German Edition: DOI: 10.1002/ange.201812489Imaging Agents Hot PaperInternational Edition: DOI: 10.1002/anie.201812489

A Fluorogenic Probe for Cell Surface Phosphatidylserine Using anIntramolecular Indicator Displacement Sensing MechanismVincent E. Zwicker, Bruno L. Oliveira, Jia Hao Yeo, Stuart T. Fraser, GonÅalo J. L. Bernardes,Elizabeth J. New, and Katrina A. Jolliffe*

Abstract: The detection of externalized phosphatidylserine(PS) on the cell surface is commonly used to distinguishbetween living, apoptotic, and necrotic cells. The tools ofchoice for many researchers to study apoptosis are annexin V-fluorophore conjugates. However, the use of this 35 kDaprotein is associated with several drawbacks, including temper-ature sensitivity, Ca2+ dependence, and slow binding kinetics.Herein, a fluorogenic probe for cell surface PS, P-IID, isdescribed, which operates by an intramolecular indicatordisplacement (IID) mechanism. An intramolecularly boundcoumarin indicator is released in the presence of cell surfacePS, leading to a fluorescence “turn-on” response. P-IIDdemonstrates superior performance when compared to annex-in V, for both fluorescence imaging and flow cytometry. Thisallows P-IID to be used in time-lapse imaging of apoptosisusing confocal laser scanning microscopy and demonstratesthe utility of the IID mechanism in live cells.

Phosphatidylserine (PS), an anionic phospholipid, isa minor, but important, component of the membrane of alleukaryotic cells.[1] In healthy cells, PS is almost exclusivelyfound on the inner (cytoplasmic-facing) leaflet of the cellmembrane.[2] The exposure of PS on the cell surface isa common marker of cell death[3] and one of the earliesthallmarks of apoptosis (programmed cell death),[4,5] whereexternalization of PS acts as a signal for phagocytes torecognize and engulf the dying cells.[6] The ability toselectively and rapidly detect apoptotic cells is crucial acrossa range of applications in molecular imaging and clinicalmedicine.[7] To distinguish cell populations undergoing apop-tosis from both living and necrotic cells, probes for thedetection of cell surface PS are commonly employed in

conjunction with nuclear stains such as propidium iodide.[8]

The most frequently used probe for detection of PS external-ization is fluorescently labeled annexin V (AnV), a 35 kDaprotein,[5, 9] which binds with high affinity to PS in a Ca2+-dependent manner.[10] However, there are a number ofproblems associated with the use of fluorescently labeledAnV derivatives to detect cell surface PS.[11] These include1) binding of AnV to PS exhibits slow binding kinetics, whichlimit its use in high-throughput drug assays,[12] and must beperformed at room temperature, precluding concomitant useof other assay systems, such as immunostaining;[13] 2) themillimolar levels of Ca2+ required for AnV binding to PS altercellular physiology and can result in scramblases translocatingmore PS to the cell surface;[14] and 3) currently availableAnV–fluorophore conjugates are not so-called “turn-on”probes, and therefore their use generally requires a washingstep to remove the unbound probe, so they are not useful forreal-time imaging of apoptosis. To circumvent the numerousissues with using AnV–fluorophore conjugates for monitoringapoptosis, a fluorogenic small molecule probe capitalizing onintramolecular indicator displacement sensing has beendeveloped.

Smith and co-workers have pioneered the use of zinc(II)dipicolylamine (ZnDPA) complexes to selectively target PS-rich membrane surfaces, typically using a bis(ZnDPA) PSbinding motif attached to a fluorophore via a linker.[15, 16]

However, the positioning of a linker between the fluorophoreand PS binding site generally means that the fluorescenceintensity is not altered upon binding, requiring a washing stepto clear unbound probe prior to imaging. It is preferable fora fluorescent probe to only exhibit fluorescence upon bindingto the analyte of interest, but fluorogenic probes for imagingapoptosis are scarce.[17,18]

Conjugating a fluorophore to the binding site (receptor)of a probe to prepare a fluorogenic probe is often a syntheticchallenge. One novel approach is to employ the highlyeffective, but rarely utilized, intramolecular indicator dis-placement (IID) mechanism[19–22] in which a fluorescentindicator is covalently attached to the receptor via a flexiblelinker. In the resting state the indicator binds to the receptorsite with quenching of its fluorescence. Displacement of theindicator from the receptor site by the analyte of interestleads to fluorescence (Figure 1).

