METHOD AND PROBE FOR MONITORING OXYGEN STATUS IN LIVE MAMMALIAN CELLS

Abstract

A method of determining oxygen concentration, metabolic activity, and/or the effects of a test substance on the metabolic activity of a live cell sample by photoluminescence quenching technique employing a photoluminescent probe that self-loads sans any loading reagent into the cells of the cell sample. The probe comprises a plurality of polymeric particles each comprising an amphiphilic cationic polymer matrix having a hydrophobic core and a hydrophilic positively charged surface provided by quaternary amino groups, and a hydrophobic oxygen-sensitive photoluminescent dye embedded in the hydrophobic core.

Claims

1. A method of determining oxygen concentration in a live mammalian cell sample by photoluminescence quenching technique, which method employs a photoluminescent probe, the method comprising the steps of: incubating the live mammalian cell sample in a suitable growth medium with the photoluminescent probe whereby the probe self-loads sans any loading reagent into the cells of the mammalian cell sample; detecting a photoluminescent signal of the probe from the probe-loaded cell sample; and correlating the detected photoluminescent signal to local oxygen concentration within the cell sample, wherein the photoluminescent probe is a plurality of polymeric particles each comprising an amphiphilic cationic polymer matrix having a hydrophobic core and a hydrophilic positively charged surface provided by quaternary amino groups, and a hydrophobic oxygen-sensitive photoluminescent dye embedded in the hydrophobic core.

2. A method as claimed in claim 1 in which the photoluminescent probe is used in the form of an aqueous suspension at a working concentration of 1 to 10 g/ml.

3. A method as claimed in claim 1 in which the amphiphilic cationic polymer matrix comprises a co-polymer of poly (ethylacrylate, methyl-metacrylate and chloro trimethyl-ammoniethyl methacrylate) containing quaternary ammonium groups.

4. A method as claimed in claim 1 in which the photoluminescent probe has low intrinsic toxicity on the cells.

5. A method as claimed in claim 1 in which the hydrophobic oxygen sensitive photoluminescent dye is selected from Pt-porphyrin, Pd-porphyrin, Pt-porphyrin-ketone or Pd-porphyrin-ketone, Pt-benzoporphyrin or Pd-benzoporphyrin, cyclometallated complex of Ir.sup.3+, Os.sup.2+ or Ru.sup.2+, or close analogs or derivatives of these dyes.

6. A method as claimed in claim 1 in which the hydrophobic oxygen sensitive photoluminescent dye is Pt-tetrakis(pentafluorophenyl) porphine (PtPFPP) dye.

7. A method as claimed in claim 1, in which the photoluminescent probe is capable of 1.5 to 15 fold quenching at ambient oxygen concentration of 21 kPa or 250 M O.sub.2.

8. A method as claimed in claim 1 in which from 0.1% to 3.0% (w/w) of the photoluminescent probe is oxygen sensitive photoluminescent dye.

9. A method as claimed in claim 1, in which the cell sample is washed prior to measurement of the photoluminescent signal to remove extracellular probe.

10. A method according to claim 1 which is used to monitor oxygen in cell populations.

11. A method according to claim 1 which is used to monitor oxygen in individual cells.

12. A method according to claim 1, wherein the photoluminescent signal is detected by time-resolved fluorometry in the microsecond domain.

13. A method according to claim 1, wherein the photoluminescent signal is detected by fluorescence microscopy imaging technique.

14. A method according to claim 1, wherein detecting the photoluminescent signal includes measuring the photoluminescence lifetime of the photoluminescent signal or a parameter related to it.

15. A method according to claim 1, wherein the photoluminescent signal is detected using ratiometric intensity based oxygen sensing or imaging and the photoluminescent probe further contains an oxygen-insensitive dye which is used as a reference or as part of a FRET pair.

16. A method of determining the metabolic activity of a cell or a cell population, which method comprises monitoring oxygen concentration of the cell or cell population by: incubating the cell or cell population in a suitable growth medium with a photoluminescent probe whereby the probe self-loads sans any loading reagent into the cell or cells of the cell population detecting a photoluminescent signal of the probe from the probe-loaded cell or cell population; correlating the detected photoluminescent signal to local oxygen concentration within the cell or cell population, and correlating the measured oxygen concentration, or changes in the oxygen concentration, of the cell or cell population to metabolic activity of the cell or cell population, wherein the photoluminescent probe is a plurality of polymeric particles each comprising an amphiphilic cationic polymer matrix having a hydrophobic core and a hydrophilic positively charged surface provided by quaternary amino groups, and a hydrophobic oxygen-sensitive photoluminescent dye embedded in the hydrophobic core.

