solubility in water

4 R2 = AcHN-WDCLDNRIGRRQCVKL-CONH2

5 R2 = AcHN-SGSGWDCLDNRIGRRQCVKL-CONH2

6 R2 = AcHN-SGSGSGSGWDCLDNRIGRRQCVKL-CONH2

R2

Figure 2 C3-symmetric tris-cyclometalated Ir complex-peptidehybrids (IPHs)

2 Bioinorganic Chemistry and Applications

(SGSG) was inserted at the N-terminus of the peptide parts of5-6 to improve their solubility e results of 27MHz quartz-crystal microbalance (QCM)measurements of DR5 with 5 and6 and costaining experiments of Jurkat cells with 5 and anti-DR5 antibody that 5 and 6 bind to DR5 at the different sitesfrom the recognition sites of anti-DR5 antibody Some cancercell lines such as Jurkat cells K562 cells and Molt-4 cells werestained with 5 for luminescence microscopic observationshowing that Ir complexes exhibit green emitting spots lo-calized inside the cell In addition it was turned out that 5induces cell death of Jurkat cells more slowly than those by 2c-d (5 requires almost 24 h to induce considerable cell deathwhile 2c 2d 3a and 3c induce cell death in a couple of hours)Binding of 5 to cancer cells and its cytotoxicity against cancercells are dependent on the DR5 expression level of cancer cellsFurther mechanistic studies suggest that cell death induced by5 is necrotic type Interestingly the treatment of cancer cellswith 5 and then with anti-DR5 antibody lowers the staininglevel on cell membrane of Jurkat cells by anti-DR5 antibodyindicating that DR5 undergoes endocytosis upon binding with5 Furthermore it was found that DR5 moved from cytoplasmto the cellmembrane or reproduced on the cellmembrane afteradditional incubation for 6 h Finally detection of Jurkat cellsspiked in bovine blood is demonstrated To the best of ourknowledge 5 is the first example of artificial luminescent deathligand that detects cancer cells and induces their necrosis-typecell death

2 Experimental Section

21 General Information All reagents and solvents werepurchased from commercial suppliers and were used withoutfurther purification unless otherwise noted MTT (3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2H-tetrazolium bromide)was purchased from Dojindo Z-VAD-FMK (Z-Val-Ala-Asp(OMe) fluoromethylketone) was purchased from the PeptideInstitute Necrostatin-1 and IM-54were purchased fromEnzoLife Sciences Anti-DR5 antibody [DR5-01-1] (phycoery-thrin) (ab55863) was purchased from Abcam TRAILApo2L(human recombinant) chloroquine diphosphate verapamilhydrochloride and nicardipine hydrochloride were pur-chased fromWako Pure Chemical Industriese oligomycincomplex was purchased from Cayman Chemical Co quin-idine and 4-aminopyridine were purchased from TCI CCCP(carbonyl cyanide 3-chlorophenylhydrazone) and bafilo-mycin A1 and amiloride hydrochloride were purchased fromSigma-Aldrich Propidium iodide and NaN3 were purchasedfrom Nacalai Tesque Annexin V-Cy3 was purchased fromBioVision Inc All aqueous solutions were prepared usingdeionized and distilled water UV spectra were recorded ona JASCO V-550 spectrophotometer equipped with a tem-perature controller unit at 25plusmn 01degC Emission spectra wererecorded on a JASCOFP-6200 and FP-6500 spectrometers IRspectra were recorded on a Perkin-Elmer FT-IR spectro-photometer (Spectrum100) 1HNMR (300MHz) spectra wererecorded on a JEOL Always 300 spectrometer Tetrame-thylsilane (TMS) was used as an internal reference for 1Hmeasurements in CDCl3 and CD3OD and 3-(Trimethylsilyl)-propionic-2233-d4 acid (TSP) sodium salt was used as an

external reference for 1H NMR measurement in D2OMass spectral measurements were performed on a JEOLJMS-SX102A and Varian TQ-FT Luminescence imagingstudies were performed using fluorescent microscope (Bio-revo BZ-9000 Keyence)in-layer chromatographies (TLC)and silica gel column chromatographies were performedusing Merck Art 5554 (silica gel) TLC plate and Fuji SilysiaChemical FL-100D respectively HPLC experiments werecarried out using a system consisting of two PU-980 in-telligent HPLC pumps (JASCO Japan) a UV-970 intelligentUV-visible detector (JASCO) a Rheodine injector (Model no7125) and a Chromatopak C-R6A (Shimadzu Japan) Foranalytical HPLC the Senshu Pak Pegasil ODS column(Senshu Scientific Co Ltd) (46φtimes 250mm No 07051001)was used For preparative HPLC the Senshu Pak Pegasil ODSSP100 column (Senshu Scientific Co Ltd) (20φtimes 250mmNo 1302014G) was used Lyophilization was performed withthe freeze-dryer FD-5N (EYELA)

22 Synthesis of Ir Complexes and Peptide Units Ir Com-plexes 1 and 7 ese complexes were synthesized accordingto our previously reported procedure [75]

Ir Complex 8 A solution of Ir complex 7 (50mg006mmol) DIEA (190 microL 108mmol) and PyBOP(283mg 054mmol) in distilled DMF (3mL) was stirredfor 10min at room temperature to which β-alanine ethylester (84mg 054mmol) was added e whole solutionwas stirred for 18 h and concentrated under reducedpressure e remaining residue was extracted withCHCl3H2O dried over Na2SO4 concentrated under re-duced pressure and purified by silica gel column chro-matography (hexanes AcOEt 2 3rarr1 2rarr1 4rarr1 6)to obtain Ir complex 8 as a yellow solid is Ir complex(30mg 0026mmol) and 5M LiOH (25mg 106mmol) inH2OTHF (11 9mL) was stirred at 60degC for 17 h and then2N HCl (pH 1) was added to form precipitate eprecipitate was filtrated and washed with H2O to give Ircomplex 8 as a yellow solid (26mg 41 from 7) IR (ATR)] 3279 2924 2579 1975 1712 1586 1527 1471 12591186 1068 1022 892 781 and 750 cmminus1 1H NMR(CD3OD 300MHz) δ 804 (d 3H J 81 Hz) 774 (m6H) 747 (d 3H J 54 Hz) 698 (t 3H J 75Hz) 669 (s3H) 359 (t 6H J 51Hz) 263 (t 6H J 69Hz) and 212(s 9H) ppm ESI-MS (mz) calcd for C48H45IrN6O9 [M]+104228773 and found 104228784

Ir Complex 9 [72] DIEA (63mg 05mmol) PyBOP(1266mg 024mmol) and mono-Boc-protected hexame-thylenediamine (103mg 05mmol) were added to a solutionof 7 (33mg 0039mmol) in distilled DMF (1mL) e re-action mixture was stirred at room temperature for 36 h andthen concentrated under reduced pressure e remainingresidue was purified by silica gel column chromatography(CHCl3MeOH 10 to 501 to 01) gel permeation chro-matography (CHCl3) and recrystallization from hex-anesCHCl3 to afford the Boc-protected 9 as a yellow solid Amixture of TMSCl (21mg 046mmol) and NaI (70mg046mmol) in CH3CN (1mL) was added to a suspension ofBoc-protected 9 (216mg 15 μmol) in CH3CN (33mL) e

Bioinorganic Chemistry and Applications 3

mixture was stirred at room temperature for 10min andsonicated for 2min e insoluble compound was centri-fuged and washed with CH3CN to give 9 as the HI salt eproduct was purified by preparative HPLC (H2O (01TFA)CH3CN (01 TFA) 8020 to 5050 (30min)tr 21min 60mLmin) followed by lyophilization to give 9as a yellow solid (41mg 62 as 3 TFA salt) IR (ATR)] 2934 2862 2035 1674 1600 1531 1472 1425 1262 11991069 892 781 and 721 cmminus1 1H NMR (D2O 300MHz)δ 804 (d 3H J 841Hz) 782ndash776 (m 6H) 769 (d 3HJ 54Hz) 709ndash705 (t 3H J 60Hz) 658 (s 3H) 338ndash333(m 6H) 301ndash296 (t 6H J 69Hz) 211 (s 9H) 168ndash162(m 12H) and 143 (m 12H) ppm ESI-MS (mz) calcd forC57H73IrN9O3 [M+H]+ 112454656 and found 112454487

Fmoc-Leu-NH-SAL-Trt(2-Cl)-resin NH2-SAL-Trt (2-Cl)-resin (500mg 054mmolg) was suspended in DMF(25mL) to which a mixture of Fmoc-Leu-OH (3 eq) DIC(4 eq) and HOBt (4 eq) was added After stirring for 2 hthe residue was filtrated washed with DMF CH2Cl2 andether and dried in vacuo e amount of Fmoc-Leu-OHloaded on the resin was determined by UV absorption ofthe Fmoc derivative at 301 nm (ε301nm 7800Mminus1middotcmminus1)after treatment with 20 (vv) piperidine in DMF Yield593mg (loading 043mmolg resin)

Cyclic peptide CP1 Fmoc-protecting group of Fmoc-Leu-NH-SAL-Trt(2-Cl)-resin (500mg 043mmol) wasdeprotected by treatment with 20 piperidine in DMFEach Fmoc-Xaa-OH (4 eq) was coupled at 45degC for 1 h tothe Fmoc-deprotected resin in the presence of DIC (4 eq)and HOBt (8 eq) in DMF (25mL) After the last depro-tection the N-terminus was acetylated using Ac2O (4 eq)and DIEA (4 eq) in DMF (25mL)e peptide was cleavedfrom the resin and also deprotected using a mixture ofTFAH2OTIPSthioanisole (85251025) After 4 h ofstirring the resin was filtered and washed with TFA Afterevaporation of TFA the precipitation was obtained byadding cooled Et2O and collected by centrifugation ecrude product was purified by preparative HPLC (H2O(01 TFA)CH3CN (01 TFA) 8020rarr5050 (30min)tr 11min 1mLmin) lyophilized to give CP1 as a linearform e linear form of CP1 was subjected to cyclizationusing 01mM aq NH4HCO3 (1mg1mL) After cyclizationreaction for 12ndash24 h the solution was concentrated and theremaining residue was purified again by preparative HPLC(H2O (01 TFA)CH3CN (01 TFA) 8020rarr5050(30min) tr 75min 1mLmin) lyophilized to give CP1 aswhite powder (217mg 26) IR (ATR) ] 3217 2992 22912167 2097 2031 1980 1637 1508 1434 1173 1118 839 800and 723 cmminus1 1H NMR (D2O 300MHz) δ 762 (m 1H)746 (m 1H) 723 (m 2H) 722 (m 1H) 559 (s 1H) 429(m 17H) 391 (s 1H) 334 (s 1H) 280 (s 8H) 255 (m 3H)249 (m 3H) 234 (m 6H) 195 (m 5H) 159 (m 7H) 141(m 31H) 129 (m 2H) and 098 (m 50H) ppm ESI-MS (mz)calcd for C85H142N30O23S6 [M+2H]2+ 100801809 Found100801709

Cyclic peptides CP2 and CP3 were prepared accordingto the same procedure described for cyclic peptide CP1

Cyclic peptide CP2 white powder (371mg 29 overtwo steps) HPLC (H2O (01 TFA)CH3CN (01

TFA) 9010rarr6040 (30min) tr 16min 1mLmin) IR(ATR) ] 3283 2939 2167 2027 1980 1651 1533 14311177 1119 1044 891 840 798 721 and 655 cmminus1 1H NMR(D2O 300MHz) δ 749 (m 1H) 738 (m 1H) 720 (m2H) 709 (m 1H) 497 (m 24H) 386 (m 12H) 317 (m24H) 209 (m 24H) 162 (m 29H) and 090 (m 38H) ppmESI-MS (mz) calcd for C95H159N34O29S2 [M+3H]3+76871559 and found 76871568

Cyclic peptideCP3 white powder (18mg 23 over twosteps) HPLC (H2O (01 TFA)CH3CN (01 TFA)

