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Development of responsive lanthanide probes for cellular applications
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Development of responsive lanthanide probes for cellular applications Elizabeth J New, David Parker, David G Smith and James W Walton Useful probes of the intracellular environment are required for a wide range of bioactive species including metal ions, oxyanions and pH. These probes need to be targeted to specific organelles (mitochondria, nucleus and lysosomes) in order to allow direct observation of the changes in these regions. Critical probe design features for luminescent lanthanide complexes are defined, together with a review of published sub-cellular localisation profiles. Cell uptake by macropinocytosis has been demonstrated for a wide range of probes and the importance of minimising perturbation of cellular homeostasis emphasised, so that cell viability, proliferation and membrane permeability are not compromised. Address Department of Chemistry, Durham University, South Road, Durham DH1 3LE, UK Corresponding author: Parker, David ([email protected])
Current Opinion in Chemical Biology 2010, 14:238–246 This review comes from a themed issue on Bioinorganic chemistry Edited by Kathy Franz and Chuan He Available online 31st October 2009 1367-5931/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2009.10.003
Introduction Non-invasive probes of the composition and structure of a cell are of key importance in enhancing the understanding of cellular processes. Amongst these, optical probes are pre-eminent as emitted light can be traced with high sensitivity and spatial resolution. The majority of optical probes are based on organic fluorophores. Responsive probes have been developed to either monitor local changes in pH or pM (M = Ca, Mg or Zn mostly) [1,2], or report on the local physical environment providing information on polarity, fluidity or geometry [3]. The majority of optical probes are best considered as cellular stains that localise to particular organelles or sub-cellular locations. The luminescence output of these stains provides information about the physical layout of the cell [4]; they report primarily structural information. Information about the function of the cell is better obtained using responsive probes, in which modulation occurs of Current Opinion in Chemical Biology 2010, 14:238–246
luminescence anisotropy, polarisation, lifetime or the ratio of emission intensity for one or more pairs of spectral bands. These parameters are independent of probe concentration and avoid problems associated with measurements of emission intensity. These include perturbations induced by changes in local probe concentration, pH, temperature, light scattering or the extent of photobleaching or local quenching. It is important to recognise that organelle-specific targeting remains a major challenge in biology and medicine [5]. Delivery of a novel therapeutic agent to its target organelle may reduce toxic side effects and enhance efficacy. For example, gene therapy drugs target nuclear DNA, whilst several anti-cancer agents exert their effects in mitochondria. From an imaging perspective, certain conditions within a specific organelle can characterise the onset or development of a particular disease. Several lysosomal storage diseases are characterised by elevated lysosomal pH whilst cytosolic pH is only slightly perturbed [6]. Similarly, an increase in mitochondrial levels of calcium, but not the total intracellular calcium concentration, has been implicated in mitochondrial dysfunction accompanying ischaemia and ageing [7]. Many other characteristic changes in cellular composition within a given organelle await discovery, because there are no probes available to report these changes selectively. Luminescent metal complexes have arisen as alternative probes to low MW organic dyes or recombinant fluorescent proteins. Two main classes may be distinguished. The first comprises d6 or d8 transition metal complexes, often based on Ru(II), Os(II), Re(I), Ir(III) and Pt(II). These involve highly conjugated ligands giving rise to intense charge transfer bands. The LMCT or MLCT excited states are relatively long-lived (10 7 to 10 4 s) allowing time-gated methods to be used to reduce background noise [8–15]. So far, these systems function primarily as cellular stains. The second set of complexes is based on luminescent lanthanide (III) complexes, for which a larger number and broader range of responsive systems has been defined [16,17,18,19,20]. Responsive probes based on the modulation of lanthanide emission intensity, lifetime or polarisation have been frequently reported over the past decade. However, these systems are rarely adapted to the constraints imposed by the complex intracellular environment. In this review, we examine these issues and highlight the critical probe design features that must be addressed in developing a practicable intracellular probe. www.sciencedirect.com
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Critical probe design features There are four key issues to consider in devising an emissive probe suitable for use in living cells. First, the probe must be able to cross the outer lipid membrane at a relatively rapid rate, preferably with a slow rate of egress. Uptake should occur by a comprehensible mechanism [21]. In addition, it should exhibit a sub-cellular localisation profile that is amenable to microscopic observation. Second, the probe must localise preferentially to a particular organelle, preferably by a transport mechanism that is understood or can be rationalised. Third, the probe should not unduly perturb cellular homeostasis. A non-invasive probe should neither inhibit cellular viability nor proliferation (often defined by an IC50 value) nor alter the mitochondrial membrane potential (MMP). Its presence should also not alter the cell’s intrinsic permeability. Several literature reports purporting to herald a selective localisation profile may be ascribed to induction of enhanced membrane permeability, caused by the presence of the probe in the cellular incubation medium [11,13,21,22]. Finally, probe integrity and performance in cellulo needs to be considered. The probe should resist enzymatic degradation and minimise the problems associated with photobleaching or photofading that may occur following excitation. It should be addressed at an accessible wavelength using lasers or powerful LEDs (e.g. 355 nm, 365 nm and 405 nm) or using two-photon excitation in the near-IR optical window 700–820 nm that best permeates cells and tissues [23,24]. The probe must also resist excited-state quenching by species that diminish its brightness [25,26]. These range from endogenous reducing agents (urate, ascorbate and glutathione) to the proteins that are involved in intracellular trafficking. These include tubulin or actin, the latter present at concentrations of the order of 0.1 mM, or those that are abundant in the organelle observed, for example the protein-dense regions of the nucleoli and ribosomes. The timescales for intracellular probe trafficking and for image data acquisition also need to be compared rationally to the timescale for modulation of the biochemical variable being monitored.