17. A method of determining the effects of a test substance on the metabolic activity of a cell or cell population, the method comprising stimulating a cell or cell population with the test substance and determining the metabolic activity of the cell or cell population by: incubating the cell or cell population in a suitable growth medium with a photoluminescent probe whereby the probe self-loads sans any loading reagent into the cell or cells of the cell population; detecting a photoluminescent signal of the probe from the probe-loaded cell or cell population using ratiometric intensity based oxygen sensing or imaging; correlating the detected photoluminescent signal to local oxygen concentration within the cell or cell population, and correlating the measured oxygen concentration, or changes in the oxygen concentration, of the cell or cell population to metabolic activity of the cell or cell population, wherein the photoluminescent probe contains an oxygen-insensitive dye which is used as a reference or as part of a FRET pair, the photoluminescent probe being a plurality of polymeric particles each comprising an amphiphilic cationic polymer matrix having a hydrophobic core and a hydrophilic positively charged surface provided by quaternary amino groups, and a hydrophobic oxygen-sensitive photoluminescent dye embedded in the hydrophobic core.

Description

BRIEF DESCRIPTION OF FIGURES

[0045] The invention will be more clearly understood from the following description of some embodiments thereof, given by example only, and with reference to accompanying figures in which:

[0046] FIG. 1. Loading of the NP probe in MEF cells in (control24 h).

[0047] FIG. 2. Effect of serum content on probe loading in HCT116 cells.

[0048] FIG. 3. Signal stability measured in HCT116 cells. Increase in intensity due to cell growth.

[0049] FIG. 4.A Toxicity of the NP probe (PtPFPP) in MEF cells.

[0050] FIG. 4.B RL100-PtTFPP concentration dependent signal in HCT116 cells loaded for 14 h.

[0051] FIG. 5. Probe calibrations in different cell lines measured by time-resolved fluorometry.

[0052] FIG. 6. Metabolic responses of MEF cells to different concentrations of the mitochondrial uncoupler (FCCP), measured with the PtPFPP-RL100 probe (10 ug/ml, 12 h loading, DMEM).

DETAILED DESCRIPTION OF THE INVENTION

[0053] The probe of the invention comprises a composition of an amphyphilic cationic polymer and a hydrophobic oxygen-sensitive dye, which is specially processed to produce a suspension of particles having pre-defined characteristics. In particular, the particles have the size in the region of 20-100 nanometers, i.e. much smaller than the size of a typical mammalian cell (10 microns), typically relatively narrow size distribution, and characteristic core-shell architecture. The hydrophobic core of the nanoparticles is used for impregnating them with hydrophobic oxygen-sensitive dyes (or a combination of several dyes to allow ratiometric O.sub.2 sensing), while the hydrophilic shell bears a number of cationic groups (preferably as part of the polymer backbone) on the surface providing strong interaction with the surface of mammalian cells which facilitates efficient loading with the probe.

[0054] Examples of polymers that can be used in the invention include poly (ethylacrylate, methyl-methacrylate, and chloro trimethyl-ammonioethyl methacrylate) copolymers having quaternary cationic groups. Examples of such copolymers include Eudragit RL-100 and RS-100 polymers produced by Degussa/Evonic Industries. Thus, Eudragit RL100 is a copolymer of poly (ethylacrylate, methyl-methacrylate, and chloro trimethyl-ammonioethyl methacrylate) containing between 8.8% and 12% of quaternary ammonium groups, M.W. is approximately 150,000 D. Eudragit RS-100 polymer has a similar chemical composition, but contains less quaternary ammonium groups. Some other polymers can also be used, for example sol-gel nanoparticles with the indicator dye embedded inside and the surface modified with quaternary cationic (i.e. ammonium) groups. These polymers can be easily processed to produce the nanoparticle probe of the invention. One such method involves dissolving the polymer and the oxygen-sensitive dye in a medium polarity solvent (e.g. acetone) and subsequent dilution of this solution with water followed by the removal of acetone (Example 1). This method has been successfully applied to produce the RL-100 nanoparticles impregnated with different O.sub.2-sensitive dyes, including platinum(II) complexes with octaethylporphyrin (PtOEP), meso-tetra(pentafluorophenyl)porphyrin (PtPFPP), octaethylporphyrin ketone (PtOEPK) and various benzoporphyrins. The resulting nanoparticles had the size of approximately 70 nm, Z-potential of +58 mV, and showed bright phosphorescence in aqueous solution which was quenched by oxygen. Impregnation of RL-100 nanoparticles with perylene and some other hydrophobic fluorescent dyes was also conducted, to mimic the corresponding O.sub.2 probes in cell loading and probe localisation studies.

