Bioluminescent Turn-On Probe for Sensing Hypochlorite in Vitro and

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A Bioluminescent Turn-On Probe for Sensing Hypochlorite in Vitro and in Tumors Peiyao Chen, Zhen Zheng, Yunxia Zhu, Yu Dong, Fuqiang Wang, and Gaolin Liang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 09 May 2017 Downloaded from http://pubs.acs.org on May 9, 2017

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Analytical Chemistry

A Biolu uminescent Tu urn-On Probe for Sen nsing Hypochllorite in n Vitro an nd in Tumors Peiyao Cheen,† Zhen Zhheng,† Yunxxia Zhu,‡ Yuu Dong,† Fuqqiang Wangg,*,†,‡ and Gaaolin Liang*,† †

CAS Key Labboratory of Sooft Matter Cheemistry, Deparrtment of Chemistry, Univeersity of Sciencce and Technoology of Chinaa, Hefei, Anhui 230026, Chinna. ‡

Analysis Cennter, Nanjing M Medical Univeersity, Nanjingg, Jiangsu 2100093, China.

ABSTRACT: Hypochlorite (ClO-) is one of the most iimportant reacttive oxygen sppecies but usinng a BL probee for its selectiive detection (or imaging) i still remains challeenging. Hereinn, we report a latent BL proobe benzoylhyddrazine luciferrin (1) for highhly selective detecction of ClO- inn vitro and imaaging ClO- in lliving cells andd tumors. In viitro tests indicaated that 1 couuld be applied for highly selectivve detection of ClO- within thhe range of 0-662.5 μM with a limit of detecttion of 0.705 μ μM. Using thesse unique featuures of 1, we succeessfully appliedd it to image CllO- in living ceells and tumorss. We envision that probe 1 m might be applieed to elucidate tthe biological rolees of ClO- in wiider physiologiical and pathollogical processes in the near ffuture.

Hypochloritee (ClO-) is onne of the mostt important reactive oxygen speciees (ROS),1-3 w which plays ann important roole in sustaining hum man innate imm munity during microbial invaasion.4 Endogenous C ClO is generatted by hydroggen peroxide (H H2O2) reacting with chloride ions (Cl-), catalyzedd by heme ennzyme myeloperoxidasse (MPO).5,6 A Abnormal levelss of hypochloriite are reported to assoociate with certtain diseases suuch as cardiovaascular diseases, neurron degeneratiion, lung injuury, atheroscleerosis, arthritis, and caancer.7-12 Thus, it is of great im mportance to dettect or image ClO- in cells or in vivvo. To date, nuumerous fluoresscence probes have beeen reported forr the detection (or imaging) off ClO.13-17 However,, most of the ffluorescent probbes suffer from m low photostability oof photobleachiing or autofluorrescence interfeerence from the detectting samples. Bioluminescent imaging (B BLI) is a reliaable, sensitive, noninvasive imagiing technique which has beeen widely used in imaging a myriad of life proccesses such as cell c proliferatioon and migration, genee expression, annd enzyme activvities.18-24 Amonng the approximate 300 known biolum minescence (BL L) systems, the firefly f luciferase-lucifferin system is the most widelly used one.25 IIn this BL system, inn the presence of adenosine triphosphate ((ATP), oxygen and m magnesium ionss (Mg2+), fireffly luciferase ((fLuc) catalyzes a tw wo-step oxidatiion of luciferinn to emit a vvisible (yellow to green) photoon.26-29 Notably, the geneerated bioluminescentt photons can eeven penetrate the tissues of intact rodents, renderring BLI very suitable for in i vivo imaginng.30,31 Moreover, com mpared with fluuorescence imaaging, BLI doees not require externaal excitation andd therefore possesses higher signals to-noise ratios. To date, a variety of BL L probes have been developed forr the detectioon (or monitooring) of impportant analytes.32-36 IIn 2015, Kojiima et al. reeported a sennsitive bioluminogenicc probe for in vvivo imaging thhe activity of hhighly reactive oxygenn species (hRO OS) including C ClO-.37 Neverthheless, this probe coulld not differentiiate ClO from oother hROS. Thhus, it still remains chhanlleging to deevelop a biolum minescence probbe for the selective deetection of ClO-.