Classification of probe sub-cellular localisation The factors and processes involved in compartmentalisation of exogenous species into sub-cellular organelles are not well understood. Trafficking processes require an appreciation of three main aspects: the ‘signal’ that triggers a specific localisation; the pathways by which the molecule enters an organelle and the mechanical process by which translocation occurs within the cell. A pragmatic first step towards understanding these processes is to examine the localisation behaviour of structurally homologous series of probe complexes and classify them in terms of this property, following observation of their localisation profile using luminescence microscopy. www.sciencedirect.com
Work at Durham University has generated a library of more than 70 europium or terbium (III) complexes, allowing preliminary conclusions to be made of the relationship between probe structure and localisation. Data are summarised in Figure 1. Four main classes can be defined. The complexes in the largest group are those that localise to the lysosomes. A cell typically contains about 300 lysosomes that are membrane-bound and of variable size, with a luminal pH between 4 and 5. Lysosomal localisation usually occurs by two mechanisms. If cellular uptake occurs by receptor-mediated endocytosis, the probe will reside in an endosome that ages to become a lysosomal compartment. Alternatively, if a cell recognises a species as being foreign, it is likely to be ubiquinated and transported to a lysosome for subsequent degradation. For a wide variety of luminescent lanthanide (III) probes, an endosomal/lysosomal localisation profile has been confirmed by co-staining experiments using commercially available dyes (LysoTrackerTM, Invitrogen). No obvious correlation with probe structure has been made [16,19,21]. A smaller group of complexes has been observed to localise to the mitochondria [24,27,28]. These organelles are the centres of aerobic respiration through the electron transport chain and oxidative phosphorylation. Critically, they are involved in generating ATP that is an essential energy source for many cellular processes. Co-staining experiments with MitoTracker GreenTM for europium (III) complexes revealed that certain probes were subsequently (typically >8 hours) transported to the lysosomes [27,29]. Indeed, simultaneous localisation in late endosomes and mitochondria has been observed for a series of complexes that contain an azaxanthone sensitising chromophore, linked to the central ligand ring through a pyrazole, pyridine or amide group [29]. The nucleolus is a sub-nuclear structure involved in ribosome assembly, with the highest local density of proteins in a cell. A series of europium complexes, including a metal-coordinated azathiaxanthone sensitising moiety (Figure 1), has been shown to localise to this region and the related extranuclear ribosomes: co-staining experiments with Syto-RNA confirmed this profile [22,30,31]. More recent experiments examining localisation over a wider range of probe concentrations and incubation times in different cell types suggest that this behaviour is characteristic of a cell under stress [21], in which the cell membranes become more permeable. For example, several complexes that exhibited a mitochondrial or lysosomal distribution were observed in the cell nucleoli and ribosomes following deliberate permeabilisation of the cell membrane. This can be achieved by the addition of surfactants, for example saponin or Triton (Figure 2). Control experiments involving coincubation with propidium iodide confirm this hypothesis. In the absence of added saponin, the outer cell Current Opinion in Chemical Biology 2010, 14:238–246
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Figure 1
Illustration of four distinct sub-cellular localisation patterns.
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Figure 2
Fluorescence microscopy images revealing the metal-based emission in Chinese hamster ovarian (CHO) cells, following incubation with the shown terbium complex (four hours, 50 mM) and propidium iodide (1 mM): (a) terbium emission control; (b) propidium fluorescence; (c) terbium emission after saponin treatment (15 min, 0.5 mg/mL); (d) propidium fluorescence after saponin treatment, revealing nuclear DNA [16,21].
membrane is impermeable to propidium iodide. Following treatment, propidium iodide is able to cross each barrier and its fluorescence is clearly visible in the nucleus. Thus, luminescence staining of the nucleolus may be linked to an increased permeability of the cell membranes that is induced by the presence of the probe. Other reports of the intra-nuclear localisation of an optical imaging probe need to heed this aspect. For example, the definition of DNA-staining with cationic complexes bearing an intercalating tetraazatriphenylene sensitising moiety only occurred at relatively high probe concentrations [20], which may have compromised the ‘normal’ permeability of the cell’s membranes. However, a recent discovery of the selective staining of the nucleus of cells undergoing division (Figure 1) has allowed selective visualisation of the DNA packaging and organisation that accompanies the progression from mesophase to telophase during mitosis. This was made with sub-micromolar concentrations of terbium complex and occurred within 5 min of addition of the probe to the cell incubation medium. It is possible that a broader range of complexes, with observed lysosomal or mitochondrial distributions, may also exhibit this behaviour at much lower concentrations. www.sciencedirect.com
Mechanism of cell uptake for lanthanide (III) optical probes It is important to understand the mechanism of cell uptake and subsequent trafficking for an imaging probe, in order to address the rational development of a responsive probe that can selectively localise in a target organelle. Several different cell uptake mechanisms exist, for which a series of inhibitors and promoters has been described (Figure 3) [32]. The major pathway of cell entry for low MW probes, present at low concentration in the incubation medium, is endocytosis. This typically occurs by invagination of the cell membrane and is energy dependent, so that it is suppressed at 58C. Membrane invagination may involve the formation of clathrincoated pits (suppressed by the addition of sucrose or chlorpromazine), caveolae (inhibited by the addition of nystatin or filipin) or macropinocytosis. The latter process involves cell surface ruffling, leads to the formation of large endocytotic vesicles that are irregular in shape and size and relatively ‘leaky’, allowing cycling of the contents of the macropinosome to other parts of the cell. This process is not receptor-mediated and involves the actin-dependent formation of lamellipodia — sheet-like plasma membrane extensions, supported by a web of actin filaments. It is suppressed by the addition of Current Opinion in Chemical Biology 2010, 14:238–246
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Figure 3
Schematic diagram illustrating the three endocytic pathways of macropinocytosis, clathrin-mediated endocytosis and caveolin-dependent endocytosis, in addition to the intracellular maturation of endosomes to lysosomes. Inhibitors and activators of each pathway are indicated [16,21,32].
amiloride or wortmannin (which inhibits PI3 kinase activity) and enhanced by the addition of phorbol esters or fatty acid glycerides. For each of the lanthanide optical probes discussed here, macropinocytosis has been identified as the predominant mechanism of cell uptake, confirmed by co-staining of the observed young macropinosomes with a 70 kDa fluorescein-labelled dextran [21].
Strategies for controlling or predicting subcellular localisation
bium probe has been defined, assuming that this moiety will enhance sensitivity for the peripheral benzodiazepine receptor found in mitochondria [28]. This targeting approach is further exemplified by certain peptide conjugates, containing an established sequence used to signal the trafficking of proteins. Conjugation of a nuclear localising sequence (NLS) has been hypothesised to cause the predominant nuclear localisation of Cys-GlyGly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly conjugated to a neutral Pt(II) amine complex, involving a lengthy PEG spacer [35].