9010rarr6040 (30min) tr 11min 1mLmin) IR (ATR)] 3287 3071 2965 2547 2045 1778 1651 1532 14401290 1155 1042 845 700 and 578 cmminus1 1H NMR (D2O300MHz) δ 751 (m 1H) 738 (m 1H) 720 (m 2H)710 (m 1H) 497 (m 31H) 386 (m 18H) 317 (m 27H)209 (m 24H) 162 (m 29H) and 090 (m 38H) ppm ESI-MS (mz) calcd for C105H175N38O35S2 [M+ 3H]3+86441581 and found 86441735

Ir Complex 4 EDC (832mg 434mmol) and NHS(500mg 434mmol) was added to a solution of 7 (120mg014mmol) in DMF (12mL) e reaction mixture wasstirred for 24 h at room temperature and concentratedunder reduced pressure After addition of CHCl3 organiclayer was washed with sat NH4Cl e combined organiclayer was washed with H2O dried over Na2SO4 filteredand concentrated under reduced pressure to give NHS esterof 7 as a yellow solid (110mg 67) IR (ATR) ] 29412162 1705 1581 1519 1380 1293 1206 1066 976 886and 648 cmminus1 1H-NMR (CDCl3 300MHz) δ 845 (s3H) 800 (d 3H J 78) 773 (t 3H J 78) 742 (d 3HJ 57) 697 (t 3H J 60) 682 (s 3H) 289 (s 12H) and242 (s 9H) ppm ESI-MS (mz) calcd for C51H39IrN6O12[M]+ 112022552 Found 112022641 NHS ester of Ircomplex 7 (1mg 08micromol) was added to a solution of CP1(539mg 26micromol) and DIEA (47microL 0026mmol) in DMF(100μL) and stirred for 24 h at room temperature in the darkAfter that 01 TFA H2O was added to the reaction mixtureand the crude product was purified by preparative HPLC (H2O(01 TFA)CH3CN (01 TFA) 8020rarr5050 (30min)tr 21min (1mLmin)) lyophilized to give 4 as a yellowpowder (38mg 43 from 7) IR (ATR) ] 32879 2320 19811638 1535 1426 1264 1201 1134 923 835 800 and722 cmminus1 1H NMR (DMSO 300MHz) δ 826 (s 3H) 822(s 7H) 815 (m 10H) 783 (m 8H) 740 (m 8H) 730 (m12H) 713 (m 21H) 696 (s 13H) 655 (s 3H) 423 (m6H) 412 (m 14H) 311 (s 3H) 251 (m 29H) 249 (m 10H)248 (m 24H) 222 (m 12H) 176 (s 7H) 147 (m 36H)13 (s 3H) and 080 (m 41) ppm ESI-MS (mz) calcdfor C294H450IrN93O72S6 [M+6H]6+ 11372104 and found113720847

Ir Complex 5 EDC (137mg 071mmol) and NHS(83mg 071mmol) was added to a solution of 8 (25mg0023mmol) in DMF (25mL) e resulting solution wasstirred for 24 h at room temperature e reaction mixturewas concentrated under reduced pressure After addition ofCHCl3 the organic layer was washed with sat NH4Cl ecombined organic layer was washed with H2O and thendried over Na2SO4 filtered and concentrated under reducedpressure to give NHS ester of 8 as a yellow solid (20mg


62) IR (ATR) ] 3743 3318 2925 1815 1780 17061471 1375 1260 1204 1131 1067 994 781 and 648 cmminus11H-NMR (CDCL3 300MHz) δ 794 (d 3H J 81) 773 (s 3H)758 (t 3H J 78) 740 (d 3H J 51) 684 (t 3H J 63)667 (s 3H) 650 (t 3H J 66) 381 (d 6H J 51) 295(t 6H J 63) 291 (s 12H) and 223 (s 9H) ESI-MS (mz)calcd for C60H54IrN9O15 [M]+ 133333686 and found133333747 NHS ester of Ir complex 8 (6mg 00044mmol)was added to a solution of CP2 (3106mg 0013mmol) andDIEA (23 microL 0134mmol) in DMF (600 μL) and stirred for24 h at room temperature in the dark e reaction mixturewas diluted with 01 TFA H2O and purified by preparativeHPLC (H2O (01 TFA)CH3CN (01 TFA) 8020rarr5050(30min) tr 10min 1mLmin) lyophilized to give 5 asa yellow powder (1545mg 27 from 8) IR (ATR) ] 32823074 2964 2054 1980 1639 1531 1472 1425 1261 1181915 799 and 720 cmminus1 1H NMR (D2O 300MHz) δ 768(s 3H) 746 (s 3H) 708 (m 6H) 689 (m 3H) 668 (s 3H)379 (m 18H) 373 (m 7H) 371 (m 11H) 325 (m 18H)323 (m 12H) 318 (m 13H) 273 (m 5H) 224 (m 193H)223 (m 20H) 200 (m 11H) 163 (m 45) 135 (m 50H) 115(m 12H) and 089 (m 74H) ppm ESI-MS (mz) calcd forC333H513IrN108O93S6 [M+6H]6+ 131694104 Found131694569

Ir complex 6 was prepared according to the sameprocedure described for 5

Ir Complex 6 yellow powder (83mg 21 from 8) HPLC(H2O (01 TFA)CH3CN (01 TFA) 9010rarr6040(30min) tr 12min 1mLmin) IR (ATR) ] 3383 29632014 1984 1638 1535 1475 1262 1200 1057 836 799 and720 cmminus1 1H NMR (D2O 300MHz) δ 772 (s 3H) 742(s 3H) 717 (m 6H) 695 (m 3H) 678 (s 3H) 386 (m 23H)371 (m 38H) 323 (m 42H) 273 (m 31H) 207 (m 12H)192 (m 70H) 162 (m 69H) 134 (m 132H) and 088(m 120H) ppm ESI-MS (mz) calcd for C363H563IrN120O111S6[M+8H]8+ 109600145 and found 109600136

23UVVisAbsorptionandLuminescenceSpectraMeasurementsUVVis spectra were recorded on a JASCO V-550 UVVisspectrophotometer equipped with a temperature controllerand emission spectra were recorded on a JASCO FP-6200spectrofluorometer at 25degC Before the luminescence mea-surements sample aqueous solutions were degassed by Arbubbling for 10min in quartz cuvettes equipped with Teflonseptum screw caps Concentrations of all the Ir complexesin stock solutions (DMSO) were determined based ona molar extinction coefficient of 380 nm (ε380nm 108plusmn007times104Mminus1middotcmminus1) Quantum yields for luminescence (Φ)were determined by comparing with the integrated correctedemission spectrum of a quinine sulfate standard whoseemission quantum yield in 01M H2SO4 was assumed to be055 (excitation at 366 nm) Equation (1) was used to cal-culate the emission quantum yields in which Φs and Φrdenote the quantum yields of the sample and referencecompounds ηs and ηr are the refractive indexes of thesolvents used for the measurements of the sample andreferenceAs andAr are the absorbance of the sample and thereference and Is and Ir stand for the integrated areas under

the emission spectra of the sample and reference re-spectively (all of the Ir compounds were excited at 366 nmfor luminescence measurements in this study)

Φs Φr η2sArIs( 1113857

η2rAsIr( 1113857 (1)

e luminescence lifetimes of sample solutions weremeasured on a TSP1000-M-PL (Unisoku Osaka Japan) in-strument by using THG (355nm) of NdYAG laser Minilite I(Continuum CA USA) at 25degC in degassed aqueous solutionse R2949 photomultiplier were used to monitor the signalsDatawere analyzed using the nonlinear least-squares procedure

24 27MHz Quartz Crystal Microbalance (QCM) AnalysisQCM analysis was performed on an Affinix-Q4 apparatus(Initium Inc Japan) e clean Au (49mm2) electrodeequipped on the quartz crystal was incubated with an aqueoussolution of 33prime-dithiodipropionic acid (3mM 4 μL) at roomtemperature for 60min After washing with distilled waterthe surface was activated by a mixture of EDCmiddotHCl (052M)andN-hydroxy succinimide (087M) for 30min washed withdistilled water and then treated with DR5 (100 μgmL 4 μL)at room temperature for 60min After washing with distilledwater an aqueous solution of 1M ethanolamine (5 μL) wasadded as a blocking reagent After washing with distilledwater cell was filled with phosphate-buffered saline (PBS)(500 μL)e apparent binding constants (Kapp) for IPHs withDR5 in PBS were calculated from the decrease in frequencye nonspecific response was subtracted from frequencydecrease curve to obtain apparent complexation constantsKapp and dissociation constants Kd (1Kapp)

25 Cell Culture All cell lines (Jurkat Molt-4 and K562 cells)were cultured in RPMI 1640 medium supplemented with 10heat-inactivated fetal calf serum (FCS) L-glutamine HEPES(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acidpKa 75) 2-mercaptoethanol and penicillinstreptomycinand monothioglycerol (MTG) in a humidified 5 CO2 in-cubator at 37degC

26 MTT Assay Jurkat cells (10times105 cellsmL) were in-cubated in 1DMSO 10 FCS RPMI 1640 medium (MTGfree) containing solution of Ir complexes 4ndash6 (0ndash75 microM)under 5 CO2 at 37degC for 1 to 24 h in 96-well plates (BDFalcon) then 05 MTT reagent in PBS buffer (10 microL) wasadded to the cells After incubation under 5 CO2 at 37degCfor 4 h formazan lysis solution (10 SDS in 001 N HCl)(100 microL) was added and incubated overnight under sameconditions followed by measurement of absorbance at570 nm by a microplate reader (Bio-Rad) MTT assay ofMolt-4 cells and K562 cells with Ir complexes was alsoperformed according to the same procedure describedabove

27 MTT Assay in the Presence of Caspase Inhibitor (Z-VAD-FMK) and Necroptosis Inhibitor (Necrostatin-1) and Oxida-tive Stress Induce Necrosis Inhibitor (IM-54) Jurkat cells


(10times105 cellsmL) were incubated in 10 FCS RPMI 1640medium (MTG-free) containing a solution Z-VAD-fmk(15microM)necrostatin-1 (30microM)IM-54 (10microM)(under 5CO2 at 37degC for 1h in 96-well plates (BD Falcon) en Ircomplexes (75microM)were added and incubated under 5CO2 at37degC for 24h and then 05MTTreagent in PBS buffer (10microL)was added to the cells and incubated under 5 CO2 at 37degC for4h A formazan lysis solution (10 SDS in 001NHCl) (100microL)was added and the resulting solution incubated overnightunder the same conditions followed by measurement of ab-sorbance at 570nm with a microplate reader (Bio-Rad)

28 FluorescentMicroscopy Studies of Jurkat Cells K562 CellsandMolt-4Cellswith IrComplexes Jurkat cells K562 cells orMolt-4 cells (10times106 cellsmL) were incubated in the absenceor presence of Ir complexes in 10 FCS RPMI 1640 medium(MTG free) for specified time under 5CO2 at 37degCe cellswere then washed twice with ice-cold PBS with 01 NaN3and 05 FCS and taken on a Greiner CELLviewtrade petri dish(35times10mm) and mounted on fluorescent microscope forobservation (Biorevo BZ-9000 Keyence) (excitation 377plusmn25 nm emission 520plusmn 35 nm FF01 filter)

29 Fluorescent Microscopy Studies of Jurkat Cells with IrComplexes in the Presence of Inhibitors Jurkat cells(10times106 cellsmL) were incubated with the given inhibitorsin RPMI 1640 medium (MTG free) with 10 FCS under 5CO2 at 37degC for 1 h and then Ir complexes were added andthen again incubated at 37degC for 24 h e cells were thenwashed twice with ice-cold PBS containing 05 FCS and01 NaN3 and incubated with PI in PBS at room tem-perature for 10ndash15min e cells were then again washedwith PBS buffer and observed by fluorescent microscope(Biorevo BZ-9000 Keyence) (excitation 377plusmn 25 nmemission 520plusmn 35 nm FF01 filter)