H Herein, we repport a BL turnn-on probe for in vitro selecttive dettection of ClO- and in vivo im maging of ClO- in tumors. As we knoow, ClO- is abble to oxidize dibenzoylhydrrazine into watterunsstable dibenzoyyl diimide.38 Thhus, as outlinedd in Scheme 1, we ratiionally designeed a latent BL probe p benzoylhhydrazine lucifeerin (1)) with the carbooxyl acid group of whose D-lucciferin motif beiing cagged by benzoylhhydrazine. Probbe 1 itself is noot the substrate for fLuuc. However, uppon ClO- oxidaation and follow w-up hydrolysiss in phyysiological conndition, probe 1 yields the aactive substrate Dlucciferin for lucifeerase to generatee a light readouut (Scheme 1). Sch heme 1. Schem matic illustrattion of ClO- oxidation o of 1 to yieeld D-luciferin ffor bioluminesccence generatioon.

W began the sstudy with the synthesis and ccharacterizationn of We proobe 1 (Scheme S1, Figure S1-S3). Then we used u D-luciferin n to scrreen out an apprropriate fLuc cooncentration to sstudy the responnse of probe 1 towardd ClO-. As show wn in Figure S44, the BL intenssity Luc concentratioon of 0.1 mg/m mL was 5.3 foldss of of D-luciferin at fL thaat at fLuc conccentration of 0.05 mg/mL. Wee thus choose 0.1 mgg/mL fLuc to cconduct the folllowing in vitrro experiment. As exppected, in the absence of C ClO-, almost noo BL signal w was gennerated by 1 mM M 1 after beingg incubated withh 0.1 mg/mL fL Luc andd 1 mM ATP inn phosphate bufffer (0.2 M, pH H 7.4) (Figure 1A A). Hoowever, additionn of 70-fold ClO O- to 1 mM 1 soolution resultedd in a ssignificant increease of BL inteensity (a 58-folld increase at 5560 nm m, Figure 1A) under u the same condition. This result suggested thaat free D-luciferrin, which respoonds for the BL L generation, w was yieelded from ClO- oxidation of 1,, as we proposed in Scheme 1.

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Figure 1. (A) B Bioluminescent wavelength scaan for 1 mM 1 aand 0.1 mg/mL fLuc in the presence (red) or aabsence (black) of 70 mM ClO- in phosphate buffe fer (0.2 M, pH 77.4). (B) Fitted ccalibration curvve for BL intenssity of 25 μM 1 at 560 nm withh ClO- concentrration in the linnear region of 0-62.5 μM. (C) Selecctivity studies oof 1: Selectivityy of 1 at 25 μM in the presencee of 25 μM ClO O- or 100 μM NO O, H2O2, hydroxxyl radical (•OH), peroxynitrite (O ONOO-), alkylpperoxyl radical (ROO•), superroxide (O2•-), siinglet oxygen (1O2), glutathionne (GSH), HCO O3-, 2NO2 , NO3 , Cl , or SO4 respeectively. The exxperiments weree performed in ttriplicate. Error bars represent relative r standardd deviations (RSD) of three indepenndent experimeents which are 11.1 × 10-3, 2.3 × 10-3, 6.9 × 10-3, 4.0 × 10-5, 1.3 × 10-3, 3.8 × 10-3, 4.2 × 10-3, 5.2 × 10-4, 0.018, 4.4 × 10-3, 8.2 × 10-3, respectivvely. Emission: 560 nm.

High performannce liquid chrom matography (HP PLC) analysis clearly c indicated that, about 75.4% of 1 was convverted to D-lucciferin under this conddition (Figure S55). After validaation of the feasibility of 1 for ClO- ddetection, a lineaar relationship (Y ( = 0.599X + 0.694, 0 R2 = 0.998) bettween the BL enhancement e (i.e., B - B0) of 225 µM 1 at 560 nm annd ClO- concenttration was obttained over the range of 0-62.5 µM ((Figure 1B). Thhe limit of detection (LOD) off ClOof this assay w was calculated to be 0.705 µM µ according tto the general 3σ meethod (3σ/s),39 comparable too those of repported methods (Tablee S1). Selectivity is one imporrtant parameteer to evaluatee the which performance off a new probe. Particularly, forr a new probe w is intentionallyy designed for biomedical appplications, its hhighly selective respoonse to the targget over other possible compeetitive species is a m must. Therefore,, we screened a wide array of o the potential compeetitive species ssuch as intracelllular reactive oxxygen and nitrogen sppecies (ROS/RN NS) (e.g., NO, H2O2, •OH, ON NOO-, 1 •ROO•, O2 , annd O2), intraceellular abundant biomolecules (e.g., GSH) and com mmon anions ((e.g., HCO3-, N NO2-, NO3-, Cl-, and 2SO4 ) to studyy the selectivity of probe 1 by comparing its B - B0 value induced by ClO- over those t of the selected species in the O• and presence of fLuuc. As shown inn Figure 1C, onlly 100 µM ROO 1 O2 showed w weak bioluminesscence after adddition to 25 µ µM 1, which was alm most negligible compared to thhat induced byy ClO(BL enhancemeent by ClO- waas 23-182 folds of those inducced by other species). Therefore, we cconcluded that probe p 1 has exccellent selectivity towaard ClO- over otther testing speccies.