Notwithstanding the paucity of knowledge of sub-cellular trafficking pathways, various strategies have emerged that strive to assist probe delivery to particular organelles. For example, encapsulation of a probe in a liposome inevitably leads to a lysosomal localisation, as the liposomes commonly are endocytosed into a vesicle that fuses with an endosome [33]. Various cationic and amphiphilic species have been found to permeate the negatively charged surface of mitochondria selectively, including lipophilic tetraguanidinium derivatives [24], the gold anti-tumour complex auranofin [34] and the simple dye rhodamine 123. Additionally, various conjugates can be considered in which a probe structural element dictates sub-cellular localisation. For example, integration of a 2chlorophenyl-1-naphthyl moiety into an emissive ter-
Amongst the family of emissive lanthanide (III) probes (Figure 1), the nature and mode of linkage of the heterocyclic or aromatic sensitising moiety dictates the facility of cell uptake and sub-cellular localisation [16,21, 27,36,37]. Furthermore, in a systematic examination of cellular behaviour of 11 derivatives of a common core probe structure, involving only variation of the nature of the sensitiser substituent [24,36], the sub-cellular localisation profile was very similar, but certain differences in behaviour were observed. These included the perturbation of the homeostasis of the cell, as revealed by measurement of cellular toxicity, modulation of the MMP and definition of certain cell-type-specific responses. An obvious conclusion from such observations
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is that non-covalent binding to particular transport proteins, including the lipophilic sensitising moiety, is important in determining sub-cellular localisation.
Perturbation of cellular homeostasis A non-invasive optical probe should not unduly perturb the structure and function of a healthy cell. It is important to perform control experiments when evaluating the utility of such a probe, examining changes to cell viability, proliferation or permeability. This is commonly assessed by monitoring changes to membrane permeability (e.g. using Trypan Blue or propidium iodide staining only compromised cells in which membrane permeability is enhanced considerably), esterase activity (e.g. using the acetomethoxy derivative of calcein, which is fluorescent
only when hydrolysed by active cytosolic enzymes) or mitochondrial redox (e.g. using tetrazolium salts such as MTT or WST-1 that assess changes to mitochondrial redox enzymes). Such methods allow measurement of IC50 values (typically in the range 1–500 mM) defining the probe concentration needed to kill 50% of the cell population. Complexes that simply localise in lysosomes/endosomes are insulated from more sensitive organelles of a cell and have IC50 values (after 24 hours incubation) in excess of 0.1 mM, considerably greater than concentrations used in practice. As noted above, the presence of certain probes may lead to enhanced membrane permeability (Figure 2) and has been associated with observation of staining of the nuclei or chromosomal DNA of a cell [21]. This may, in part, explain observations
Figure 4
Examples of cell permeable responsive emissive lanthanide probes: (upper) for singlet oxygen [39]; (centre) for bicarbonate in mitochondria; (bottom) for pH [31]. www.sciencedirect.com
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reported for the staining of nucleoli using a covalently linked Ru-bipyridyl-phenanthridine conjugate [38], or the change in localisation to an oligo-Arg-Ru-bipyridyl conjugate in HeLa cells induced by introduction of a fluorescein moiety [11]. Indeed, in the latter report, dead cells were ‘excluded’ from the quantitative work, yet loss of outer and nuclear membrane integrity is a common step in the pathway to necrotic cell death. The distinction between cell death induced by necrosis versus apoptosis has been reliably identified for certain lanthanide probes, using flow cytometric methods [24]. For probes that localise to mitochondria, the perturbation of the MMP needs to be examined and compared to IC50 values derived from analysis of mitochondrial redox enzyme activity. The lanthanide probes described in Figure 1 [27], for example, possess high IC50 values (100 mM) and do not significantly change the MMP, auguring favourably for their application as probes of this organelle.
Intracellular performance of responsive probes Very few studies have described the function of a responsive luminescent lanthanide probe within a specified cell (Figure 4) [30,31,39,40]. At best, only one such system reports non-invasively on a particular organelle [30,31]. Ultimately, this must be the goal of this research, yet this is difficult to predict from prior in vitro characterisation studies. A key issue is the sensitivity of the optical probe to excited-state quenching by endogenous species such as urate, ascorbate and glutathione, present at near millimolar concentrations in most cell types. Furthermore, the impact of non-covalent binding to endogenous macromolecules (such as proteins and nucleic acids) needs to be considered, and the extent to which this may modulate sensitivity to quenching assessed [25,27,36,37,41]. Certain lanthanide complexes have been identified, for example, which are taken into cells efficiently (intracellular concentration of the order of 50 mM, deduced by ICPMS analysis of [Ln], coupled with cell counting by flow cytometry) yet are barely visible by microscopy because they are so sensitive to quenching by urate and ascorbate [41]. A second obvious key issue is that the probe must be cell permeable and not gain entry under conditions that may perturb cellular homeostasis. The promising irreversible Eu(III) probe for singlet oxygen was shown to localise in HeLa cells, but only in the presence of a porphyrin photosensitiser that was added to the growth medium to artificially enhance local 1DO2 levels. Its presence may well have enhanced membrane permeability, leading to the observed nuclear localisation profile [39]. Similarly, a zinc probe has been demonstrated to give rise to modulation of Eu(III) luminescence in HeLa cells. The europium complex, however, was Current Opinion in Chemical Biology 2010, 14:238–246
injected directly into the observed cell and relatively weak emission observed by time-resolved microscopy [40]. The complex possessed a substituted quinoline sensitising moiety that has a long wavelength absorption band at 320 nm, but absorbs light very poorly at the excitation wavelength used (360 nm). This contrasts with the characteristics of a pH sensitive probe (Figure 4) with an azathiaxanthone sensitising moiety (lmax 385 nm) that absorbs strongly at the common laser lines of 365 nm or 405 nm and has a pKa of 7.1 under simulated intracellular conditions. This complex was shown to localise to the ribosomes/nucleoli revealing a local pH of 7.4 following ratiometric analysis of europium emission [30,31]. It is desirable to devise an analogue of this system that localises to lysosomes with a pKa of around 4.5, so that perturbations in lysosomal pH can be investigated. A final obvious issue is that the working dynamic range of the responsive probe for the target analyte should correspond to the local concentration of the species under study. Calibration studies in diluted serum solutions or in macerated cellular media offer a partial solution [42] to simulate the intracellular fluid composition.