210 Propidium Iodide (PI) Staining e cells were in-cubated with Ir complexes for specified time washed withPBS buffer and then incubated with PI in PBS buffer at roomtemperature for 10ndash15 minutes e cells were then againwashed with PBS buffer and observed by fluorescent mi-croscope (Biorevo BZ-9000 Keyence) (excitation 540plusmn25 nm emission 605plusmn 55 nm TRICT filter) or analyzed byflow cytometer (Beckman Coulter Gallios Flow Cytometerdetector FL2 excitation 488 nm emission 575plusmn 20 nm)

211 Anti-DR5 Antibody Staining Given cells wereincubated with anti-DR5 antibody at 4degC for 15min on icee cells were then washed twice with ice-cold PBS con-taining 05 FCS and 01 NaN3 and were mounted onfluorescent microscope (Biorevo BZ-9000 Keyence) (ex-citation 540plusmn 25 nm emission 605plusmn 55 nm TRICT filter)or analyzed by flow cytometer (Beckman Coulter GalliosFlow Cytometer detector FL2 excitation 488 nm emission575plusmn 20 nm)

212 Annexin V-Cy3 Staining Jurkat cells were incubatedwith Ir complexes for 2 h washed with PBS buffer and thensuspended in 1X binding buffer Annexin V-Cy3 wasadded to the cell suspension and then incubated at roomtemperature in the dark for 5ndash10 minutes e cells werethen washed with PBS buffer and observed by fluorescentmicroscope (Biorevo BZ-9000 Keyence) (excitation540 plusmn 25 nm emission 605 plusmn 55 nm TRICT filter)

213 Flow Cytometry Analysis of Staining and Cell DeathInduction Assay Jurkat cells K562 cells or Molt-4 cells(30times105 cells) were incubated in the absence or the pres-ence of Ir complexes in 10 FCS RPMI 1640 medium (MTGfree) for the specified time under 5 CO2 at 37degC After thatthe cells were washed twice with ice-cold FACS buffer andthen suspended in 450 microl FACS buffer e cells were an-alyzed by flow cytometer (Beckman Coulter Gallios FlowCytometer detector FL2 excitation 488 nm emission575plusmn 20 nm) to detect PI staining or anti-DR5 antibodystaining (detector FL10 excitation 405 nm emission 550plusmn40 nm) to detect Ir complexes staining)

3 Results and Discussion

31 Design and Synthesis of Ir Complex-Peptide Hybrids(IPHs) Synthesis of the Ir complex-peptide hybrids (IPHs)4ndash6 is shown in Figure 3 e Vilsmeier reaction of 1 (fac-Ir(tpy)3) and the following Pinnick oxidation [72 73 75] gave7 Condensation of 7 with β-alanine ethyl ester hydro-chloride and the following ester hydrolysis yielded 8 Both 7and 8 were converted to the corresponding N-hydroxysuccinimide (NHS) esters and then reacted with the peptideunits CP1 CP2 and CP3 that had been prepared by Fmocsolid-phase peptide synthesis to afford 4ndash6 respectivelyBecause 4 is poorly soluble in water a hydrophilic Ser-Gly-Ser-Gly (H2N-SGSG-CO) sequence was incorporated to theN-terminus of cyclic peptide CP1 to afford CP2-3 All the Ircomplexes 4ndash6 were purified by reversed-phase HPLCcolumn with a continuous gradient of H2O (01TFA)CH3CN (01TFA) and lyophilized to give yellowpowders as the corresponding TFA salts It should bementioned that negligible conversion of the facial form of4ndash6 to the corresponding meridional form was observedduring their synthesis and the following biological assays

32 UVVis and Luminescence Spectra of IPHs UVVis andluminescence spectra of Ir complexes 2c 4 5 and 6 (10 microM)in DMSO at 25degC are shown in Figure 4 and their photo-physical data are summarized in Table 1 e concentrationsof the Ir complexes in stock solutions (DMSO) were de-termined by the molar extinction coefficient at 380 nm(ε380nm 143plusmn 003times104middotMminus1middotcmminus1) of 4 5 and 6 whichare almost identical to those of typical Ir(tpy)3 derivativeshaving peptides 2andashf and 3andashc (Figure 1) as we previouslyreported [72 73] e strong absorption bands at 270ndash300 nm were assigned to the 1π-πlowast transition of tpy ligandsand weak shoulder bands at 320ndash450 nm were assigned asspin-allowed singlet-to-singlet metal-to-ligand charge


transfer (1MLCT) transitions spin-forbidden singlet-to-triplet (3MLCT) transitions and 3π-πlowast transitions Stronggreen emission of 4 5 and 6 is observed with emissionmaxima at ca 506 nm which are almost same as that of 2c(Figure 4(b)) e luminescence quantum yields (Φ) of 4 5and 6 were determined to be 039 033 and 036 re-spectively and their luminescence lifetimes (τ) are11ndash13 micros which are almost same as those of 1ndash3

33 Determination of Complexation Properties of IPHs with DR5First complexation of IPHs with DR5 was checked by27MHz quartz-crystal microbalance (QCM) DR5 wasimmobilized on a sensor chip to which IPHs were added ona certain time interval Upon addition of IPHs frequency

change was observed (Figure 5) indicating the interactionof IPHs with DR5 e apparent complexation constants(Kapp) (and dissociation constant Kd) for TRAIL 5 and 6with DR5 were determined to be (23plusmn 005)times 108Mminus1(Kd 43plusmn 01 nM) (38plusmn 01)times 105Mminus1 (Kd 27plusmn 01 microM)and (40plusmn 02)times 105Mminus1 (Kd 25plusmn 01 microM) respectivelyassuming 1 1 complexation (Table 2) Negligible interactionwas observed for 9 which lacks the receptor-binding peptide(Figure 6) and 2c that contains a KKGG peptide (Figure 1)[72 73]

34 Cancer Cell Death Induced by IPHs as Evaluated byMTTAssay and Fluorescence Microscopy Next cell death in-ducing activity of Ir complexes 4ndash6 against Jurkat cells was

1

[75]

HO

OO

PyBop DIEA DMF

NIr

3 38

41 (from 7)

4 (43 over 2 steps from 7)


CP2or

CP3

DIEA DMF

CP1 AcHN-WDCLDNRIGRRQCVKL-CONH2

CP2 AcHN-SGSGWDCLDNRIGRRQCVKL-CONH2

CP3 AcHN-SGSGSGSGWDCLDNRIGRRQCVKL-CONH2

H2N

H2N

H2N


7

Me

O

N

NIr

CP1

DIEA DMF

3

3

O

OO

OO O

OO

Me

Me

N

N

NHS

EDC DMF8

7NHS

EDC DMF

Ir

NH

HO

O O

N

N

HIr

Me(1) EtO NH2HCl

(2) LiOH H2OTHFfac-Ir(tpy)3

Figure 3 Synthesis of the Ir complex-peptide hybrids (IPHs)


evaluated by MTT assay (MTT 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2H-tetrazolium bromide) Jurkatcells were incubated with the given concentrations of 5 or 6in 10 FCS (fetal calf serum) RPMI 1640 (MTG free)medium at 37degC and treated with MTT reagent It wasobserved that the induction of cell death of Jurkat cells by 5

and 6 is much slower than that by our previous Ir complexes2c 2d and 3c which induce cell death of Jurkat cells in 1 hBecause both 5 and 6 interact with DR5 to the same extentas described in Figure 5 and Table 2 the following assayswere carried out with 5 Jurkat cells were observed byluminescence microscopy after incubation with 5 for 1 h6 h 12 h and 24 h and stained with propidium iodide (PI)to check their cell death As summarized in Figures 7 and 85 requires ca 24 h to induce considerable cell death

Table 1 Photophysical properties of Ir complexes 2c 4 5 and 6 indegassed DMSO at 25degC ([Ir complex] 10microM excitation at 366nm)

Compound λmax (absorption)(nm)

λmax (emission)(nm) Φa τb (micros)

2c 280 362 509 055 174 285 363 505 039 115 285 361 506 033 136 286 360 506 036 12aQuinine sulfate in 01M H2SO4 (Φ 055) was used as a reference bLi-fetime of luminescence emission

Table 2 Complexation constants of IPHs (assuming 1 1complexation)

Analyte Kapp (Mminus1) Kd

TRAIL (23plusmn 005)times 108 43plusmn 01 nm5 (38plusmn 01)times 105 27plusmn 01 μM6 (40plusmn 02)105 25plusmn 01 μM9 lt104 gt100 μM2c lt104 gt100 μM

Me

Ir

9

3

N6 HH2N

N

O

Figure 6 Ir complex having no peptide

1

08

06

04Abso

rban

ce

02

0270 300 350 400 450

Wavelength (nm)500 550 600

2c456

(a)

Wavelength (nm)

2000

1600

1200

800

400

0400 450 500 550 600

2c

45

6

650 700

Emiss

ion

inte

nsity

Abs

366

nm (a

u)

(b)

Figure 4 UVVis spectra of (a) 2c (dashed curve) 4 (bold curve)5 (plain curve) and 6 (bold dashed curve) Emission spectra of (b)2c (dashed curve) 4 (bold curve) 5 (plain curve) and 6 (bolddashed curve) in degassed DMSO at 25degC ([Ir complex] 10 μMexcitation at 366 nm) (au is the arbitrary unit)

0 30 60Time (min)

90

ndash8000

ndash6000

ndash4000

ΔF (H

z)

ndash2000

0

+2000

Figure 5 Time course of frequency change (ΔF (Hz)) of IPHs-DR5 complexation Conditions temperature 25degC solventphosphate-buered saline (PBS) An aliquot of solutions ofTRAIL (red curve) (200 microgmL) 5 (green curve) 6 (blue curve) 9(orange curve) and 2c (black curve) ([Ir complex] 10mM so-lution in DMSO) was added to DR5 xed on the sensor chip Plainarrows indicate the time when solutions of these analytes wereadded to DR5


(ca 50 cell death at [5] 72 microM) For comparison neg-ligible 4-induced cell death even after incubation for 16 hwas possibly due to its low solubility in water (Figure S1 inSupplementary Materials)

35 Luminescence Staining of Jurkat Cells with IPHsSlow induction of cell death by IPHs allowed us toconduct luminescence staining experiments of Jurkatcells Figures 9(a)ndash9(c) and 9(d)ndash9(f ) indicate that

100

80

60

40

20

00 20 40 60

[5] (μM)80 100

24 h

12 h

6 h

1 h

Cel

l via

bilit

y (

)

Figure 7 e results of MTT assay cell viability of Jurkat cells after incubation in the presence of 5 (5ndash75microM) for 1h (lled circles) 6h (lleddiamonds) 12h (lled triangles) and 24h (lled squares) at 37degC

(f) (g) (h) (i) (j)

(k) (l) (m) (n) (o)

(p) (q) (r) (s) (t)

Bright field

(a) (b)

Emission image(emission from 5)

(c)

Emission image(emission from PI)

(d)

Overlay(emission images)

(e)

Overlay(emission imageswith bright field)

Figure 8 Time lapse luminescence microscopy images (Biorevo BZ-9000 Keyence) of Jurkat cells (times40) treated with 5 (75 microM) at 37degC Celldeaths were conrmed by staining with propidium iodide (PI) (andashe) after incubation for 1 h (fndashj) after incubation for 6 h (kndasho) afterincubation for 12 h and (pndasht) after incubation for 24 h Scale bar (white) 10 microm


5 (5 and 10 microM) binds to Jurkat cells after incubation at37degC for 1 h Passive uptake of 5 through the cell membraneis unlikely due to the facts that negligible emission wasobserved after incubation at 4degC for 1 h ([5] 20 microM)(Figures 9(g)ndash9(i)) and that the pretreatment with NaN3 (ametabolic inhibitor) at 4degC for 15min ([NaN3] 5mMa concentration required to inhibit metabolic activity)exhibits the localization of 5 on the cell membrane (Fig-ures 9(j)ndash9(l))