[0055] Furthermore, it is demonstrated herein that the oxygen probes having the above characteristic features are highly efficient in loading various mammalian cells by passive means. In one aspect, loading is achieved by simply incubating the cells with the probe under standard culturing conditions (i.e. growth medium, CO.sup.2 atmosphere, additives, 37 C.). After the exposure to a relatively low probe concentration (1-10 g/ml), relatively high loading of the cells is achieved in 1-6 h, and after 12-24 h the cells produce very high phosphorescent signals. Furthermore, the probes of the invention load efficiently in different cells types (both adherent and suspension), and after loading they remain inside the cell over long periods without significant leaching, and work very reliably and reproducibly. Unlike many other cell-penetrating probes, loading with these probes is not blocked by high serum content in the medium. Such simple and efficient loading of cells and high brightness of the probe make it easy to measure intracellular oxygen by phosphorescence quenching and conduct various physiological experiments with mammalian cells and tissue.

[0056] For the latter application, the preferred probe of the invention comprises Eudragit RL-100 nanoparticles impregnated with PtPFPP dye (suitably 1% w/w), and typically diluted to a working concentration of 1-10 g/ml in an aqueous solvent. Such probe provides excellent brightness and photostability under intense illumination (important for imaging applications), optimal sensitivity to oxygen, long emission lifetime and convenient spectral properties. It is well compatible with both PMT-based detectors and imaging devices (fluorescent microscopes), and allows phosphorescence lifetime based sensing of oxygen on standard fluorometers, time-resolved fluorescence readers or more sophisticated fluorescence lifetime imaging (FLIM) systems. This probe IS easy to produce and it can be stored over long periods of time without deterioration.

[0057] For the sensing of intracellular oxygen and physiological experiments with cells it is important to control the intracellular location of the probe and avoid possible effects of the probe (and assay) on cellular function. Many existing probes suffer from undefined or uncontrolled sub-cellular localisation, transport to undesirable compartments such as cell nucleus, significant intrinsic toxicity and interference with cellular function. We demonstrate in this invention that the self-loading nanoparticle probes show similar localisation pattern in different cell types and low intrinsic toxicity. Thus, in MEF cells the probe penetrates inside, but does not go into the nucleus and localises close to it. Probe localisation pattern resembles that of periplasmic vesicles.

[0058] Another aspect of the invention is the method of measuring cellular oxygen concentration using the above probes. The method involves growing test cells, exposing them to the probe for a reasonable period of time under normal culturing conditions to achieve efficient cell loading by passive means. After that the cells are generally normally washed with medium and subject to fluorescence/phosphorescence measurements. The latter is generally conducted under constant temperature (37 C.) and external gas composition (normoxia21% O.sub.2 or hypoxiareduced atmospheric p O.sub.2) by luminescence intensity, lifetime or phase measurements. Measured phosphorescent signal (or signal profile) is converted into O.sub.2 concentration using pre-determined calibration of the intracellular probe. The new nanoparticle probes make the assay simple and accurate. The core-shell structure also ideally shield the O.sub.2-sensitive dye from interfering specie that may occur in the sample.

[0059] In the preferred embodiment the phosphorescent signal from the cells loaded with probe is measured on a sensitive fluorescent reader which supports time-resolved fluorescence or lifetime measurement mode. This allows simple quantification of local oxygen levels within a monolayer of adherent cells or a slice of respiring tissue (i.e. cell population studies), and real-time monitoring of changes in cell respiration and metabolic activity. The sample can also be analysed on a fluorescent microscope to analyse O.sub.2 levels in individual cells or sections of a samples.