Concluding remarks The design of luminescent lanthanide sensory systems with a tailored affinity and selectivity profile for use in vitro can now be undertaken with reasonable confidence [16,17,18]. Recently, promising systems that report on changes to species such as potassium [43], lactate and citrate [44] have been defined. Critical design features need to be engineered into the probe from the outset to permit successful use of such probes in cellulo. The most important of these relate to the need to minimally perturb the cell under observation and to target the organelle of interest, where the analyte observed is to be found. Such studies are actively being addressed and should provide new tools to uncover the role and potential diagnostic value of intracellular changes to key bioactive, low MW species.
Acknowledgements We thank EPRSC, CISbio and the Association of Commonwealth Universities for support.
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24. Kielar F, Congreve A, Law G-L, New EJ, Parker D, Wong K-L, Castren˜o P, de Mendoza J: Two-photon microscopy study of the intracellular compartmentalisation of emissive terbium complexes and their oligo-arginine and oligo-guanidinium conjugates. Chem Commun 2008: 2435-2437. Using a titanium sapphire laser, excitation of complexes at 720 nm allows visualisation of probe uptake by confocal microscopy. Flow cytometry studies were used to distinguish cell death pathways induced by necrosis (membrane destabilisation pathway) and apoptosis.
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25. Kielar F, Montgomery CP, New EJ, Parker D, Poole RA, Richardson SL, Stenson PA: A mechanistic study of the dynamic quenching of the excited state of europium (III) and terbium (III) macrocyclic complexes by charge or energy transfer. Org Biomol Chem 2007, 5:2055-2062. 26. Law G-L, Parker D, Richardson SL, Wong K-L: The mechanism of quenching of the lanthanide excited state for optical probes using sensitised emission. Dalton Trans. 2009, 8481–8484. Direct laser excitation of Eu and Tb complexes allows population of the metal excited state; under these conditions the lanthanide excited state is not quenched by added urate, ascorbate or iodide. Formation of an exciplex between the sensitising moiety and the electron-rich quencher leads to dynamic quenching of metal-based emission. 27. Murray BS, New EJ, Pal R, Parker D: Critical evaluation of five emissive europium(III) complexes as optical probes: correlation of cytotoxicity, anion and protein affinity with complex structure, stability and intracellular localisation profile. Org Biomol Chem 2008, 6:2085-2094. 28. Manning HC, Smith SM, Haviland MS, Bai S, Cederquist MFK, Stella N, Bornhop DJ: A peripheral benzodiazepine receptor targeted agent for in vitro imaging and screening. Bioconjug Chem 2006, 17:735-740. 29. Montgomery CP, New EJ, Palsson LO, Parker D, Batsanov AS, Lamarque L: Emissive and cell permeable pyridyl and pyrazoyl1-azaxanthone lanthanide complexes and their behaviour in cellulo. Helv Chim Acta in press; doi: 10.1002/hlca200900122. 30. Pal R, Parker D: A single component ratiometric pH probe with long wavelength excitation of europium emission. Chem Commun 2007, 2007:474-476. 31. Pal R, Parker D: A ratiometric optical imaging probe for intracellular pH based on modulation of europium emission. Org Biomol Chem 2008, 6:1020-1033. A europium complex localises to the nucleoli and ribosomes within mouse skin cells and the pH was determined by microscopy to be 7.4 within these regions. 32. Khalil IA, Kogwe K, Akita H, Harashima H: Uptake pathways and subsequent intracellular trafficking in non-viral gene delivery. Pharmacol Rev 2006, 58:32-45. 33. Huth US, Schubert R, Peschka-Su¨ss R: Investigating the uptake and intracellular fate of pH-sensitive liposomes by flow cytometry and spectral bio-imaging. J Controlled Release 2006, 110:490-504. 34. Barnard PJ, Berners-Price SJ: Targeting the mitochondrial cell death pathway with gold compounds. Coord Chem Rev 2007, 251:1889-1902. Current Opinion in Chemical Biology 2010, 14:238–246
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Changes in intracellular ionic zinc concentration were monitored using a zinc-selective binding moiety integrated into an emissive europium complex. The complex was injected into the observed cell. 41. Poole RA, Montgomery CP, New EJ, Congreve A, Parker D, Botta M: Identification of emissive lanthanide complexes suitable for cellular imaging that resist quenching by endogenous anti-oxidants. Org Biomol Chem 2007, 5:2055-2062. A set of 16 lanthanide complexes was examined and their susceptibility to quenching by urate and ascorbate compared to their affinity for albumin, as a model protein. 42. Bretonniere Y, Cann MJ, Parker D, Slater R: Design, synthesis and evaluation of ratiometric probes for hydrogen carbonate based on europium emission. Org Biomol Chem 2004, 2:1624-1632. A series of emissive europium complexes, absorbing at 405 nm, exhibits large changes in emission spectral form selectively with bicarbonate, with Kd values in the range 2–20 mM. These complexes only localise to the lysosomes, so far 43. Thibon A, Pierre VC: A highly selective luminescent sensor for the time-gated detection of potassium. J Am Chem Soc 2009, 131:434-435. A potassium selectivity over sodium of nearly 100 coupled with a luminescence enhancement of a factor of 25 (0–15 mM K+), augurs well for this terbium-based azaxanthone macrocyclic probe. Tuning of the affinity to the intracellular range and devising a comparable Na+ probe are obvious next steps. 44. Pal R, Parker D, Costello LC: A europium luminescence assay of lactate and citrate in biological fluids. Org Biomol Chem 2009, 7:1525-1528. Ratiometric analysis of europium (III) emission in seminal and prostatic fluid allows the measurement of citrate and lactate in microlitre samples of fluid. Changes to levels of citrate in prostatic fluid samples may provide a more accurate diagnostic marker for the onset and progression of prostrate cancer.