Herein, a novel ZnDPA-based probe, P-IID, for thedetection of apoptosis by sensing cell surface PS using a “turn-on” fluorescence IID activation mechanism is described. P-IID incorporates three components on a peptide backbone:a bis(ZnDPA) binding motif for PS;[23] a 6,7-dihydroxycou-marin indicator positioned such that in the resting state the

[*] V. E. Zwicker, Dr. J. H. Yeo, Prof. E. J. New, Prof. K. A. JolliffeUniversity of Sydney, School of ChemistrySydney NSW 2006 (Australia)E-mail: kate.jolliffe@sydney.edu.au

Dr. B. L. Oliveira, Dr. G. J. L. BernardesUniversity of Cambridge, Department of ChemistryLensfield Road, CB2 1EW Cambridge (UK)

Dr. B. L. Oliveira, Dr. G. J. L. BernardesUniversidade de Lisboa, Instituto de Medicina Molecular Jo¼o LoboAntunes, Faculdade de MedicinaAvenida Professor Egas Moniz, 1649-028 Lisboa (Portugal)

Dr. J. H. Yeo, Dr. S. T. FraserUniversity of Sydney, School of Medical SciencesCamperdown NSW 2050 (Australia)

Supporting information and the ORCID identification number(s) forthe author(s) of this article can be found under:https://doi.org/10.1002/anie.201812489.

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dye coordinates to one of the ZnDPA moieties, whichquenches its fluorescence;[21, 22] and a stearic acid membraneanchor to reduce cellular uptake of the probe, therebypreventing the imaging of PS present on the inner leaflet ofthe cell membrane.[24] In the presence of externalized PS, thefluorophore is displaced from the ZnDPA site by the anionicPS headgroup, which leads to restoration of the coumarinfluorescence (Figure 1).

The synthesis of P-IID was readily achieved usingstandard solid-phase peptide synthesis methods, with on-resin attachment of both the coumarin fluorophore and DPAmoieties (see the Supporting Information for details). Com-plexation of the purified peptide with two equivalents of Zn2+

led to fluorescence quenching (Supporting Information,Figure S1). This was attributed to the coordination of thecatechol to one of the ZnDPA arms with the associateddeprotonation of the catechol moiety, as indicated byDFT calculations (Supporting Information, Figure S2)and confirmed by mass spectrometry (m/z 723.7886;C71H93N15O10Zn2

2+).The ability of P-IID to detect PS-rich membranes was

demonstrated using vesicle titration experiments. Zwitter-ionic vesicles composed of 100 % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and anionic vesicles com-posed of 50% POPC and 50 % 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) were prepared by stan-dard extrusion techniques. Subsequent titrations of P-IID(10 mm, HEPES buffer pH 7.4) with the zwitterionic POPCvesicles resulted in negligible modulations in the fluorescencespectrum (Figure 2A), indicating no significant binding to themembrane surface, whereas the addition of anionic POPC/

POPS vesicles caused an approximately four-fold fluores-cence increase at around 530 nm (Figure 2 B), attributed tothe displacement of the coumarin indicator from the ZnDPAbinding site by PS.

Having demonstrated that P-IID recognizes PS witha fluorigenic response, we set out to visualize apoptosis inboth fixed and live cell fluorescence imaging studies. Initialexperiments were conducted using HeLa cells in whichapoptosis was induced by treatment with camptothecin(CPT, 40 mm, 12 h). Following CPT treatment, cells werestained with P-IID (20 mm, 20 min) and a commercial cellstain (SYTO85 Orange, 25 nm, 20 min). Confocal microscopyimages show staining of apoptotic cells by P-IID, whilehealthy control cells remained unstained (Figure 3). Subse-quent experiments in HCC-1806 cells showed that theobserved membrane staining pattern was maintained acrossdifferent cell lines (Supporting Information, Figure S3). Two

Figure 1. Chemical structure of the probe P-IID and proposed bindingmechanism with PS. The NO3

@ counterions have been omitted forclarity.

Figure 2. A), B) Fluorescence spectra of P-IID (10 mm, HEPES buffer,pH 7.4, lex =405 nm) upon the incremental addition of (A) zwitterionicvesicles (100% POPC) or (B) anionic vesicles (50% POPC/50%POPS), and change in fluorescence intensity (F.I.) at 530 nm asa function of phospholipid concentration.

Figure 3. Confocal microscopy images of untreated and CPT-treatedHeLa cells (focused on single cell). Cells were stained simultaneouslywith SYTO85 Orange (cell stain) and P-IID. Scale bars: 10 mm.

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analogues of P-IID that lack the membrane anchor, bearingeither a coumarin that does not coordinate to the ZnDPAbinding site (and is therefore always “on”; probe 6, structurein Supporting Information), or the same fluorophore as P-IID(probe 8, structure in Supporting Information) were used ascontrols; neither of these showed the membrane stainingobserved with P-IID (Supporting Information, Figure S3).