Figure 2. Tiime-course bioluminescence images of fLuctransfected MD DA-MB-231 cells incubated with 50 μM 1 (topp row), pre-incubated w with 5 μM exoggenous ClO- and then with 50 μM 1 (middle row), ppre-incubated w with 5 μM exoggenous ClO- folllowed by 25 mg/mL ClO- scavengeer taurine and then with 50 μM 1 (bottom row) aacquired at 0, 15, 30, 50, 70, 990, 110, 130 annd 150 min in serum-frree culture meddium at 37 °C.

W With its good in vitro propeerty, probe 1 w was subsequenntly appplied for BL imaging ClO- in living ceells. Before thhat, cyttotoxicity of 1 aand ClO- was eevaluated on MDA-MB-231 ceells (noon-luciferase traansfected) withh 3-(4,5-dimethyylthiazol-2-yl) 2,5 dipphenyltetrazpliuum bromide (MT TT) assay. As shhown in Figure S6, 90..0% of the cellss survived afterr 24 h incubatioon with 100 µM M 1, sugggesting that proobe 1 under 1000 µM is safe forr live cell imagiing. Forr ClO-, 95% off the cells surviived after 4 h inncubation with 20 µM M ClO- (Figure S7), S suggesting that ClO- concentration underr 20 µM M is safe for livve cell imagingg. In this work, we chose 1 at 50 µM M and ClO- at 5 µM to conduct the following ccell imaging stuudy. FL Luc–transfected MDA-MB-231 cells were pre--incubated withh or witthout 5 µM ClO O- in serum-freee culture mediuum at 37 °C for 30 minn, and washed with phosphatee buffered salinne (PBS) for thhree tim mes to remove eextracellular ClO O-. Then the ceells were placedd in thee 96-well plate aat 1 × 106 cells/w /well. BL signalls generated by the cellls were recordeed with a small animal imagingg system in a reealtim me manner. Geneerally, as shownn in Figure 2, affter the additionn of proobe 1 to each w well, the BL siignals graduallyy increased to the maaximum at arouund 90 min, foollowed by a ddecrease, and thhen maaintained at com mparable valuees over the obsservation winddow (Fiigure S8). To bee specific, the bbrightest BL siggnal was observved froom the cells pree-incubated withh ClO- (middle row in Figure 2), sugggesting that prrobe 1 has goood cell permeabbility, and can be oxiidized by ClO- to yield D-lucifferin inside cells. However, whhen thee cells were incuubated with 25 mg/mL ClO- sccavenger taurinne40 forr 5 min before thhe addition of 1 to the ClO- pree-incubation ceells, theeir BL signal (bottom row in Figure 2) was significanntly deccreased compaared to that of ClO--treaated cells. T This dem monstrated that ClO- could triggger 1 to releasee of D-luciferin for BL L generation in cells. For the cells without pre-incubation of ClO O-, weak BL siignal could be detected (top row in Figure 2). This could be expllained that the iintracellular prooteases hydrolyzzed thee amide bond off 1 to yield D-luuciferin to geneerate the weak BL beccause MDA-MB-231 cells themselves do nnot generate CllO-. HP PLC analysis inddicated that, aftter the MDA-M MB-231 cells (noonlucciferase transfeccted) were incubbated with 200 μM 1 for 30 m min, 15..4% of uptakenn 1 was proteoolysed to yield D-luciferin inside cellls (Figure S9). For the ClO---pretreated cellss, incubation w with proobe 1 resulted iin a three-time increased amoount of D-lucifeerin insside cells, comppared with thoose cells withouut ClO- treatm ment (Fiigure S9). This further comfirm med that most oof the intracelluular D-lu uciferin was geenerated by 1 thhat reacted withh ClO- for the BL