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Development of responsive lanthanide probes for cellular applications Elizabeth J New, David Parker, David G Smith and James W Walton Useful probes of the intracellular environment are required for a wide range of bioactive species including metal ions, oxyanions and pH. These probes need to be targeted to specific organelles (mitochondria, nucleus and lysosomes) in order to allow direct observation of the changes in these regions. Critical probe design features for luminescent lanthanide complexes are defined, together with a review of published sub-cellular localisation profiles. Cell uptake by macropinocytosis has been demonstrated for a wide range of probes and the importance of minimising perturbation of cellular homeostasis emphasised, so that cell viability, proliferation and membrane permeability are not compromised. Address Department of Chemistry, Durham University, South Road, Durham DH1 3LE, UK Corresponding author: Parker, David ([email protected])
Current Opinion in Chemical Biology 2010, 14:238–246 This review comes from a themed issue on Bioinorganic chemistry Edited by Kathy Franz and Chuan He Available online 31st October 2009 1367-5931/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2009.10.003
Introduction Non-invasive probes of the composition and structure of a cell are of key importance in enhancing the understanding of cellular processes. Amongst these, optical probes are pre-eminent as emitted light can be traced with high sensitivity and spatial resolution. The majority of optical probes are based on organic fluorophores. Responsive probes have been developed to either monitor local changes in pH or pM (M = Ca, Mg or Zn mostly) [1,2], or report on the local physical environment providing information on polarity, fluidity or geometry [3]. The majority of optical probes are best considered as cellular stains that localise to particular organelles or sub-cellular locations. The luminescence output of these stains provides information about the physical layout of the cell [4]; they report primarily structural information. Information about the function of the cell is better obtained using responsive probes, in which modulation occurs of Current Opinion in Chemical Biology 2010, 14:238–246
luminescence anisotropy, polarisation, lifetime or the ratio of emission intensity for one or more pairs of spectral bands. These parameters are independent of probe concentration and avoid problems associated with measurements of emission intensity. These include perturbations induced by changes in local probe concentration, pH, temperature, light scattering or the extent of photobleaching or local quenching. It is important to recognise that organelle-specific targeting remains a major challenge in biology and medicine [5]. Delivery of a novel therapeutic agent to its target organelle may reduce toxic side effects and enhance efficacy. For example, gene therapy drugs target nuclear DNA, whilst several anti-cancer agents exert their effects in mitochondria. From an imaging perspective, certain conditions within a specific organelle can characterise the onset or development of a particular disease. Several lysosomal storage diseases are characterised by elevated lysosomal pH whilst cytosolic pH is only slightly perturbed [6]. Similarly, an increase in mitochondrial levels of calcium, but not the total intracellular calcium concentration, has been implicated in mitochondrial dysfunction accompanying ischaemia and ageing [7]. Many other characteristic changes in cellular composition within a given organelle await discovery, because there are no probes available to report these changes selectively. Luminescent metal complexes have arisen as alternative probes to low MW organic dyes or recombinant fluorescent proteins. Two main classes may be distinguished. The first comprises d6 or d8 transition metal complexes, often based on Ru(II), Os(II), Re(I), Ir(III) and Pt(II). These involve highly conjugated ligands giving rise to intense charge transfer bands. The LMCT or MLCT excited states are relatively long-lived (10 7 to 10 4 s) allowing time-gated methods to be used to reduce background noise [8–15]. So far, these systems function primarily as cellular stains. The second set of complexes is based on luminescent lanthanide (III) complexes, for which a larger number and broader range of responsive systems has been defined [16,17,18,19,20]. Responsive probes based on the modulation of lanthanide emission intensity, lifetime or polarisation have been frequently reported over the past decade. However, these systems are rarely adapted to the constraints imposed by the complex intracellular environment. In this review, we examine these issues and highlight the critical probe design features that must be addressed in developing a practicable intracellular probe. www.sciencedirect.com
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Critical probe design features There are four key issues to consider in devising an emissive probe suitable for use in living cells. First, the probe must be able to cross the outer lipid membrane at a relatively rapid rate, preferably with a slow rate of egress. Uptake should occur by a comprehensible mechanism [21]. In addition, it should exhibit a sub-cellular localisation profile that is amenable to microscopic observation. Second, the probe must localise preferentially to a particular organelle, preferably by a transport mechanism that is understood or can be rationalised. Third, the probe should not unduly perturb cellular homeostasis. A non-invasive probe should neither inhibit cellular viability nor proliferation (often defined by an IC50 value) nor alter the mitochondrial membrane potential (MMP). Its presence should also not alter the cell’s intrinsic permeability. Several literature reports purporting to herald a selective localisation profile may be ascribed to induction of enhanced membrane permeability, caused by the presence of the probe in the cellular incubation medium [11,13,21,22]. Finally, probe integrity and performance in cellulo needs to be considered. The probe should resist enzymatic degradation and minimise the problems associated with photobleaching or photofading that may occur following excitation. It should be addressed at an accessible wavelength using lasers or powerful LEDs (e.g. 355 nm, 365 nm and 405 nm) or using two-photon excitation in the near-IR optical window 700–820 nm that best permeates cells and tissues [23,24]. The probe must also resist excited-state quenching by species that diminish its brightness [25,26]. These range from endogenous reducing agents (urate, ascorbate and glutathione) to the proteins that are involved in intracellular trafficking. These include tubulin or actin, the latter present at concentrations of the order of 0.1 mM, or those that are abundant in the organelle observed, for example the protein-dense regions of the nucleoli and ribosomes. The timescales for intracellular probe trafficking and for image data acquisition also need to be compared rationally to the timescale for modulation of the biochemical variable being monitored.