Having shown the suitability of P-IID for fluorescenceimaging of PS expression in apoptosis, we next assessed itsperformance in detecting apoptotic PS expression in HCC-1806 and MCF-7 cells, as compared to annexin V, using flowcytometry. Following induction of apoptosis by CPT treat-ment, cells were stained with P-IID (15 mm). Co-staining withpropidium iodide (PI, 1 mgmL@1) was performed to identifyhealthy cells (PI@/P-IID@ , Q3), early apoptotic cells (PI@/P-IID+, Q4), late apoptotic cells (PI+/P-IID+, Q1), and deadcells/debris (PI+/P-IID@ , Q2) in the flow cytometry profiles(Figure 4A, left two graphs; see Supporting Information for

staining conditions). This is comparable to the commonlyemployed method of double labeling with AnV and PI.[25] Inparallel experiments, cells were stained with commerciallyobtained AnV–Alexa Fluor 647 (2.5 mg mL@1) and PI. Theflow cytometric profiles of apoptotic cells stained with P-IIDresembled those stained with the AnV–Alexa Fluor 647conjugate when the recommended standard conditions forAnV staining (15 min, 2.5 mm Ca2+, 25 88C, washing step afterstaining) were used (Supporting Information, Figures S5 andS16, left two graphs), indicating that there is little differencein the frequency of cells binding to either AnV or P-IID,under these conditions.

To determine whether P-IID would be effective inconditions under which AnV staining is problematic, suchas when the labeling of apoptotic cells is required in theabsence of Ca2+, without washing steps, or at low temper-atures (i.e. on ice), a range of experiments were conducted inwhich the standard conditions were modified accordingly, and

the results were quantita-tively compared to thoseobtained with AnV (Fig-ure 4A–E for HCC-1806cells, Supporting Informa-tion, Figures S5–S10 forMCF-7 cells). Figure 4Ashows representative flowcytometric profiles ofhealthy, and apoptoticHCC-1806 cells stainedwith P-IID and PI, in thepresence, and absence of2.5 mm Ca2+. No significantdifference in the frequencyof cell populations wasobserved between CPT-treated cells stained withP-IID in either the pres-ence or absence of 2.5 mmCa2+ (Figure 4B), indicat-ing that P-IID can effec-tively detect apoptotic cellpopulations in the absenceof Ca2+ ions. Similar resultswere obtained when theexperiment was performedwith MCF-7 cells (Support-ing Information, Fig-ure S5). In contrast, AnVonly stained apoptotic cellpopulations in the presenceof Ca2+ ions (Figure 4C forHCC-1806 cells; Support-ing Information, Figure S6for MCF-7 cells).

We next evaluated ifa washing step was requiredafter staining either HCC-1806 or MCF-7 cells with P-IID or AnV. P-IID stained

Figure 4. A) Representative flow cytometry analysis of untreated and CPT-treated HCC-1806 cells stained withpropidium iodide (PI) and P-IID, in the presence and absence of 2.5 mm Ca2+. B) Quantification of (A)showing the frequency of CPT-treated HCC-1806 cells stained with PI+/P-IID+ (late apoptotic) and PI@/P-IID+

(early apoptotic), with and without 2.5 mm Ca2+. C) Staining of CPT-treated HCC-1806 cells with AnV, withand without 2.5 mm Ca2+. The frequency of cells stained PI+/AnV+ (late apoptotic) and PI@/AnV+ (earlyapoptotic) is shown. D), E) Staining of CPT-treated HCC-1806 cells with P-IID or AnV (D) with or withouta washing step and (E) at 25 88C and at 4 88C. Data for (B–E) are represented as the mean:SEM from threeindependent experiments; ns, not significant; *P,0.05; **P,0.01 (Student’s t-test). F) Representative flowcytometry histograms of untreated and CPT-treated MCF-7 cells stained with P-IID or AnV for 1 min, 5 min,and 15 min. G) Quantification of (F) showing the geometric mean fluorescence intensity (gMFI) of CPT-treated cells stained with P-IID and AnV after 1 min, 5 min, and 15 min. Data are represented as themean:SEM from three independent experiments; ns, not significant; **P,0.01 (one-way ANOVA).

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apoptotic cells equally well, regardless of either the cell typeand whether or not a washing step had been performed(Figure 4D, left graph for HCC-1806 cells and SupportingInformation, Figure S7 for MCF-7 cells). This was ascribed tothe “turn-on” nature of the probe. In contrast, while there wasno significant difference in AnV staining of HCC-1806 cellswhen compared to the standard conditions (Figure 4D, rightgraph), with MCF-7 cells “no-wash” AnV staining showedhigher numbers of early apoptotic as well as late apoptoticcells (Supporting Information, Figure S8), suggesting thatnon-specific staining had occurred due to the “always-on”state of the AnV–fluorophore conjugate. This demonstratesthat P-IID can be used without a washing step, which isa significant advantage for real-time imaging and high-throughput assays.