Competitive staining of Jurkat cells with 5 and DR5binding peptide [20ndash22] (CP1 Figure 3) was conductedJurkat cells were incubated with CP1 for 1 h at 37degC to which5 was added and incubated again for 1 h at 37degC Greenemission from the cells was considerably reduced by thepretreatment with CP1 (100 microM) than those treated withonly 5 (Figure S2 in Supplementary Materials) Similarresults were obtained by ow cytometry (Figure S3 inSupplementary Materials) implying that 5 competes withCP1 for DR5

36 Costaining of JurkatCells by IPHs andAnti-DR5AntibodyandObservation of theMovement ofDR5 betweenCell Surface

and Cytoplasm In Figures 11(aa)ndash11(ee) red emission wasobserved on the cell membrane of Jurkat cells that weretreated with anti-DR5 antibody (conjugated with a red coloruorochrome) at 4degC for 15min (Method a in Figure 10)Next we observed luminescence images of Jurkat cells thatwere treated with anti-DR5 antibody at 4degC for 15minand then incubated with 5 at 37degC for 1 h (Method b inFigure 10) In Figures 11(bb) and 11(bc) green emissionfrom 5 and red emission from anti-DR5 antibody wereobserved respectively e overlays of Figures 11(bb) and11(bc) show yellow spots (Figures 11(bd) and 11(be))suggesting that anti-DR5 antibody and 5 stain the same orsimilar area on the cells Besides it was found that there isnegligible dierence between emission intensity (fromanti-DR5 antibody) of Jurkat cells treated with only anti-DR5 antibody and then 5 (Figure S4 in SupplementaryMaterials) suggesting that both of 5 and anti-DR5 antibodybind with DR5 possibly at the dierent sites

Next the order of the treatment of Jurkat cells withanti-DR5 antibody and 5 was reversed In this experimentJurkat cells were treated with 5 (5 microM) and then withanti-DR5 antibody (Method c in Figure 10) In this casevery weak red emission was observed from Jurkat cells(Figures 11(ca)ndash11(ce)) suggesting that the expression levelof DR5 on cell membrane was decreased by the treatmentwith 5 e increase in the concentration of 5 to 10 microMresulted in the considerable decrease in the red emissionfrom anti-DR5 antibody (Figures 11(da)ndash11(de)) Similarresults were observed in ow cytometry assay in whichJurkat cells treated with only anti-DR5 antibody exhibithigh emission intensity (red curve in Figure S5 in Sup-plementary Materials)

In the previous experiments it was hypothesized thatDR5 is internalized from the cell membrane to the cellcytoplasm after binding with IPH To check this hypothesisJurkat cells were incubated with 5 at 37degC for 1 h washedwith fresh medium and incubated again in fresh medium at37degC for 1 or 6 h (Method d in Figure 10) (Figures 11(ea)ndash11(ee) for 1 h incubation and Figures 11(fa)ndash11(fe) for 6 hincubation) Interestingly the red emission from anti-DR5antibody was restored after an additional incubation for 6 has displayed in Figures 11(fa)ndash11(fe) showing a goodcontrast to Figures 11(da)ndash11(de) obtained without in-cubation in fresh medium for 6 h In ow cytometry assayred emission intensity from anti-DR5 antibody in cells wasreduced after 1 h incubation with 5 and then anti-DR5antibody in comparison to Jurkat cells treated with onlyanti-DR5 antibody After incubation of Jurkat cells with 5and then again in fresh medium for 6 h emission from anti-DR5 antibody was restored (Figure S6 in SupplementaryMaterials)

Our assumption based on the results of these experi-ments is summarized in Figure 12 (i) Upon incubationof Jurkat cells with IPH at 37degC for 1 h IPH-DR5 complexundergoes internalization into the cytoplasm from thecell surface (Figure 12(a)) (ii) As a result the binding ofanti-DR5 antibody to the cell membrane becomes weak(Figure 12(b)) (iii) After incubation of Jurkat cells (inwhich IPH-DR5 complexes is internalized in the cytoplasm)

(c)

Overlay

(b)

Emission imageBright field

(a)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

Figure 9 Luminescence microscopy images (Biorevo BZ-9000Keyence) of Jurkat cells (times40) stained with 5 (andashc) Jurkat cellsafter incubation with 5 (5microM) at 37degC for 1h (dndashf ) Jurkat cells afterincubation with 5 (10microM) at 37degC for 1h (gndashi) Jurkat cells afterincubation with 5 (20microM) at 4degC for 1h (jndashl) Jurkat cells after in-cubation with NaN3 (5mM) at 4degC for 15min and then with 5 (5microM)at 37degC for 1h Scale bar (white) 10microm


in fresh medium at 37degC for 1 or 6 h a considerableamount of DR5 is restored on the cell membrane (Figure 12(c))and then (iv) anti-DR5 antibody is able to bind DR5(Figure 12(d))

To assess the anotnity of IPH to DR5 expressed on cancercell membrane we carried out cell staining of dierent typesof cancer cell lines that express dierent levels of DR5 DR5expression of Jurkat cells K562 cells and Molt-4 cells wasevaluated by staining with anti-DR5 antibody and owcytometer analysis It was found that Jurkat cells expresscomparatively high level of DR5 than K562 cells and Molt-4cells as conrmed by staining with anti-DR5 antibody (thesecond left in Figure 13) ese three cell lines were in-cubated with 5 (5 microM and 10 microM) at 37degC for 1 h and thenanalyzed by ow cytometer Figure 13 shows that Jurkatcells are highly stained by 5 while K562 cells andMolt-4 cellsare weakly stained Luminescence microscopic observationof dierent types of cancer cell lines also show similar results(Figures S7(a)ndashS7(i) in Supplementary Materials) and Ircomplex 9 having no peptide [72] (Figure 6) negligibly stainsJurkat cells (Figures S7(j)ndashS7(l) in Supplementary Mate-rials) Together with the results of the aforementioned QCMexperiments (Figure 2) these facts strongly suggest that thebinding of 5 is dependent on the DR5 expression level ofcancer cells

Relationship between cell death inducing activity of IPHand DR5 expression of cancer cells was studied Flowcytometry assay was conducted with Jurkat cells K562 cellsand Molt-4 cells that express dierent level of DR5 asmentioned above ese three cell lines were incubated with5 (2575 microM) at 37degC for 24 h to which PI (30 microM)was addedto stain the dead cells for analysis by ow cytometer

As summarized in Figure 14 cell death induction of thesethree cell lines is parallel to expression level of DR5

37 Mechanistic Studies of Cell Death Induced by IPHe aforementioned results strongly suggest that 5 is ableto detect Jurkat cells and induce their cell death viacomplexation with DR5 e stability of 5 in RPMI 1640(MTG Free) medium (Incubation at 37degC for 24 h) waschecked as shown in Figure S8 in the SupplementaryMaterials Since it is well established that DR5 initiatesapoptosis signal after binding with TRAIL or other re-ported articial TRAIL mimics it was initially presumedthat the cell death induced by 5 would be apoptosis MTTassay of Jurkat cells with 5 was carried out in the presenceof Z-VAD-FMK (15 microM) which is a broad caspase inhibitor[76] necrostation-1 (30 microM) which is a necroptosis in-hibitor [77] and IM-54 (10 microM) which is an inhibitor ofoxidative stress induced necrosis [78] However these threedrugs negligibly inhibited the cell death induced by 5(Figures S9 and S10 in Supplementary Materials) On theother hand TRAIL-induced cell death was inhibited byZ-VAD-FMK almost completely (Figures S9 and S10 inSupplementary Materials) In addition the staining ex-periments of the dead Jurkat cells with annexin V-Cy3[79 80] which is a well-known reagent to detect apoptosisdisclosed that only few cells were stained by annexinV-Cy3 ese facts have allowed us to conclude that it isunlikely that 5 induces apoptosis of Jurkat cells

Aforementioned studies suggest that 5 internalizesinto cells by DR5-mediated endocytosis erefore 5 mayinteract with cytoplasmic organelles or interfere andor

Method (a)

Method (b)

Jurkat cells Microscopic observation(Figures 11(aa)ndash11(ae))

Anti-DR5antibody

At 4degCfor 15 min

Jurkat cells Microscopic observation(Figures 11(ba)ndash11(be))

Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1h

Method (c)

Jurkat cells Microscopic observation(Figures 11(ca)ndash11(ce) and

11(da)ndash11(de))

Anti-DR5antibody

At 4degCfor 15 min

5 (5 and 10 μM)

At 37degCfor 1h

Method (d)

Jurkat cells

Microscopic observation(Figures 11(ea)ndash11(ee) for 1 h

incubation and Figures 11(fa)ndash11(fe)for 6 h incubation)

Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1 h

Fresh medium(RPMI 1640)

At 37degCfor 1 or 6 h

Figure 10 Costaining assay protocol


activate any cellular events to induce cell death In order toexamine this mechanism we evaluated the cell death in thepresence of the inhibitors of several cellular events or me-tabolisms channel blockers or receptor antagonist namelycarbonyl cyanide 3-chlorophenylhydrazone (CCCP) (amitochondrial uncoupling reagent) [81 82] amiloride(Na+ channel blocker inhibitors of macropinocytosis) [83]chloroquine (inhibitors of autophagy) [84] balomycin A1(an inhibitor of vacular ATPase (V-ATPase) [85] oligomycin

(an inhibitor of the mitochondrial F1F0-ATP synthase tocause ATP depletion) [86] 4-aminopyridine (K+ channelblocker) verapamil (L-type voltage-operated Ca2+ channelblocker) [87ndash89] nicardipine (L-type voltage-operatedCa2+ channel blocker) [87ndash89] and quinidine (Na+ andK+ channel blocker antagonist of α-adrenergic receptorsand an inhibitor of the mitochondrial uptake of Ca2+)[90ndash94] according to our previous studies [72 73] Jurkatcells were incubated with these inhibitors at their

(ab)


(ac)

Emission image(emission from

anti-DR5 antibody)

(ad)


(ae)


Bright field

(aa)

(bb) (bc) (bd) (be)(ba)

(cb) (cc) (cd) (ce)(ca)

(db) (dc) (dd) (de)(da)

(eb) (ec) (ed) (ee)(ea)

() (fc) (fd) (fe)(fa)

Figure 11 Luminescence microscopic images of (Biorevo BZ-9000 Keyence) of Jurkat cells stained with 5 and anti-DR5 antibody obtainedby protocol presented in Figure 10 (aandashae) Jurkat cells incubated with anti-DR5 antibody (15 microgmL) at 4degC for 15min (bandashbe) Jurkat cellsincubated with anti-DR5 antibody (15 microgmL) at 4degC for 15min and then with 5 (5 microM) at 37degC for 1 h (candashce) Jurkat cells incubated with 5(5 microM) at 37degC for 1 h and then with anti-DR5 antibody (15 microgmL) at 4degC for 15min (dandashde) Jurkat cells incubated with 5 (10 microM) at 37degCfor 1 h and then with anti-DR5 antibody (15 microgmL) at 4degC for 15min (eandashee) Jurkat cells incubated with 5 (10 microM) at 37degC for 1 h and thenin fresh medium for 1 h (2 h in total) and then with anti-DR5 antibody (15 microgmL) at 4degC for 15min (fandashfe) Jurkat cells incubated with 5(10 microM) at 37degC for 1 h and then in fresh medium for 6 h (7 h in total) and then with anti-DR5 antibody (15 microgmL) at 4degC for 15min Scalebar (white) 10 microm


Trimeric iridiumcomplex-peptidehybrids (IPHs)

Incubationfor 1 h

(a) (b)

(d) (c)

DR5

Cell membranecytoplasm




IPH-DR5 complex

Binding of IPH with DR5 andinternalization into cytoplasm

DR5 decreases oncell membrane

DR5 is restored oncell membrane

Anti-DR5 antibody isable to bind with DR5

Anti-DR5antibody

Anti-DR5antibody

Incubationfor 1 or 6 h

IPH-DR5complex

IPH-DR5complex

IPH-DR5complex

DR5DR5

Figure 12 Schematic presentation of the behavior of DR5 after complexation with IPH