Classification of probe sub-cellular localisation The factors and processes involved in compartmentalisation of exogenous species into sub-cellular organelles are not well understood. Trafficking processes require an appreciation of three main aspects: the ‘signal’ that triggers a specific localisation; the pathways by which the molecule enters an organelle and the mechanical process by which translocation occurs within the cell. A pragmatic first step towards understanding these processes is to examine the localisation behaviour of structurally homologous series of probe complexes and classify them in terms of this property, following observation of their localisation profile using luminescence microscopy. www.sciencedirect.com
Work at Durham University has generated a library of more than 70 europium or terbium (III) complexes, allowing preliminary conclusions to be made of the relationship between probe structure and localisation. Data are summarised in Figure 1. Four main classes can be defined. The complexes in the largest group are those that localise to the lysosomes. A cell typically contains about 300 lysosomes that are membrane-bound and of variable size, with a luminal pH between 4 and 5. Lysosomal localisation usually occurs by two mechanisms. If cellular uptake occurs by receptor-mediated endocytosis, the probe will reside in an endosome that ages to become a lysosomal compartment. Alternatively, if a cell recognises a species as being foreign, it is likely to be ubiquinated and transported to a lysosome for subsequent degradation. For a wide variety of luminescent lanthanide (III) probes, an endosomal/lysosomal localisation profile has been confirmed by co-staining experiments using commercially available dyes (LysoTrackerTM, Invitrogen). No obvious correlation with probe structure has been made [16,19,21]. A smaller group of complexes has been observed to localise to the mitochondria [24,27,28]. These organelles are the centres of aerobic respiration through the electron transport chain and oxidative phosphorylation. Critically, they are involved in generating ATP that is an essential energy source for many cellular processes. Co-staining experiments with MitoTracker GreenTM for europium (III) complexes revealed that certain probes were subsequently (typically >8 hours) transported to the lysosomes [27,29]. Indeed, simultaneous localisation in late endosomes and mitochondria has been observed for a series of complexes that contain an azaxanthone sensitising chromophore, linked to the central ligand ring through a pyrazole, pyridine or amide group [29]. The nucleolus is a sub-nuclear structure involved in ribosome assembly, with the highest local density of proteins in a cell. A series of europium complexes, including a metal-coordinated azathiaxanthone sensitising moiety (Figure 1), has been shown to localise to this region and the related extranuclear ribosomes: co-staining experiments with Syto-RNA confirmed this profile [22,30,31]. More recent experiments examining localisation over a wider range of probe concentrations and incubation times in different cell types suggest that this behaviour is characteristic of a cell under stress [21], in which the cell membranes become more permeable. For example, several complexes that exhibited a mitochondrial or lysosomal distribution were observed in the cell nucleoli and ribosomes following deliberate permeabilisation of the cell membrane. This can be achieved by the addition of surfactants, for example saponin or Triton (Figure 2). Control experiments involving coincubation with propidium iodide confirm this hypothesis. In the absence of added saponin, the outer cell Current Opinion in Chemical Biology 2010, 14:238–246
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Figure 1
Illustration of four distinct sub-cellular localisation patterns.
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Figure 2
Fluorescence microscopy images revealing the metal-based emission in Chinese hamster ovarian (CHO) cells, following incubation with the shown terbium complex (four hours, 50 mM) and propidium iodide (1 mM): (a) terbium emission control; (b) propidium fluorescence; (c) terbium emission after saponin treatment (15 min, 0.5 mg/mL); (d) propidium fluorescence after saponin treatment, revealing nuclear DNA [16,21].
membrane is impermeable to propidium iodide. Following treatment, propidium iodide is able to cross each barrier and its fluorescence is clearly visible in the nucleus. Thus, luminescence staining of the nucleolus may be linked to an increased permeability of the cell membranes that is induced by the presence of the probe. Other reports of the intra-nuclear localisation of an optical imaging probe need to heed this aspect. For example, the definition of DNA-staining with cationic complexes bearing an intercalating tetraazatriphenylene sensitising moiety only occurred at relatively high probe concentrations [20], which may have compromised the ‘normal’ permeability of the cell’s membranes. However, a recent discovery of the selective staining of the nucleus of cells undergoing division (Figure 1) has allowed selective visualisation of the DNA packaging and organisation that accompanies the progression from mesophase to telophase during mitosis. This was made with sub-micromolar concentrations of terbium complex and occurred within 5 min of addition of the probe to the cell incubation medium. It is possible that a broader range of complexes, with observed lysosomal or mitochondrial distributions, may also exhibit this behaviour at much lower concentrations. www.sciencedirect.com
Mechanism of cell uptake for lanthanide (III) optical probes It is important to understand the mechanism of cell uptake and subsequent trafficking for an imaging probe, in order to address the rational development of a responsive probe that can selectively localise in a target organelle. Several different cell uptake mechanisms exist, for which a series of inhibitors and promoters has been described (Figure 3) [32]. The major pathway of cell entry for low MW probes, present at low concentration in the incubation medium, is endocytosis. This typically occurs by invagination of the cell membrane and is energy dependent, so that it is suppressed at 58C. Membrane invagination may involve the formation of clathrincoated pits (suppressed by the addition of sucrose or chlorpromazine), caveolae (inhibited by the addition of nystatin or filipin) or macropinocytosis. The latter process involves cell surface ruffling, leads to the formation of large endocytotic vesicles that are irregular in shape and size and relatively ‘leaky’, allowing cycling of the contents of the macropinosome to other parts of the cell. This process is not receptor-mediated and involves the actin-dependent formation of lamellipodia — sheet-like plasma membrane extensions, supported by a web of actin filaments. It is suppressed by the addition of Current Opinion in Chemical Biology 2010, 14:238–246
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Figure 3
Schematic diagram illustrating the three endocytic pathways of macropinocytosis, clathrin-mediated endocytosis and caveolin-dependent endocytosis, in addition to the intracellular maturation of endosomes to lysosomes. Inhibitors and activators of each pathway are indicated [16,21,32].
amiloride or wortmannin (which inhibits PI3 kinase activity) and enhanced by the addition of phorbol esters or fatty acid glycerides. For each of the lanthanide optical probes discussed here, macropinocytosis has been identified as the predominant mechanism of cell uptake, confirmed by co-staining of the observed young macropinosomes with a 70 kDa fluorescein-labelled dextran [21].
Strategies for controlling or predicting subcellular localisation
bium probe has been defined, assuming that this moiety will enhance sensitivity for the peripheral benzodiazepine receptor found in mitochondria [28]. This targeting approach is further exemplified by certain peptide conjugates, containing an established sequence used to signal the trafficking of proteins. Conjugation of a nuclear localising sequence (NLS) has been hypothesised to cause the predominant nuclear localisation of Cys-GlyGly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly conjugated to a neutral Pt(II) amine complex, involving a lengthy PEG spacer [35].
Notwithstanding the paucity of knowledge of sub-cellular trafficking pathways, various strategies have emerged that strive to assist probe delivery to particular organelles. For example, encapsulation of a probe in a liposome inevitably leads to a lysosomal localisation, as the liposomes commonly are endocytosed into a vesicle that fuses with an endosome [33]. Various cationic and amphiphilic species have been found to permeate the negatively charged surface of mitochondria selectively, including lipophilic tetraguanidinium derivatives [24], the gold anti-tumour complex auranofin [34] and the simple dye rhodamine 123. Additionally, various conjugates can be considered in which a probe structural element dictates sub-cellular localisation. For example, integration of a 2chlorophenyl-1-naphthyl moiety into an emissive ter-
Amongst the family of emissive lanthanide (III) probes (Figure 1), the nature and mode of linkage of the heterocyclic or aromatic sensitising moiety dictates the facility of cell uptake and sub-cellular localisation [16,21, 27,36,37]. Furthermore, in a systematic examination of cellular behaviour of 11 derivatives of a common core probe structure, involving only variation of the nature of the sensitiser substituent [24,36], the sub-cellular localisation profile was very similar, but certain differences in behaviour were observed. These included the perturbation of the homeostasis of the cell, as revealed by measurement of cellular toxicity, modulation of the MMP and definition of certain cell-type-specific responses. An obvious conclusion from such observations
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is that non-covalent binding to particular transport proteins, including the lipophilic sensitising moiety, is important in determining sub-cellular localisation.