Finally, to compare the binding kinetics of P-IID withAnV, flow cytometric profiles of cell populations stained withP-IID or AnV, either at 4 88C or with shorter staining timeswere compared. Staining efficacy remained constant for P-IID with a reduction in temperature from 25 88C to 4 88C,whereas lowering the temperature greatly reduced theefficacy of AnV staining (Figure 4E, Supporting Figures S9and S10). Similarly, when the AnV staining time was reducedfrom 15 min to 5 min or 1 min, distinction between the AnV+

and AnV@ population in either CPT-treated MCF-7 cells(Figure 4F, quantification in Figure 4G) or HCC-1806 cells(Supporting Information, Figure S17) became difficult,reflecting the slow binding kinetics of AnV to PS. In contrast,identical staining patterns were observed upon incubatingMCF-7 and HCC-1806 cells with P-IID for either 15 min,5 min, or 1 min. This indicated that staining with P-IID can beperformed extremely quickly and without the need forincubation periods, suggesting rapid binding kinetics of P-IID to PS.

Having determined that P-IID binds rapidly to cellsurface PS with a fluorogenic response, we assessed the utilityof P-IID in real time imaging of cells undergoing apoptosis.Using confocal microscopy, we imaged HCC-1806 cells towhich both P-IID (15 mm) and CPT (40 mm) were added underan atmosphere of 5% CO2 at 37 88C over the course of 9 h.(Figure 5A). A net fluorescence enhancement in the P-IIDchannel was observed, with an increase in mean fluorescence

intensity (MFI) commencing approximately 3 h after additionof CPT and plateauing after 7 h (Figure 5B). Healthy controlcells imaged in the presence of P-IID did not showa fluorescence increase over the same time period (Figure 5Cand Supporting Information, Figure S18). These results con-firm that P-IID can be used for time-lapse imaging of drug-induced apoptosis in cell populations.

In conclusion, a novel fluorogenic probe for cell surfacephosphatidylserine has been synthesized and evaluated. P-IID senses cell surface phosphatidylserine by fluorescence“turn-on” using an intramolecular indicator displacement(IID) mechanism. This is the first time an IID probe has beenused in live cells. P-IID detects cell surface phosphatidylser-ine on apoptotic cells using both confocal microscopy andflow cytometry, and enabled time-lapse imaging of cellsundergoing apoptosis. P-IID circumvents some of the majordrawbacks associated with AnV by reliably detecting cellsurface phosphatidylserine in the absence of Ca2+, at 4 88C, andwithout the need for a wash step. Furthermore, P-IID bindingto phosphatidylserine is extremely rapid (1 min vs. 15 min forAnV). P-IID is readily synthesized using a modular approachthat will allow the incorporation of alternative fluorophores,and our current studies are focussed on development ofa suite of analogues. Taken together, these characteristicsmake P-IID a powerful tool for imaging cell surfacephosphatidylserine. It is superior to fluorescent conjugatesof annexin V for the differentiation of living, apoptotic, andnecrotic cells in co-staining experiments with propidiumiodide.

Acknowledgements

We thank the Australian Research Council (DP140100227 toK.A.J., and DP180101353 to K.A.J. and E.J.N.); the Univer-sity of Sydney (IPRS and John A. Lamberton scholarships toV.E.Z.; Prof. N.G.W. and Mrs. Ann Macintosh Memorialscholarship to J.H.Y.; NWG Macintosh Grant 2018 to S.T.F.);the Royal Society (URF to G.J.L.B., UF110046 andURF\R\180019), FCT Portugal (iFCT to G.J.L.B., IF/00624/2015), ERC StG (GA No. 676832), the European Commis-sion (Marie Sklodowska-Curie ITN ProteinConjugates GA

Figure 5. A) Time-lapse imaging of a single field of HCC-1806 cells simultaneously treated with CPT (40 mm) and stained with P-IID (15 mm) overthe course of 6 h (5 % CO2 at 37 88C). B) Quantification of the change in the mean fluorescence intensity (MFI) over the total imaging time of 9 h.C) Change in MFI when untreated HCC-1806 cells were incubated with P-IID (15 mm) for 9 h. Scale bar: 30 mm. Data are represented as themean:SEM from three independent experiments.

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No. 675007 to G.J.L.B. and Marie Sklodowska-Curie IEF GANo. 702574 to B.L.O.) and the EPSRC (EP/M003647/1 toG.J.L.B.) for funding. We also acknowledge the AustralianCentre for Microscopy & Microanalysis (ACMM) and theBosch Live Cell Analysis Facility (LCAF) for access tofacilities.

Conflict of interest

The authors declare no conflict of interest.

Keywords: annexin V · apoptosis · fluorescent probes ·imaging agents · phosphatidylserine

How to cite: Angew. Chem. Int. Ed. 2019, 58, 3087–3091Angew. Chem. 2019, 131, 3119–3123

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Manuscript received: October 31, 2018Accepted manuscript online: November 22, 2018Version of record online: December 13, 2018

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