[0060] Another embodiment describes the method of invention in which the intracellular probe contains an additional fluorescent dye which produces a distinct emission. In this case, the O.sub.2 sensitive dye produces a photoluminescence intensity signal which is dependent on O.sub.2 concentration, whereas the second dye emission is O.sub.2-insensitive. Such probe and method allow monitoring of cell oxygenation and metabolic responses by ratiometric intensity measurements. The mode is simpler and more common than phosphorescence lifetime measurements, and supported by many standard microscopes and spectrometers. Examples of such pairs of dyes include PtPFPP and a perylene dye, PtPFPP and a coumarin dye.

[0061] The method of invention using the new nanoparticle probes can be used to study the effects of drugs and various stimuli on cell metabolism. This is conducted by preparing the cells, loading them with the nanoparticle probe, washing and equilibrating in the desired medium, temperature and gas composition. Under these conditions probe signal is recorded which corresponds to the respiratory activity of resting cells (basal oxygen concentration). After that the cells are stimulated with a drug or effector added to the sample, and changes in probe signal are recorded. The resulting changes in probe signal, if occur, are indicative to the changes in cell metabolism. Thus, increased probe signal (intensity or lifetime) is indicative to increased respiration activity which lowers intracellular O.sub.2 concentration, while decreased probe signal reflects inhibition of respiration which brings cellular O.sub.2 levels closer to those of bulk medium. In this application, it is very important to ensure partial deoxygenation of cell microenvironment due to respiration. This can be achieved by optimising the density/numbers, respiratory activity of test cells under resting conditions and/or external O.sub.2 levels (hypoxia). Contribution of diffusion processes which influence the shape of the phosphorescent signal (Zhdanov et al.Integr. Biol., 2010) should be considered.

[0062] Overall, compared to the existing conventional O.sub.2 probes, including the molecular probes with self-loading capabilities, the nanoparticle O.sub.2 probes of the invention have multiple advantages. They are relatively large in size and can be heavily loaded with the dye (up to 3% w/w), thus providing high brightness. The production and impregnation of the probe are simple and allow the use of oxygen-sensitive dyes with different spectral characteristics, chemical structures, including the highly photostable dyes such as PtPFPP. These probes load the cells passively, under mild conditions, with high efficiency and speed, low cell-specificity and minimal impact on cellular function. They have a defined sub-cellular localisation and low intrinsic cyto-, geno- and photo-toxicity. The probes provide simple real-time monitoring of cellular O.sub.2 concentration in adherent and suspension cells and responses to stimulation. Large cell populations (monolayers, tissue slices) and individual cells can be analysed.

[0063] The invention is demonstrated with the following non-limiting examples.

Example 1. Fabrication of the Nanoparticle O.SUB.2 .Probe

[0064] 1.5 g of RL-100 polymer and 22.5 mg of PtTFPP were dissolved in 750 g of acetone. The solution was placed in a SL beaker to which 4 L of deionized water were added over 20 s under rigorous stirring. Acetone was subsequently removed under reduced pressure and aqueous dispersion of the beads was further concentrated to approximately 75 mL. Traces of aggregates were removed by filtration through a paper filter. The resulting solution was filtered through a sterile filter to produce stock of O.sub.2 probe for intracellular use. It was aliquoted and stored in a dark place at +4 C. for several months for further use.

Example 2. Assessment and Optimisation of Cell Loading

[0065] Mouse embryonic fibroblast (MEF) cells were cultured in 96-well plates using DMEM medium supplemented with 10% of foetal calf serum, CO.sub.2 incubator at 37 C., until they reach high confluence (periodically changing medium, if required). Stock solution of the PtPFPP-RL100 probe was diluted to the desired concentration with appropriate growth medium (determined by the cells and culturing conditions) and added to the wells with growing cells at the required final concentration (usually 1-10 g/ml). Cells were incubated with probe, typically for 6-24 h, and then washed two times with fresh medium. Similar experiments were conducted with other cells, probe concentrations and incubation times. The efficiency of cell loading was assessed by measuring phosphorescence intensity signals in each well on a fluorescent reader Victor2 (Perkin Elmer) in time-resolved mode under the following settings: excitation340 nm, emission642 nm, delay time30 us, gate time100 us, number of flashes1000, temperature37 C. The plate with loaded cells and necessary controls (wells without probe or without cells) was pre-incubated for15-20 min (to achieve temperature and gas equilibration), and then measured repetitively over 20-30 min taking reading every 2 min.