Perturbation of cellular homeostasis A non-invasive optical probe should not unduly perturb the structure and function of a healthy cell. It is important to perform control experiments when evaluating the utility of such a probe, examining changes to cell viability, proliferation or permeability. This is commonly assessed by monitoring changes to membrane permeability (e.g. using Trypan Blue or propidium iodide staining only compromised cells in which membrane permeability is enhanced considerably), esterase activity (e.g. using the acetomethoxy derivative of calcein, which is fluorescent
only when hydrolysed by active cytosolic enzymes) or mitochondrial redox (e.g. using tetrazolium salts such as MTT or WST-1 that assess changes to mitochondrial redox enzymes). Such methods allow measurement of IC50 values (typically in the range 1–500 mM) defining the probe concentration needed to kill 50% of the cell population. Complexes that simply localise in lysosomes/endosomes are insulated from more sensitive organelles of a cell and have IC50 values (after 24 hours incubation) in excess of 0.1 mM, considerably greater than concentrations used in practice. As noted above, the presence of certain probes may lead to enhanced membrane permeability (Figure 2) and has been associated with observation of staining of the nuclei or chromosomal DNA of a cell [21]. This may, in part, explain observations
Figure 4
Examples of cell permeable responsive emissive lanthanide probes: (upper) for singlet oxygen [39]; (centre) for bicarbonate in mitochondria; (bottom) for pH [31]. www.sciencedirect.com
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reported for the staining of nucleoli using a covalently linked Ru-bipyridyl-phenanthridine conjugate [38], or the change in localisation to an oligo-Arg-Ru-bipyridyl conjugate in HeLa cells induced by introduction of a fluorescein moiety [11]. Indeed, in the latter report, dead cells were ‘excluded’ from the quantitative work, yet loss of outer and nuclear membrane integrity is a common step in the pathway to necrotic cell death. The distinction between cell death induced by necrosis versus apoptosis has been reliably identified for certain lanthanide probes, using flow cytometric methods [24]. For probes that localise to mitochondria, the perturbation of the MMP needs to be examined and compared to IC50 values derived from analysis of mitochondrial redox enzyme activity. The lanthanide probes described in Figure 1 [27], for example, possess high IC50 values (100 mM) and do not significantly change the MMP, auguring favourably for their application as probes of this organelle.
Intracellular performance of responsive probes Very few studies have described the function of a responsive luminescent lanthanide probe within a specified cell (Figure 4) [30,31,39,40]. At best, only one such system reports non-invasively on a particular organelle [30,31]. Ultimately, this must be the goal of this research, yet this is difficult to predict from prior in vitro characterisation studies. A key issue is the sensitivity of the optical probe to excited-state quenching by endogenous species such as urate, ascorbate and glutathione, present at near millimolar concentrations in most cell types. Furthermore, the impact of non-covalent binding to endogenous macromolecules (such as proteins and nucleic acids) needs to be considered, and the extent to which this may modulate sensitivity to quenching assessed [25,27,36,37,41]. Certain lanthanide complexes have been identified, for example, which are taken into cells efficiently (intracellular concentration of the order of 50 mM, deduced by ICPMS analysis of [Ln], coupled with cell counting by flow cytometry) yet are barely visible by microscopy because they are so sensitive to quenching by urate and ascorbate [41]. A second obvious key issue is that the probe must be cell permeable and not gain entry under conditions that may perturb cellular homeostasis. The promising irreversible Eu(III) probe for singlet oxygen was shown to localise in HeLa cells, but only in the presence of a porphyrin photosensitiser that was added to the growth medium to artificially enhance local 1DO2 levels. Its presence may well have enhanced membrane permeability, leading to the observed nuclear localisation profile [39]. Similarly, a zinc probe has been demonstrated to give rise to modulation of Eu(III) luminescence in HeLa cells. The europium complex, however, was Current Opinion in Chemical Biology 2010, 14:238–246
injected directly into the observed cell and relatively weak emission observed by time-resolved microscopy [40]. The complex possessed a substituted quinoline sensitising moiety that has a long wavelength absorption band at 320 nm, but absorbs light very poorly at the excitation wavelength used (360 nm). This contrasts with the characteristics of a pH sensitive probe (Figure 4) with an azathiaxanthone sensitising moiety (lmax 385 nm) that absorbs strongly at the common laser lines of 365 nm or 405 nm and has a pKa of 7.1 under simulated intracellular conditions. This complex was shown to localise to the ribosomes/nucleoli revealing a local pH of 7.4 following ratiometric analysis of europium emission [30,31]. It is desirable to devise an analogue of this system that localises to lysosomes with a pKa of around 4.5, so that perturbations in lysosomal pH can be investigated. A final obvious issue is that the working dynamic range of the responsive probe for the target analyte should correspond to the local concentration of the species under study. Calibration studies in diluted serum solutions or in macerated cellular media offer a partial solution [42] to simulate the intracellular fluid composition.
Concluding remarks The design of luminescent lanthanide sensory systems with a tailored affinity and selectivity profile for use in vitro can now be undertaken with reasonable confidence [16,17,18]. Recently, promising systems that report on changes to species such as potassium [43], lactate and citrate [44] have been defined. Critical design features need to be engineered into the probe from the outset to permit successful use of such probes in cellulo. The most important of these relate to the need to minimally perturb the cell under observation and to target the organelle of interest, where the analyte observed is to be found. Such studies are actively being addressed and should provide new tools to uncover the role and potential diagnostic value of intracellular changes to key bioactive, low MW species.
Acknowledgements We thank EPRSC, CISbio and the Association of Commonwealth Universities for support.