12

10

Rela

tive i

nten

sity

8

6

4

2

0Blank

Molt-4 cellK562 cellJurkat cell

Anti-DR5antibody

5 (5 μm) 9 (5 μm)5 (10 μm)

Figure 13 Summary of ow cytometry assay of DR5 expression of Molt-4 cells (blue bar) K562 cells (orange bar) and Jurkat cells (red bar)e cells were stained with anti-DR5 antibody (15 microgmL) at 4degC for 15min 5 (510 microM) and 9 (5 microM) at 37degC for 1 he relative intensity isthe ratio of the geometric mean values of luminescence intensity to the blank


recommended concentrations (Figure S11 in Supplemen-tary Materials) at 37degC for 1 h to which 5 was added andincubated again at the same temperature for 24 h e cellswere then washed with PBS buer and stained with pro-pidium iodide (PI) for microscopic observation In-terestingly it was found that only voltage-operated Ca2+channel blocker (nicardipine and verapamil) considerablyinhibited cell death induced by 5 erefore we conclude thatthe cell death induced by 5 can be considered as necrosis-typecell death via Ca2+-mediated intracellular signaling pathway

38Detectionof JurkatCells inBovineBlood edetection ofcancer cells by use of IPHs was carried out Jurkat cells(10times105 cells) were suspended in bovine blood (100 microL) andthen incubated with 5 (10 microM) in medium (RPMI 1640) at37degC for 6 h resulting in the successful detection of Jurkatcells in bovine blood as shown in Figure 15

4 Conclusions

In this study we report on the design and synthesis of Ircomplex-peptide hybrids (4ndash6) consisted of C3-symmetrictris-cyclometalated Ir complexes equipped with DR5binding cyclic peptides Among these complexes 5 and 6were found to be cytotoxic against Jurkat cells Studiesdemonstrate that 5 binds with DR5 on the cell membrane(by 27MHz QCM and costaining experiments with anti-DR5 antibody) and their complex is internalized into thecytoplasm by DR5-mediated endocytosis Our previous Ircomplexes 2ndash3 having basic peptides such as KKGG se-quence induce Jurkat cell death in a few hours and exhibitstrong emission from dead cells In contrast the IPHs

6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)

Figure 14 Summary of cell death assay (PI staining of dead cells) ofMolt-4 cells (blue bar) K562 cells (orange bar) and Jurkat cells (redbar) e relative intensity is the ratio of the geometric mean valuesof the luminescence intensity to the blank

(a)

(b)

(c)

Figure 15 Luminescence microscopy images (times40) (Biorevo BZ-9000 Keyence) of Jurkat cells spiked in bovine blood in presenceof 5 (andashc) Jurkat cells spiked in bovine blood after incubationwith 5 (10 microM) at 37degC for 6 h (a) bright eld image (b) emissionimage and (c) overlay image of (a) and (b) Scale bar (white) 10 microm


reported in this study (5 and 6) detect Jurkat cells first andthen induce their death very slowly (after 16 to 24 h)Moreover DR5 is transferred andor reproduced on the cellsurface after additional incubation Jurkat cells can be de-tected even in bovine blood which may offer an easy andconvenient method for cancer diagnosis To the best of ourknowledge 5 is the first example of the artificial compoundthat achieve imaging and cell death induction of cancer cellsassociated with DR5 due to its moderate cytoxicity and slowcell death induction property

e aforementioned results postulate that IPHs can begood candidates to study death receptor biology cancer cellimaging induction of cancer cell death and understandingof the mechanism of cell death mediated by death receptorAdditionally IPHs induce slow cell death which mayprovide new approaches in the treatment of cancer andrelated diseases Improvement of the anticancer activity ofIPHs and attempts at controlling cell death types are now inprogress

Abbreviations

DIC NNprime-diisopropylcarbodiimideDIEA NN-diisopropylethylamineDMF NN-dimethylformamideEDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimideHOBt 1-HydroxybenzotriazoleNHS N-hydroxy succinimidePyBOP Benzotriazol-1-yl-oxytripyrrolidinophosphonium

hexafluorophosphateTFA Trifluoroacetic acidTHF TetrahydrofuranTIPS TriisopropylsilaneTMSCl Trimethylsilyl chloride

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

is work was supported by grants-in-aid from the Min-istry of Education Culture Sports Science and Technology(MEXT) of Japan (nos 22890200 24890256 26860016 and16K18851 to Yosuke Hisamatsu and nos 1965902622390005 24590025 24650011 24640156 and 15K00408to Shin Aoki) the Uehara Memorial Foundation (TokyoJapan) to Yosuke Hisamatsu the Tokyo Biochemical Re-search Foundation (Tokyo Japan) to Shin Aoki ldquoAcademicFrontiersrdquo Project for Private Universities (matching fundsubsidy from MEXT) and TUS (Tokyo University ofScience) fund for strategic research areas e authorsthank Prof Dr Takeshi Inukai (Department of PediatricsSchool of Medicine University of Yamanashi) for thevaluable suggestion e authors sincerely acknowledgeMs Fukiko Hasegawa and Ms Noriko Sawabe (Faculty of

Pharmaceutical Sciences Tokyo University of Science) formeasurement of mass spectra and NMR

Supplementary Materials

Figure S1 the results of MTT assay Figure S2 microscopicimages of competitive staining Figure S3 flow cytometryassay results of competitive staining Figures S4ndashS6 flowcytometry assay results of costaining Figure S7 microscopicimages of Jurkat K562 and Molt-4 cells staining Figure S8HPLC charts of 5 after incubation in RPMI 1640 mediumFigures S9 and S10 the results of MTT assay in presence ofinhibitors Figure S11 microscopic images of staining inpresence of inhibitors Figure S12ndash13 1H NMR and ESImass charts of NHS ester of Ir complex 7 Figures S14ndashS151H NMR and ESI mass charts of Ir complex 8 Figures S16and S17 1H NMR and ESI mass charts of NHS ester of Ircomplex 8 Figures S18 and S19 1H NMR and ESI masscharts of Ir complex 9 Figures S20ndashS22 HPLC 1H NMRand ESI mass charts of CP1 Figures S23ndashS25 HPLC 1HNMR and ESI mass charts of CP2 Figures S26ndashS28 HPLC1H NMR and ESI mass charts of CP3 Figure S29ndashS31HPLC 1H NMR and ESI mass charts of Ir complex 4Figures S32ndashS34 HPLC 1H NMR and ESI mass charts of Ircomplex 5 Figures S35ndashS37 HPLC 1H NMR and ESI masscharts of Ir complex 6 (Supplementary Materials)

References

[1] A Ashkenazi and V M Dixit ldquoDeath receptors signaling andmodulationrdquo Science vol 281 no 5381 pp 1305ndash1308 1998

[2] G Pan K OrsquoRourke A M Chinnaiyan et al ldquoe receptorfor the cytotoxic ligand TRAILrdquo Science vol 276 no 5309pp 111ndash113 1997

[3] HWalczak M A Delgi-Esposti R S Johnson et al ldquoTRAIL-R2 a novel apoptosis-mediating receptor for TRAILrdquo EMBOJournal vol 16 no 17 pp 5386ndash5397 1997

[4] G Pan J Ni Y F Wei G Yu R Gentz and V M Dixit ldquoAnantagonist decoy receptor and a death domain-containingreceptor for TRAILrdquo Science vol 277 no 5327 pp 815ndash8181997

[5] M A Delgi-Esposti P J Smolak H Walczak et al ldquoCloningand characterization of TRAIL-R3 a novel member of theemerging TRAIL receptor familyrdquo Journal of ExperimentalMedicine vol 186 no 7 pp 1165ndash1170 1997

[6] M A Delgi-Esposti W C Dougall P J Smolak J Y WaughC A Smith and R G Goodwin ldquoe novel receptor TRAIL-R4 induces NF-kB and protects against TRAIL-mediatedapoptosis yet retains an incomplete death domainrdquo Immu-nity vol 7 no 6 pp 813ndash820 1997

[7] W S Simonet D L Lacey C R Dunstan et al ldquoOsteo-protegerin a novel secreted protein involved in the regulationof bone densityrdquo Cell vol 89 no 2 pp 309ndash319 1997

[8] S G Hymowitz H W Christinger G Fuh et al ldquoTriggeringcell death the crystal structure of Apo2LTRAIL in a complexwith death receptor 5rdquo Molecular Cell vol 4 no 4pp 563ndash571 1999

[9] S G Hymowitz M OrsquoConnell M H Ultsch et al ldquoA uniquezinc-binding site revealed by a high-resolution X-ray struc-ture of homotrimeric Apo2LTRAILrdquo Biochemistry vol 39no 4 pp 633ndash640 2000


[10] S W Fesik ldquoInsight into programmed cell death throughstructural biologyrdquo Cell vol 103 no 2 pp 273ndash282 2000

[11] A Ashkenazi R C Pai S Fong et al ldquoSafety and anti-tumoractivity of recombinant soluble Apo2 ligandrdquo Journal ofClinical Investigation vol 104 no 2 pp 155ndash162 1999

[12] H Walczak R E Miller K Ariall et al ldquoTumoricidal ac-tivity of tumor necrosis factor-related apoptosis-inducingligand in vivordquo Nature Medicine vol 5 no 2 pp 157ndash1631999

[13] S K Kelly and A Ashkenazi ldquoTargeting death receptors incancer with Apo2LTRAILrdquo Current Opinion in Pharma-cology vol 4 no 4 pp 333ndash339 2004

[14] A Ashkenazi P Holland and S G Eckhardt ldquoLigand-basedtargeting of apoptosis in cancer the potential of recombinanthuman apoptosis ligand 2tumor necrosis factor-relatedapoptosis-inducing ligand (rhApo2LTRAIL)rdquo Journal ofClinical Oncology vol 26 no 21 pp 3621ndash3630 2008

[15] K Ichikawa W Liu L Zhao et al ldquoTumoricidal activity ofa novel anti-human DR5 monoclonal antibody without he-patocyte cytotoxicityrdquo Nature Medicine vol 7 no 8pp 954ndash960 2001

[16] H Jin R Yang S Fong et al ldquoApo2 ligandtumor necrosisfactor-related apoptosis-inducing ligand cooperates withchemotherapy to inhibit orthotopic lung tumor growth andimprove survivalrdquo Cancer Research vol 64 no 14 pp 4900ndash4905 2004

[17] D R Camidge ldquoApomab an agonist monoclonal antibodydirected against death receptor 5TRAIL-receptor 2 for use inthe treatment of solid tumorsrdquo Expert Opinion on BiologicalLerapy vol 8 no 8 pp 1167ndash1176 2008

[18] J Li D A Knee Y Wang et al ldquoLBY135 a novel anti-DR5agonistic antibody induces tumor cellndashspecific cytotoxic ac-tivity in human colon tumor cell lines and xenograftsrdquo DrugDevelopment Research vol 69 no 2 pp 69ndash82 2008

[19] J Wiezorek P Holland and J Graves ldquoDeath receptor ag-onists as a targeted therapy for cancerrdquo Clinical Cancer Re-search vol 16 no 6 pp 1701ndash1708 2011

[20] Y M Angell A Bhandari A Chakrabarti et al ldquoDiscoveryand optimization of a TRAIL R2 agonist for cancer therapyrdquoin Understanding Biology Using Peptides S E Blondelle Edpp 405-406 Springer New York NY USA 2006

[21] Y M Angell A Bhandari M N De Francisco et alldquoPeptides for youthrdquo in Advances in Experimental Medicineand Biology S Del Valle E Escher and W D Lubell Edspp 101ndash103 Springer New York NY USA 2009

[22] Y M Angell M Bhandari M N De Francisco et alldquoDiscovery and optimization of a TRAIL R2 agonist for cancertherapyrdquo Advances in Experimental Medicine and Biologyvol 611 pp 101ndash103 2009

[23] V Pavet J Beyrath C Pardin et al ldquoMultivalent DR5peptides activate the TRAIL death pathway and exerttumoricidal activityrdquo Cancer Research vol 70 no 3pp 1101ndash1110 2010