[0066] Similar experiments were conducted on loading the cells with RL100-perylene fluorescent nanoparticles. After incubation with the probe, loaded adherent cells were trypsinised and analysed by flow-cytometry using 488 nm laser and 560 nm emission filter.

[0067] FIG. 1 shows that the PtPFPP-RL100 probe provides high and rapid loading, acceptable threshold levels of TR-F intensity signal (30,000 cps) which allow reliable lifetime based sensing of O.sub.2 were achieved even after 1 h. RL100-perylene probe also shows a similar loading pattern as the PtPFPP-RL100 probe (measured by flow cytometry). Loading in media with high serum content remains pretty good (FIG. 2), and signal intensity remains high over 72 h for PtPFPP-RL100 probe loaded HCT116 cells(FIG. 3). Therefore, the probe can be used in long-term experiments with cells.

Example 3. Analysis of Probe Cytotoxicity and Localisation

[0068] MEF, PC12, HepG2 and HCT116 cells were loaded with 10 g/ml of PtPFPP-RL100 probe as described in Example 2. Probe cytotoxicity was assessed by measuring total ATP levels in loaded cells and comparing them with unloaded cells. FIG. 4A shows that the probe has low intrinsic toxicity, particularly when used at low concentrations (10 g/ml) and short loading times (<18 h). FIG. 4B shows the concentration dependence of specific signal from HCT116 cells loaded with R1100-PtTFPP for 14 h and background (non-specific binding to plate). Concentrations higher than 25 ug/ml had a toxic effect on cells.

Example 4. Calibration of the Nanoparticle Probe for the Quantification of Intracellular O.SUB.2 .Concentration

[0069] MEF, PC12, HepG2 and HCT116 cells were grown in standard 96-well plates in growth medium containing 10% FBS, and then loaded with PtPFPP-RL100 probe as described in Example 2 (10 g/ml, 12 h). TR-F reader Victor3 on which the measurements were carried out was placed in a hypoxia chamber (Coy Scientific) pre-set at constant p0.sub.2 (ranging between 0 and 21%). Antimycin A was added to the wells with loaded cells (to block their respiration and formation of local O.sub.2 gradients in the wells), and the plate was then placed in the reader compartment. After a period of temperature and gas equilibration (20-40 min) the plate was measured repetitively over a period of 20-40 min, taking TR-F intensity values at two different delay times (30 us and 70 us, respectively) which were used to calculate the phosphorescence lifetime. After the measurements were completed, the hypoxia chamber was reset to a new pO.sub.2 value and the cycle (equilibration and measurement) was repeated. Kinetic profiles of phosphorescence lifetime of the probe were processed to determine average values at different external pO.sub.2. These values were used to construct probe calibration which is shown for MEF and PC12 in FIG. 5. This calibration can be used to determine O.sub.2 levels in respiring cells, both at rest and upon stimulation.

Example 5. Monitoring Cellular Responses to Metabolic Stimulation

[0070] MEF cells were cultured in a standard 96-well plate, (typically 24-36 wells, 20,000 cells/well) in DMEM medium, loaded with the nanoparticle O.sub.2 probe as described (10 ug/ml, 12 hours), washed 2 times and covered with 90 uL of medium. The plate was then measured on a Victor 2 reader as described in Example 4. Initially it was monitored for 30 min to reach O.sub.2 and temperature equilibrium and obtain basal signals, then quickly withdrawn from the reader, compounds were added to the cells (10 ul of 10 stock solution in each well) and monitoring was resumed. Measured TR-F intensity signals for each sample well were converted into phosphorescence lifetime (t) values and plotted. Control samples and repetitive additions were incorporated as appropriate. FIG. 6 shows the responses to stimulation with different concentrations of uncoupler FCCP (added at 30 min, which is known to increase cellular respiration. Increases in probe lifetime reflect (in real time) the reductions in cellular O.sub.2 levels due to increased respiration.

[0071] The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.