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25. Kielar F, Montgomery CP, New EJ, Parker D, Poole RA, Richardson SL, Stenson PA: A mechanistic study of the dynamic quenching of the excited state of europium (III) and terbium (III) macrocyclic complexes by charge or energy transfer. Org Biomol Chem 2007, 5:2055-2062. 26. Law G-L, Parker D, Richardson SL, Wong K-L: The mechanism of quenching of the lanthanide excited state for optical probes using sensitised emission. Dalton Trans. 2009, 8481–8484. Direct laser excitation of Eu and Tb complexes allows population of the metal excited state; under these conditions the lanthanide excited state is not quenched by added urate, ascorbate or iodide. Formation of an exciplex between the sensitising moiety and the electron-rich quencher leads to dynamic quenching of metal-based emission. 27. Murray BS, New EJ, Pal R, Parker D: Critical evaluation of five emissive europium(III) complexes as optical probes: correlation of cytotoxicity, anion and protein affinity with complex structure, stability and intracellular localisation profile. Org Biomol Chem 2008, 6:2085-2094. 28. Manning HC, Smith SM, Haviland MS, Bai S, Cederquist MFK, Stella N, Bornhop DJ: A peripheral benzodiazepine receptor targeted agent for in vitro imaging and screening. Bioconjug Chem 2006, 17:735-740. 29. Montgomery CP, New EJ, Palsson LO, Parker D, Batsanov AS, Lamarque L: Emissive and cell permeable pyridyl and pyrazoyl1-azaxanthone lanthanide complexes and their behaviour in cellulo. Helv Chim Acta in press; doi: 10.1002/hlca200900122. 30. Pal R, Parker D: A single component ratiometric pH probe with long wavelength excitation of europium emission. Chem Commun 2007, 2007:474-476. 31. Pal R, Parker D: A ratiometric optical imaging probe for intracellular pH based on modulation of europium emission. Org Biomol Chem 2008, 6:1020-1033. A europium complex localises to the nucleoli and ribosomes within mouse skin cells and the pH was determined by microscopy to be 7.4 within these regions. 32. Khalil IA, Kogwe K, Akita H, Harashima H: Uptake pathways and subsequent intracellular trafficking in non-viral gene delivery. Pharmacol Rev 2006, 58:32-45. 33. Huth US, Schubert R, Peschka-Su¨ss R: Investigating the uptake and intracellular fate of pH-sensitive liposomes by flow cytometry and spectral bio-imaging. J Controlled Release 2006, 110:490-504. 34. Barnard PJ, Berners-Price SJ: Targeting the mitochondrial cell death pathway with gold compounds. Coord Chem Rev 2007, 251:1889-1902. Current Opinion in Chemical Biology 2010, 14:238–246
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35. Aronov O, Horowitz AT, Gabizon A, Fuertes MA, Pe´rez JM, Gibson D: Nuclear localization signal-targeted poly(ethylene glycol) conjugates as potential carriers and nuclear localizing agents for carboplatin analogues. Bioconjug Chem 2004, 15:814-823. 36. Kielar F, Law G-L, New EJ, Parker D: The nature of the sensitiser substituent determines quenching sensitivity and protein affinity and influences the design of emissive lanthanide complexes as optical probes for intracellular use. Org Biomol Chem 2008, 6:2256-2258. 37. New EJ, Parker D, Peacock RD: Comparative study of the constitution and chiroptical properties of emissive lanthanide complexes with a common tetraazatriphenylene sensitiser: the nature of the sensitiser determines quenching sensitivity and cellular uptake. Dalton Trans 2009, 2009:672-679. 38. O’Connor NA, Stevens N, Samaroo D, Solomon MR, Marti AA, Dyer J, Vishwasrao H, Atkins DL, Kandel ER, Turro NJ: A covalently linked phenanthridine–ruthenium(II) complex as a RNA probe. Chem Commun 2009:2640-2642. The title complex exhibits enhanced metal-based emission in the presence of RNA in vitro and shows a localisation to the nucleoli of certain breast cancer cells that could also be explained by the enhancement of membrane permeability induced by the presence of the complex 39. Song B, Wang G, Tan M, Yuan J: A europium(III) complex as an efficient singlet oxygen luminescence probe. J Am Chem Soc 2006, 128:13442-13450. HeLa cells were incubated with an 0.4 mM solution containing the singlet oxygen sensitive complex but cell uptake was only observed in the presence of a porphyrin photosensitiser (10 mM), used to generate singlet O2 in situ. The nuclear localisation profile suggests that the presence of the porphyrin enhances cellular permeability: no toxicity studies were reported. 40. Hanaoka K, Kikuchi K, Kobayashi S, Nagano T: Time-resolved long-lived luminescence imaging method employing luminescent lanthanide probes with a new microscopy system. J Am Chem Soc 2007, 129:13502-13509.
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Changes in intracellular ionic zinc concentration were monitored using a zinc-selective binding moiety integrated into an emissive europium complex. The complex was injected into the observed cell. 41. Poole RA, Montgomery CP, New EJ, Congreve A, Parker D, Botta M: Identification of emissive lanthanide complexes suitable for cellular imaging that resist quenching by endogenous anti-oxidants. Org Biomol Chem 2007, 5:2055-2062. A set of 16 lanthanide complexes was examined and their susceptibility to quenching by urate and ascorbate compared to their affinity for albumin, as a model protein. 42. Bretonniere Y, Cann MJ, Parker D, Slater R: Design, synthesis and evaluation of ratiometric probes for hydrogen carbonate based on europium emission. Org Biomol Chem 2004, 2:1624-1632. A series of emissive europium complexes, absorbing at 405 nm, exhibits large changes in emission spectral form selectively with bicarbonate, with Kd values in the range 2–20 mM. These complexes only localise to the lysosomes, so far 43. Thibon A, Pierre VC: A highly selective luminescent sensor for the time-gated detection of potassium. J Am Chem Soc 2009, 131:434-435. A potassium selectivity over sodium of nearly 100 coupled with a luminescence enhancement of a factor of 25 (0–15 mM K+), augurs well for this terbium-based azaxanthone macrocyclic probe. Tuning of the affinity to the intracellular range and devising a comparable Na+ probe are obvious next steps. 44. Pal R, Parker D, Costello LC: A europium luminescence assay of lactate and citrate in biological fluids. Org Biomol Chem 2009, 7:1525-1528. Ratiometric analysis of europium (III) emission in seminal and prostatic fluid allows the measurement of citrate and lactate in microlitre samples of fluid. Changes to levels of citrate in prostatic fluid samples may provide a more accurate diagnostic marker for the onset and progression of prostrate cancer.
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