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Analytical Chemistry

augmentation. We also conduucted cell imagging experimentt with the cells pre-inncubated with 1 before ClO- adddition. As shoown in Figure S10, thee brightest BL signal was also observed from the cells with ClO- addition. How wever, the BL siignal increased to the maximum mucch quicker (15 min after ClO O- addition) andd then decreased graddually (Figure S S11). This coulld be explainedd that, upon the additiion of ClO-, proobe 1 was turned into D-lucifeerin in the medium, reesulting in sim milar cell imaginng pattern to thhat of cells directly incubated withh D-luciferin.32 The above rresults O- in living cellss. suggest that proobe 1 is suitablee for BLI of ClO We then appplied 1 for BLI of ClO in livinng animals. Thee nude mice, xenograffted with fLuc-ttransfected MD DA-MB-231 tum mor in the right thigh for each, were randomly dividded into three groups g (one group for the injection off ClO- followedd by injection oof 1 as the experimental group, one ggroup for the injjection of 1 onlly and t successive injections i of CllO-, taurine, andd 1 as one group for the the two controll groups). BL ssignal generatedd from the tum mors in the nude mice was then recorrded in real-tim me by a small animal a imaging system m. As shown in Figure 3, even without ClOinjection, tumors of 1-injectedd mice also exhibbited weak BL signal (top row in Figgure 3). This coould be explaineed that small am mount of 1 was hydroolyzed by intraacellular proteasses of tumor ceells to yield D-luciferrin for BL ggeneration. Hoowever, withinn the observation tim me (i.e., 20-2220 min), the BL B intensity oof the experimental grroup remained tthe highest (midddle row in Figgure 3). It increased too the peak at 70 7 min, and then decreased sslowly thereafter (Figuure S12), sugggesting that, unnder ClO- oxiddation, most of 1 yieldded D-luciferin for the strong BL. B ClO--scaveenging control experim ment indicated that the taurine-treated mice had obviously loweer BL intensity than that of thee experimental group (bottom row inn Figure 3). Thiis further affirm med that above strong s BL from experiimental group w was resulted froom ClO- oxidation of 1 to yield D-luuciferin. These in vivo resultts demonstratedd that probe 1 could be applied forr imaging ClO- in tumors of living animals.

tauurine at 50 mg/m mL 5 min laterr and 100 μL 1 at 2.5 mM 5 m min lateer (bottom row)) in PBS at 20, 70, 7 120, 170, annd 220 min. I summary, we have succcessfully devveloped a lattent In biooluminescent prrobe 1 for highhly selective dettection of ClO- in vitrro and imagingg ClO- in tum mors of living m mice. Upon C ClOoxiidation and thee follow-up hyydrolysis, 1 waas converted to Dlucciferin for BL generation in thee presence of fL Luc. This propeerty of 1 was successsfully applied ffor highly selecctive detection of ClO O- within 0-62.5 μM and a L LOD of 0.705 μM in vitro. T The feaasibility of this assay for imaging ClO- in liiving cells andd in tum mors was also vvalidated. Withh these unique features of 1, we envvision that it m might be appliedd to elucidate thhe biological rooles of ClO- in wider physiological aand pathologicaal processes in the neaar future.

AS SSOCIATED D CONTENT T Su upporting Infformation Geeneral methods; Syntheses and characteerizations of 1; Suppporting schem me, figures, andd tables (Schem me S1, Figure S1S S122, and Table S1-S3). This maaterial is availabble free of charrge viaa the Internet at http://pubs.acs..org.

AU UTHOR INFORMATION N Co orresponding g Authors *E-mail: wangfq@ @njmu.edu.cn *E-mail: [email protected]

No otes The authors declarre no competingg financial interrests.

AC CKNOWLED DGMENT This work was suupported by Coollaborative Innnovation Centerr of Suzzhou Nano Sccience and Tecchnology, Hefeei Science Cennter CA AS (2016HSC-IIU010), Ministrry of Science annd Technologyy of Chhina (2016YFA A0400904), andd the National Natural Sciennce Fouundation of Chiina (Grants 21675145 and U15532144).

RE EFERENCES

me-course biolum minescence im maging of nude mice Figure 3. Tim xenografted w with fLuc-transffected MDA-M MB-231 tumors after intraperitoneal injection of 1000 μL 1 at 2.5 m mM (top row), 100 μL M followed by 100 μL 1 at 22.5 mM 5 minn later ClO- at 1 mM (middle row), or 100 μL CllO- at 1 mM ffollowed by 1000 μL

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