[24] G Lamanna C R Smulski N Chekkat et al ldquoMulti-merization of an apoptogenic TRAIL-mimicking peptide byusing adamantane-based dendronsrdquo Chemistry-A EuropeanJournal vol 19 no 5 pp 1762ndash1768 2013

[25] K Pulka-Ziach V Pavet N Chekkat et al ldquoioether an-alogues of disulfide-bridged cyclic peptides targeting deathreceptor 5 conformational analysis dimerisation and con-sequences for receptor activationrdquo ChemBioChem vol 16no 2 pp 293ndash301 2015

[26] B Valldorf H Fittler L Deweid et al ldquoAn apoptosis-inducing peptidic heptad that efficiently clusters death

receptor 5rdquo Angewandte Chemie International Editionvol 55 no 16 pp 1ndash6 2016

[27] C Kaga M Okochi M Nakanishi H Hayashi R Kato andH Honda ldquoScreening of a novel octamer peptideCNSCWSKD that induces caspase-dependent cell deathrdquoBiochemical and Biophysical Research Communicationsvol 362 no 4 pp 1063ndash1068 2007

[28] G Wang X Wang H Yu et al ldquoSmall-molecule activation ofthe TRAIL receptor DR5 in human cancer cellsrdquo NatureChemical Biology vol 9 no 2 pp 84ndash89 2013

[29] S Lamansky P Djurovich D Murphy et al ldquoHighlyphosphorescent bis-cyclometalated iridium complexes syn-thesis photophysical characterization and use in organic lightemitting diodesrdquo Journal of the American Chemical Societyvol 123 no 18 pp 4304ndash4312 2001

[30] M A Baldo D F OrsquoBrien Y You et al ldquoHighly efficientphosphorescent emission from organic electroluminescentdevicesrdquo Nature vol 395 no 6698 pp 151ndash154 1998

[31] G M Farinola and R Ragni ldquoElectroluminescent materialsfor white organic light emitting diodesrdquo Chemical SocietyReviews vol 40 no 7 pp 3467ndash3482 2011

[32] H Yersin Highly Efficient OLEDs with Phosphorescent Ma-terials Wiley-VCH Weinheim Germany 2008

[33] C Ulbricht B Beyer C Friebe AWinter and U S SchubertldquoRecent developments in the application of phosphorescentiridium(iii) complex systemsrdquo Advanced Materials vol 21no 44 pp 4418ndash4441 2009

[34] A B Tamayo B D Alleyne P I Djurovich et al ldquoSyn-thesis and characterization of facial and meridional tris-cyclometalated iridium(III) complexesrdquo Journal of theAmerican Chemical Society vol 125 no 24 pp 7377ndash73872003

[35] K Dedeian P I Djurovich F O Garces G Carlson andR J Watts ldquoA new synthetic route to the preparation ofa series of strong photoreducing agents fac-tris-ortho-metalated complexes of iridium(III) with substituted 2-phe-nylpyridinesrdquo Inorganic Chemistry vol 30 no 8 pp 1685ndash1687 1991

[36] M S Lowry and S Bernhard ldquoSynthetically tailored excitedstates phosphorescent cyclometalated iridium(III) com-plexes and their applicationsrdquo Chemistry-A European Journalvol 12 no 31 pp 7970ndash7977 2006

[37] L Flamigni A Barbieri C Sabatini B Ventura andF Barigelletti ldquoPhotochemistry and photophysics of co-ordination compounds iridiumrdquo Topics in Current Chemis-try vol 281 pp 143ndash203 2007

[38] R C Evans P Douglas and C J Winscom ldquoCoordinationcomplexes exhibiting room-temperature phosphorescenceevaluation of their suitability as triplet emitters for light-emitting diodesrdquo Coordination Chemistry Reviews vol 250no 15-16 pp 2093ndash2126 2006

[39] Y Chi and P T Chou ldquoTransition-metal phosphors withcyclometalating ligands fundamentals and applicationsrdquoChemical Society Reviews vol 39 no 2 pp 638ndash655 2010

[40] G D Marco M Lanza A Mamo et al ldquoLuminescentmononuclear and dinuclear iridium(III) cyclometalatedcomplexes immobilized in a polymeric matrix as solid-stateoxygen sensorsrdquo Analytical Chemistry vol 70 no 19pp 5019ndash5023 1998

[41] M C DeRosa P J Mosher G P A Yap K S FocsaneanuR J Crutchley and C E B Evanc ldquoSynthesis character-ization and evaluation of [Ir(ppy)2(vpy)Cl] as a polymer-bound oxygen sensorrdquo Inorganic Chemistry vol 42 no 16pp 4864ndash4872 2003


[42] M Licini and J A G Williams ldquoIridium(iii) bis-terpyridinecomplexes displaying long-lived pH sensitive luminescencerdquoChemical Communications no 19 pp 1943-1944 1999

[43] K J ArmW Leslie and J A GWilliams ldquoSynthesis and pH-sensitive luminescence of bis-terpyridyl iridium(III) com-plexes incorporating pendent pyridyl groupsrdquo InorganicaChimica Acta vol 359 no 4 pp 1222ndash1232 2006

[44] M L Ho F M Hwang P N Chen et al ldquoDesign andsynthesis of iridium(III) azacrown complex application asa highly sensitive metal cation phosphorescence sensorrdquoOrganic and Biomolecular Chemistry vol 4 no 1 pp 98ndash1032006

[45] K Konishi H Yamaguchi and A Harada ldquoSynthesis ofa water-soluble iridium(iii) complex with pH andmetal cationsensitive photoluminescencerdquo Chemistry Letters vol 35no 7 pp 720-721 2006

[46] M Schmittel and H Lin ldquoLuminescent iridium phenan-throline crown ether complex for the detection of silver(I)ions in aqueous mediardquo Inorganic Chemistry vol 46 no 22pp 9139ndash9145 2007

[47] Q Zhao T Cao F Li et al ldquoA highly selective and multi-signaling optical-electrochemical sensor for Hg2+ based ona phosphorescent iridium(III) complexrdquo Organometallicsvol 26 no 8 pp 2077ndash2081 2007

[48] K K W Lo M W Louie and K Y Zhang ldquoDesign ofluminescent iridium(III) and rhenium(I) polypyridine com-plexes as in vitro and in vivo ion molecular and biologicalprobesrdquo Coordination Chemistry Reviews vol 254 no 21-22pp 2603ndash2622 2010

[49] K K W Lo S P Y Li and K Y Zhang ldquoDevelopment ofluminescent iridium(III) polypyridine complexes as chemicaland biological probesrdquo New Journal of Chemistry vol 35no 2 pp 265ndash287 2011

[50] S K Leung K Y Kwok K Y Zhang and K K W LoldquoDesign of luminescent biotinylation reagents derived fromcyclometalated iridium(III) and rhodium(III) bis(pyr-idylbenzaldehyde) complexesrdquo Inorganic Chemistry vol 49no 11 pp 4984ndash4995 2010

[51] K K W Lo and S P Y Li ldquoUtilization of the photophysicaland photochemical properties of phosphorescent transitionmetal complexes in the development of photofunctionalcellular sensors imaging reagents and cytotoxic agentsrdquo RSCAdvances vol 4 no 21 pp 10560ndash10585 2014

[52] K Y Zhang S P Y Li N Zhu et al ldquoStructure photophysicaland electrochemical properties biomolecular interactions andintracellular uptake of luminescent cyclometalated iridium-(III) dipyridoquinoxaline complexesrdquo Inorganic Chemistryvol 49 no 5 pp 2530ndash2540 2010

[53] P K Lee W H T Law H W Liu and K K W LoldquoLuminescent cyclometalated iridium(III) polypyridinedi-2-picolylamine complexes synthesis photophysicselectrochemistry cation binding cellular internalizationand cytotoxic activityrdquo Inorganic Chemistry vol 50no 17 pp 8570ndash8579 2011

[54] K Y Zhang H W Liu T T H Fong X G Chen andK K W Lo ldquoLuminescent dendritic cyclometalated iridium(III) polypyridine complexes synthesis emission behaviorand biological propertiesrdquo Inorganic Chemistry vol 49no 12 pp 5432ndash5443 2010

[55] Q Zhao C Huang and F Li ldquoPhosphorescent heavy-metalcomplexes for bioimagingrdquo Chemical Society Reviews vol 40no 5 pp 2508ndash2524 2011

[56] Q Zhao M Yu L Shi et al ldquoCationic iridium(III) complexeswith tunable emission color as phosphorescent dyes for live

cell imagingrdquo Organometallics vol 29 no 5 pp 1085ndash10912010

[57] C Li M Yu Y Sun Y Wu C Huang and F Li ldquoAnone-missive iridium(III) complex that specifically lights-up thenuclei of living cellsrdquo Journal of the American ChemicalSociety vol 133 no 29 pp 11231ndash11239 2011

[58] Y You S Lee T Kim et al ldquoPhosphorescent sensor forbiological mobile zincrdquo Journal of the American ChemicalSociety vol 133 no 45 pp 18328ndash18342 2011

[59] Y Wu H Jing Z Dong Q Zhao H Wu and F LildquoRatiometric phosphorescence imaging of hg(II) in living cellsbased on a neutral iridium(III) complexrdquo Inorganic Chem-istry vol 50 no 16 pp 7412ndash7420 2011

[60] L Murphy A Congreve L O Palsson and J A G Williamsldquoe time domain in co-stained cell imaging time- resolvedemission imaging microscopy using a protonatable lumi-nescent iridium complexrdquoChemical Communications vol 46no 46 pp 8743ndash8745 2010

[61] E Baggaley J A Weinstein and J A G Williams ldquoLightingthe way to see inside the live cell with luminescent transitionmetal complexesrdquo Coordination Chemistry Reviews vol 256no 15-16 pp 1762ndash1785 2012

[62] Y You ldquoPhosphorescence bioimaging using cyclometalated ir(III) complexesrdquo Current Opinion in Chemical Biologyvol 17 no 4 pp 699ndash707 2013

[63] R Cao J Jia X Ma M Zhou and H Fei ldquoMembrane lo-calized iridium(III) complex induces endoplasmic reticulumstress and mitochondria-mediated apoptosis in human cancercellsrdquo Journal of Medicinal Chemistry vol 56 no 9pp 3636ndash3644 2013

[64] Y Zhou J Jia W Li H Fei and M Zhou ldquoLuminescentbiscarbene iridium(III) complexes as living cell imaging re-agentsrdquo Chemical Communications vol 49 no 31pp 3230ndash3232 2013

[65] S Zhang M Hosaka T Yoshihara et al ldquoPhosphorescentlightndashemitting iridium complexes serve as a hypoxia-sensingprobe for tumor imaging in living animalsrdquo Cancer Researchvol 70 no 11 pp 4490ndash4498 2010

[66] S Tobita and T Yoshihara ldquoIntracellular and in vivo oxygensensing using phosphorescent iridium(III) complexesrdquoCurrent Opinion in Chemical Biology vol 33 pp 39ndash45 2016

[67] Y Hisamatsu and S Aoki ldquoDesign and synthesis of blue-emitting cyclometalated iridium-(III) complexes based onregioselective functionalizationrdquo European Journal of In-organic Chemistry vol 2011 no 35 pp 5360ndash5369 2011

[68] S Moromizato Y Hisamatsu T Suzuki Y Matsuo R Abeand S Aoki ldquoDesign and synthesis of a luminescent cyclo-metalated iridium(III) complex having NN-diethyl aminogroup that stains acidic intracellular organelles and inducescell death by photoirradiationrdquo Inorganic Chemistry vol 51no 23 pp 12697ndash12706 2012

[69] A Nakagawa Y Hisamatsu S Moromizato M Kohno andS Aoki ldquoSynthesis and photochemical properties of pH re-sponsive tris-cyclometalated iridium(III) complexes thatcontain a pyridine ring on the 2-phenylpyridine ligandrdquoInorganic Chemistry vol 53 no 1 pp 409ndash422 2014

[70] A Kando Y Hisamatsu H Ohwada S MoromizatoM Kohno and S Aoki ldquoPhotochemical properties of red-emitting tris(cyclometalated) iridium(III) complexes havingbasic and nitro groups and application to pH sensing andphoto induced cell deathrdquo Inorganic Chemistry vol 54 no 11pp 5342ndash57 2015

[71] S Kumar Y Hisamatsu Y Tamaki O Ishitani and S AokildquoDesign and synthesis of heteroleptic cyclometalated iridium


(III) complexes containing quinoline-type ligands that exhibitdual phosphorescencerdquo Inorganic Chemistry vol 55 no 8pp 3829ndash3843 2016

[72] Y Hisamatsu A Shibuya N Suzuki R Abe and S AokildquoDesign and synthesis of amphiphilic and luminescent tris-cyclometalated iridium(III) complexes containing cationicpeptides as inducers and detectors of cell death via a calcium-dependent pathwayrdquo Bioconjugate Chemistry vol 26 no 5pp 857ndash879 2015

[73] Y Hisamatsu N Suzuki A Masum et al ldquoCationic amphi-philic tris-cyclometalated iridium(iii) complexes induce cancercell death via interaction with Ca2+-calmodulin complexrdquoBioconjugate Chemistry vol 28 no 2 pp 507ndash523 2017

[74] K Yokoi Y Hisamatsu K Naito and S Aoki ldquoDesign synthesisand anticancer activity of cyclometalated tris(ppy) iridium(III)complexes having cationic peptides at the 4rsquo-position of the ppyligand (Ppy2-phenylpyridine)rdquo European Journal of InorganicChemistry vol 2017 no 44 pp 5295ndash5309 2017

[75] S Aoki Y Matsuo S Ogura et al ldquoRegioselective aromaticsubstitution reactions of cyclometalated ir(III) complexessynthesis and photochemical properties of substituted ir(III)complexes that exhibit blue green and red color lumines-cence emissionrdquo Inorganic Chemistry vol 50 pp 806ndash8182011

[76] E A Slee H Zhu S C Chow M MacFarlaneD W Nicholson and G M Cohen ldquoBenzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethylketone (Z-VAD-FMK) inhibitsapoptosis by blocking the processing of CPP32rdquo BiochemicalJournal vol 315 no 1 pp 21ndash24 1996

[77] A Degterev J Hitomi M Germscheid et al ldquoIdentification ofRIP1 kinase as a specific cellular target of necrostatinsrdquoNature Chemical Biology vol 4 no 5 pp 313ndash321 2008

[78] K Dodo M Katoh T Shimizu M Takahashi andM Sodeoka ldquoInhibition of hydrogen peroxide-induced ne-crotic cell death with 3-amino-2-indolylmaleimide de-rivativesrdquo Bioorganic andMedicinal Chemistry Letters vol 15no 12 pp 3114ndash3118 2005

[79] G Koopman C P Reutelingsperger G A KuijtenR M Keehnen S T Pals and M H van Oers ldquoAnnexin V forflow cytometric detection of phosphatidylserine expression onB cells undergoing apoptosisrdquo Blood vol 84 pp 1415ndash1420 1994

[80] IVermesCHaanenH Steffens-Nakken andCReutellingspergerldquoA novel assay for apoptosis Flow cytometric detection ofphosphatidylserine expression on early apoptotic cells usingfluorescein labelled Annexin Vrdquo Journal of ImmunologicalMethods vol 184 no 1 pp 39ndash51 1995

[81] P-K Lee H-W Liu S-M Yiu M M-W Louie andK K-W Lo ldquoLuminescent cyclometallated iridium(III) bis(quinolylbenzaldehyde) diimine complexesndashsynthesis pho-tophysics electrochemistry protein cross-linking propertiescytotoxicity and cellular uptakerdquo Dalton Transactions vol 40no 10 pp 2180ndash2189 2011

[82] S P Y Li T S M Tang K S M Yiu and K K W LoldquoCyclometalated iridium(III)-polyamine complexes with in-tense and long-lived multicolor phosphorescence synthesiscrystal structure photophysical behavior cellular uptake andtransfection propertiesrdquo Chemistry-A European Journalvol 18 no 42 pp 13342ndash13354 2012

[83] V Z Sun Z Li T J Deming and D T Kamei ldquoIntracellularfates of cell-penetrating block copolypeptide vesiclesrdquo Bio-macromolecules vol 12 no 1 pp 10ndash13 2011

[84] T Kimura Y Takabatake T Takahashi and Y IsakaldquoChloroquine in cancer therapy a double-edged sword ofautophagyrdquo Cancer Research vol 73 no 1 pp 3ndash7 2013

[85] S Drose and K Altendorf ldquoBafilomycins and concanamycinsas inhibitors of V-ATPases and P-ATPasesrdquo Journal of Ex-perimental Biology vol 200 pp 1ndash8 1997

[86] V Novohradsky Z Liu M Vojtiskova P J Sadler V Brabecand J Kasparkova ldquoMechanism of cellular accumulation of aniridium(III) pentamethyl cyclopentadienyl anticancer com-plex containing a CN-chelating ligandrdquo Metallomics vol 6no 3 pp 682ndash690 2014

[87] W A Catterall and J Striessnig ldquoReceptor sites for Ca2+

channel antagonistsrdquo Trends in Pharmacological Sciencesvol 13 pp 256ndash262 1992

[88] G H Hockerman B Z Peterson B D Johnson andW A Catterall ldquoMolecular determinants of drug binding andaction on l-type calcium channelsrdquo Annual Review of Phar-macology and Toxicology vol 37 no 1 pp 361ndash396 1997

[89] D J Triggle ldquoCalcium channel antagonists clinical uses pastpresent and futurerdquo Biochemical Pharmacology vol 74 no 1pp 1ndash9 2007

[90] H J Motulsky A S Maisel M D Snavely and P A InselldquoQuinidine is a competitive antagonist at alpha 1- and alpha 2-adrenergic receptorsrdquo Circulation Research vol 55 no 3pp 376ndash381 1984

[91] K Shibata A Hirasawa R Foglar S Ogawa andG Tsujimoto ldquoEffects of quinidine and verapamil on humancardiovascular α1-adrenoceptorsrdquo Circulation vol 97 no 13pp 1227ndash1230 1998

[92] G Schreiber A Barak and M Sokolovsky ldquoDisopyramideand quinidine bind with inverse selectivity to muscarinicreceptors in cardiac and extra cardiac rat tissuesrdquo Journal ofCardiovascular Pharmacology vol 7 no 2 pp 390ndash393 1985

[93] J A Harrow andN S Dhalla ldquoEffects of quinidine on calciumtransport activities of the rabbit heart mitochondria andsarcotubular vesiclesrdquo Biochemical Pharmacology vol 25no 8 pp 897ndash902 1976

[94] W Van Driessche ldquoPhysiological role of apical potassium ionchannels in frog skinrdquo Journal of Physiology vol 356 no 1pp 79ndash95 1984


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Submit your manuscripts atwwwhindawicom

receptor binders having TRAIL-like functionalities are re-ported Representative examples include DR5 bindingpeptides [20ndash26] zinc-binding site peptide of TRAIL(RNSCWSKD that was screened out from TRAIL (227ndash234))[27] and small molecular TRAIL mimics (bioymi) [28]

Meanwhile cyclometalated iridium (Ir(III)) complexessuch as fac-Ir(tpy)31 (tpy 2-(4-tolyl)pyridine) (Figure 1) drawincreasing attention as one of the imaging tools to study extra-and intracellular events in addition to organic light-emittingdiodes (OLEDs) such as phosphorescent emitters [29ndash33]because of their signicant stability and excellent photophysicalproperties under physiological conditions [31 33ndash39] Suchtypes of Ir complex analogues have been widely applied tooxygen sensors [40 41] chemosensors [42ndash47] and lumi-nescent probes for biological systems [48ndash66] ese advan-tages originate from their high-luminescence quantum yieldsthe long luminescence lifetimes (τsimmicros) that can eliminate theshort-lived autouorescence (τsimns) from biological samples incellular imaging and signicant stokes shift that minimizesself-quenching process [29ndash39] We previously reported someexamples of Ir complexes that can be functionalized asbluesimgreensimred and white color emitters [67ndash71] pH sensors[68ndash70] photosensitizers [68ndash70] and cell death inducer ofcancer cells [68ndash70] Recently we have reported on Ir com-plexes (2a-f and 3a-c) having cationic peptides (typically H2N-KKGG-) (Figure 1) as inducers and detectors of cell death ofJurkat cells (a human T-lymphoma cell line) [72ndash74] eseresults suggest that Ir complexes are potential agents for thediagnosis and treatment of cancer and related diseases and evenfor mechanistic study of cell death processes

Because cyclometalated iridium (Ir(III)) complexes suchas fac-Ir(tpy)3 1 possess a C3-symmetric structure likeTRAIL (the top of Figure 2) it is hypothesized that thesecomplexes could be good scaolds to mimic TRAIL In thismanuscript we report on the design and synthesis of somenew C3-symmetric tris-cyclometalated Ir complexes havingcyclic peptides (Figure 2) [20ndash22] which had been reported

to be able to bind DR5 for selective staining and induction ofcell death of cancer cells e Ir complexes 4ndash6 were syn-thesized by regioselective substitution reactions reported by us[75] and the successive coupling reactions with cyclic peptides[20ndash22] Due to low solubility of 4 in water Ser-Gly-Ser-Gly

MeMe

Me

Me Me

Me

Ir Ir

IrR1

Ir

N

3

3 3

ONH

2a (n = 2)2b (n = 4)2c (n = 6)2d (n = 8)2e (n = 12)2f (n = 16)

3a (R1 = H2N-D-K-D-KGG-)3b (R1 = H2N-KKG-)3c (R1 = H2N-KKKGG-)

H2N-KKGG N

N

NN

nH

N6 HN

N

H

O O O

1 (fac-Ir(tpy)3)

Figure 1 Ir complexes having cationic peptides

TRAIL homotrimer

Receptor binding site

LinkerIr core

Ir complex-peptide homotrimer(structurally similar to TRAIL)

O Me

Ir

N

3

HN

HN

HN

HN

HN

O

O

Inserted for better solubility in water

4 R2 = AcHN-WDCLDNRIGRRQCVKL-CONH2

5 R2 = AcHN-SGSGWDCLDNRIGRRQCVKL-CONH2

6 R2 = AcHN-SGSGSGSGWDCLDNRIGRRQCVKL-CONH2

R2

Figure 2 C3-symmetric tris-cyclometalated Ir complex-peptidehybrids (IPHs)



























η2rAsIr( 1113857 (1)






















1

[75]

HO

OO

PyBop DIEA DMF

NIr

3 38

41 (from 7)



CP2or

CP3

DIEA DMF




H2N

H2N

H2N


7

Me

O

N

NIr

CP1

DIEA DMF

3

3

O

OO

OO O

OO

Me

Me

N

N

NHS

EDC DMF8

7NHS

EDC DMF

Ir

NH

HO

O O

N

N

HIr

Me(1) EtO NH2HCl













Me

Ir

9

3

N6 HH2N

N

O


1

08

06

04Abso

rban

ce

02

0270 300 350 400 450


2c456

(a)

Wavelength (nm)

2000

1600

1200

800

400

0400 450 500 550 600

2c

45

6

650 700

Emiss

ion

inte

nsity

Abs

366

nm (a

u)

(b)


0 30 60Time (min)

90

ndash8000

ndash6000

ndash4000

ΔF (H

z)

ndash2000

0

+2000





100

80

60

40

20

00 20 40 60

[5] (μM)80 100

24 h

12 h

6 h

1 h

Cel

l via

bilit

y (

)


(f) (g) (h) (i) (j)

(k) (l) (m) (n) (o)

(p) (q) (r) (s) (t)

Bright field

(a) (b)


(c)


(d)


(e)











(c)

Overlay

(b)


(a)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)









Method (a)

Method (b)


Anti-DR5antibody

At 4degCfor 15 min


Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1h

Method (c)


11(da)ndash11(de))

Anti-DR5antibody

At 4degCfor 15 min

5 (5 and 10 μM)

At 37degCfor 1h

Method (d)

Jurkat cells



Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1 h







(ab)


(ac)


anti-DR5 antibody)

(ad)


(ae)


Bright field

(aa)





() (fc) (fd) (fe)(fa)




Incubationfor 1 h

(a) (b)

(d) (c)

DR5





IPH-DR5 complex





Anti-DR5antibody

Anti-DR5antibody


IPH-DR5complex

IPH-DR5complex

IPH-DR5complex

DR5DR5


12

10

Rela

tive i

nten

sity

8

6

4

2

0Blank


Anti-DR5antibody

5 (5 μm) 9 (5 μm)5 (10 μm)





4 Conclusions


6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)


(a)

(b)

(c)





Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





























η2rAsIr( 1113857 (1)






















1

[75]

HO

OO

PyBop DIEA DMF

NIr

3 38

41 (from 7)



CP2or

CP3

DIEA DMF




H2N

H2N

H2N


7

Me

O

N

NIr

CP1

DIEA DMF

3

3

O

OO

OO O

OO

Me

Me

N

N

NHS

EDC DMF8

7NHS

EDC DMF

Ir

NH

HO

O O

N

N

HIr

Me(1) EtO NH2HCl













Me

Ir

9

3

N6 HH2N

N

O


1

08

06

04Abso

rban

ce

02

0270 300 350 400 450


2c456

(a)

Wavelength (nm)

2000

1600

1200

800

400

0400 450 500 550 600

2c

45

6

650 700

Emiss

ion

inte

nsity

Abs

366

nm (a

u)

(b)


0 30 60Time (min)

90

ndash8000

ndash6000

ndash4000

ΔF (H

z)

ndash2000

0

+2000





100

80

60

40

20

00 20 40 60

[5] (μM)80 100

24 h

12 h

6 h

1 h

Cel

l via

bilit

y (

)


(f) (g) (h) (i) (j)

(k) (l) (m) (n) (o)

(p) (q) (r) (s) (t)

Bright field

(a) (b)


(c)


(d)


(e)











(c)

Overlay

(b)


(a)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)









Method (a)

Method (b)


Anti-DR5antibody

At 4degCfor 15 min


Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1h

Method (c)


11(da)ndash11(de))

Anti-DR5antibody

At 4degCfor 15 min

5 (5 and 10 μM)

At 37degCfor 1h

Method (d)

Jurkat cells



Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1 h







(ab)


(ac)


anti-DR5 antibody)

(ad)


(ae)


Bright field

(aa)





() (fc) (fd) (fe)(fa)




Incubationfor 1 h

(a) (b)

(d) (c)

DR5





IPH-DR5 complex





Anti-DR5antibody

Anti-DR5antibody


IPH-DR5complex

IPH-DR5complex

IPH-DR5complex

DR5DR5


12

10

Rela

tive i

nten

sity

8

6

4

2

0Blank


Anti-DR5antibody

5 (5 μm) 9 (5 μm)5 (10 μm)





4 Conclusions


6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)


(a)

(b)

(c)





Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls



















1

[75]

HO

OO

PyBop DIEA DMF

NIr

3 38

41 (from 7)



CP2or

CP3

DIEA DMF




H2N

H2N

H2N


7

Me

O

N

NIr

CP1

DIEA DMF

3

3

O

OO

OO O

OO

Me

Me

N

N

NHS

EDC DMF8

7NHS

EDC DMF

Ir

NH

HO

O O

N

N

HIr

Me(1) EtO NH2HCl













Me

Ir

9

3

N6 HH2N

N

O


1

08

06

04Abso

rban

ce

02

0270 300 350 400 450


2c456

(a)

Wavelength (nm)

2000

1600

1200

800

400

0400 450 500 550 600

2c

45

6

650 700

Emiss

ion

inte

nsity

Abs

366

nm (a

u)

(b)


0 30 60Time (min)

90

ndash8000

ndash6000

ndash4000

ΔF (H

z)

ndash2000

0

+2000





100

80

60

40

20

00 20 40 60

[5] (μM)80 100

24 h

12 h

6 h

1 h

Cel

l via

bilit

y (

)


(f) (g) (h) (i) (j)

(k) (l) (m) (n) (o)

(p) (q) (r) (s) (t)

Bright field

(a) (b)


(c)


(d)


(e)











(c)

Overlay

(b)


(a)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)









Method (a)

Method (b)


Anti-DR5antibody

At 4degCfor 15 min


Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1h

Method (c)


11(da)ndash11(de))

Anti-DR5antibody

At 4degCfor 15 min

5 (5 and 10 μM)

At 37degCfor 1h

Method (d)

Jurkat cells



Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1 h







(ab)


(ac)


anti-DR5 antibody)

(ad)


(ae)


Bright field

(aa)





() (fc) (fd) (fe)(fa)




Incubationfor 1 h

(a) (b)

(d) (c)

DR5





IPH-DR5 complex





Anti-DR5antibody

Anti-DR5antibody


IPH-DR5complex

IPH-DR5complex

IPH-DR5complex

DR5DR5


12

10

Rela

tive i

nten

sity

8

6

4

2

0Blank


Anti-DR5antibody

5 (5 μm) 9 (5 μm)5 (10 μm)





4 Conclusions


6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)


(a)

(b)

(c)





Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls













Me

Ir

9

3

N6 HH2N

N

O


1

08

06

04Abso

rban

ce

02

0270 300 350 400 450


2c456

(a)

Wavelength (nm)

2000

1600

1200

800

400

0400 450 500 550 600

2c

45

6

650 700

Emiss

ion

inte

nsity

Abs

366

nm (a

u)

(b)


0 30 60Time (min)

90

ndash8000

ndash6000

ndash4000

ΔF (H

z)

ndash2000

0

+2000





100

80

60

40

20

00 20 40 60

[5] (μM)80 100

24 h

12 h

6 h

1 h

Cel

l via

bilit

y (

)


(f) (g) (h) (i) (j)

(k) (l) (m) (n) (o)

(p) (q) (r) (s) (t)

Bright field

(a) (b)


(c)


(d)


(e)











(c)

Overlay

(b)


(a)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)









Method (a)

Method (b)


Anti-DR5antibody

At 4degCfor 15 min


Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1h

Method (c)


11(da)ndash11(de))

Anti-DR5antibody

At 4degCfor 15 min

5 (5 and 10 μM)

At 37degCfor 1h

Method (d)

Jurkat cells



Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1 h







(ab)


(ac)


anti-DR5 antibody)

(ad)


(ae)


Bright field

(aa)





() (fc) (fd) (fe)(fa)




Incubationfor 1 h

(a) (b)

(d) (c)

DR5





IPH-DR5 complex





Anti-DR5antibody

Anti-DR5antibody


IPH-DR5complex

IPH-DR5complex

IPH-DR5complex

DR5DR5


12

10

Rela

tive i

nten

sity

8

6

4

2

0Blank


Anti-DR5antibody

5 (5 μm) 9 (5 μm)5 (10 μm)





4 Conclusions


6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)


(a)

(b)

(c)





Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






100

80

60

40

20

00 20 40 60

[5] (μM)80 100

24 h

12 h

6 h

1 h

Cel

l via

bilit

y (

)


(f) (g) (h) (i) (j)

(k) (l) (m) (n) (o)

(p) (q) (r) (s) (t)

Bright field

(a) (b)


(c)


(d)


(e)











(c)

Overlay

(b)


(a)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)









Method (a)

Method (b)


Anti-DR5antibody

At 4degCfor 15 min


Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1h

Method (c)


11(da)ndash11(de))

Anti-DR5antibody

At 4degCfor 15 min

5 (5 and 10 μM)

At 37degCfor 1h

Method (d)

Jurkat cells



Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1 h







(ab)


(ac)


anti-DR5 antibody)

(ad)


(ae)


Bright field

(aa)





() (fc) (fd) (fe)(fa)




Incubationfor 1 h

(a) (b)

(d) (c)

DR5





IPH-DR5 complex





Anti-DR5antibody

Anti-DR5antibody


IPH-DR5complex

IPH-DR5complex

IPH-DR5complex

DR5DR5


12

10

Rela

tive i

nten

sity

8

6

4

2

0Blank


Anti-DR5antibody

5 (5 μm) 9 (5 μm)5 (10 μm)





4 Conclusions


6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)


(a)

(b)

(c)





Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls











(c)

Overlay

(b)


(a)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)









Method (a)

Method (b)


Anti-DR5antibody

At 4degCfor 15 min


Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1h

Method (c)


11(da)ndash11(de))

Anti-DR5antibody

At 4degCfor 15 min

5 (5 and 10 μM)

At 37degCfor 1h

Method (d)

Jurkat cells



Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1 h







(ab)


(ac)


anti-DR5 antibody)

(ad)


(ae)


Bright field

(aa)





() (fc) (fd) (fe)(fa)




Incubationfor 1 h

(a) (b)

(d) (c)

DR5





IPH-DR5 complex





Anti-DR5antibody

Anti-DR5antibody


IPH-DR5complex

IPH-DR5complex

IPH-DR5complex

DR5DR5


12

10

Rela

tive i

nten

sity

8

6

4

2

0Blank


Anti-DR5antibody

5 (5 μm) 9 (5 μm)5 (10 μm)





4 Conclusions


6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)


(a)

(b)

(c)





Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls










Method (a)

Method (b)


Anti-DR5antibody

At 4degCfor 15 min


Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1h

Method (c)


11(da)ndash11(de))

Anti-DR5antibody

At 4degCfor 15 min

5 (5 and 10 μM)

At 37degCfor 1h

Method (d)

Jurkat cells



Anti-DR5antibody

At 4degCfor 15 min

5

At 37degCfor 1 h







(ab)


(ac)


anti-DR5 antibody)

(ad)


(ae)


Bright field

(aa)





() (fc) (fd) (fe)(fa)




Incubationfor 1 h

(a) (b)

(d) (c)

DR5





IPH-DR5 complex





Anti-DR5antibody

Anti-DR5antibody


IPH-DR5complex

IPH-DR5complex

IPH-DR5complex

DR5DR5


12

10

Rela

tive i

nten

sity

8

6

4

2

0Blank


Anti-DR5antibody

5 (5 μm) 9 (5 μm)5 (10 μm)





4 Conclusions


6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)


(a)

(b)

(c)





Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






(ab)


(ac)


anti-DR5 antibody)

(ad)


(ae)


Bright field

(aa)





() (fc) (fd) (fe)(fa)




Incubationfor 1 h

(a) (b)

(d) (c)

DR5





IPH-DR5 complex





Anti-DR5antibody

Anti-DR5antibody


IPH-DR5complex

IPH-DR5complex

IPH-DR5complex

DR5DR5


12

10

Rela

tive i

nten

sity

8

6

4

2

0Blank


Anti-DR5antibody

5 (5 μm) 9 (5 μm)5 (10 μm)





4 Conclusions


6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)


(a)

(b)

(c)





Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





Incubationfor 1 h

(a) (b)

(d) (c)

DR5





IPH-DR5 complex





Anti-DR5antibody

Anti-DR5antibody


IPH-DR5complex

IPH-DR5complex

IPH-DR5complex

DR5DR5


12

10

Rela

tive i

nten

sity

8

6

4

2

0Blank


Anti-DR5antibody

5 (5 μm) 9 (5 μm)5 (10 μm)





4 Conclusions


6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)


(a)

(b)

(c)





Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






4 Conclusions


6

5

Rela

tive i

nten

sity 4

3

2

1

0Blank


5 (25 μm) 5 (75 μm)


(a)

(b)

(c)





Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






Abbreviations





Acknowledgments





References












































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





































































































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls




LuminescentIridiumComplex-PeptideHybrids(IPHs)for...

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Transcript of LuminescentIridiumComplex-PeptideHybrids(IPHs)for...