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ABSTRACT For detection of biological events in vitro, sensors using 129Xe NMR of trapped hyperpolarized noble gas can become a way provided the approach can cross the gap in sensitivity required. Here constructs based on the non selective grafting of cryptophane precursors on a protein interacting with cell surface receptors are proposed. The study of their interaction with cell suspensions via fluorescence microscopy and 129Xe NMR indicates interesting features such as a lack
KEYWORDS. Hyperpolarization ; Xenon ; Biosensor ; cell surface receptors.
Working on live cell suspensions through NMR is not an easy task, as the required number of cells per volume unit limits their lifetime. A number of intracellular biological events could however be studied if the sensitivity of the analysis method could be raised.
Hyperpolarized xenon is now widely recognized as a powerful MR sensor of molecular or biological events, due not only to the huge signal obtained by the prior optical pumping or dynamic nuclear polarization step, but also to the wide chemical shift range spanned by this nucleus having a big deformable electron cloud. In 2001, an approach where the hyperpolarized noble gas is vectorized toward biological receptors via functionalized host systems was proposed by the group of Alexander Pines.1 When encapsulated in the core of such transporters made by cage-molecules2 or nanoparticles,3 xenon takes a specific chemical shift, which enables its discrimination from free xenon in spectroscopic imaging.4 Whereas the proof-of-concept of this approach has been successfully achieved in isotropic solution by several groups,5-9 it has never been tested in vitro (and furthermore in vivo) by performing hyperpolarized 129Xe NMR experiments directly with cell suspensions.
Here we detect the interaction of a 129Xe NMR-based biosensor with transferrin receptors (TfR) via hyperpolarized 129Xe NMR on cell suspensions. This system has been chosen for the following reasons: i) it is a well described system : indeed Transferrin (Tf), a naturally existing protein, has already received considerable attention in the area of drug targeting since it is biodegradable, non-toxic, and nonimmunogenic,10 ii) the biological targets are cell surface receptors, thus easily reachable by dissolved xenon, iii) they can be abundant (till 105) for some cell lines, iv) endocytosis is susceptible to further increase the local density of xenon receptors. The affinity of holo-transferrin for the transferrin receptor being 800 times larger than this of apo-transferrin, it seemed to us important to work with the former, despite the relaxation induced on xenon by the paramagnetism. But if the situation had become too critical (too short lifetime of the hyperpolarization), iron atoms could have been replaced by gallium or indium atoms for a similar affinity.ref
RESULTS AND DISCUSSION
Conception and synthesis of the TfR sensors
It is known that the molecular systems for which xenon exhibits the highest affinity are cryptophanes, aromatic cage-molecules made of two cyclotriveratrylene bowls joigned by aliphatic linkers.ref Brotin, Dutasta, Chem Rev Consequently they will constitute the core of our 129Xe NMR-based biosensor. The strategy we have chosen consists in firstly building precursors where a tether constituted by a polyethylene spacer ended by a succinidimic group is grafted on the cryptophane (see Materials and Methods). The choice of four OCH2 groups to constitute the spacer gives flexibility to the cryptophane moiety, which is expected to be sufficient to avoid broad lines for encapsulated xenon that would be due to too fast transverse relaxation.11 This spacer is also expected to be long enough to avoid multiplicity of the signals of encapsulated xenon for the racemic mixture.
The succinimidic group belonging to the amine reactive derivatives reacts to the amine groups of the lysine sidechains in a non specific coupling.ref Such a construction presents many advantages: i) the cryptophane-PEG-succinimide can be a precursor for many biosensors, i. e. it can be non-specifically grafted on a protein provided that this protein exhibits lysine residues on its external side (and far from its active site), ii) several cryptophane cores can be grafted on a protein, multiplying the sites for xenon and therefore possibly increasing the signal-to-noise ratio, iii) under a certain number of cryptophanes grafted on the protein it is expected that the affinity of the assembly for the biological receptors is maintained. Indeed the grafting of a small entity on a big protein is a priori less susceptible to modify its affinity for these receptors.
Transferrin is a globular protein of molecular weight ~80,000. It possesses 54 Lys residues, 23 of them located on its external surface, and its holo form contains two iron atoms (insert of Figure 1). Labeling of the protein with the cryptophane precursor was made using the procedure detailed in Materials and Methods. In first step different stoichiometries of the cryptophane : Tf mixtures were prepared in phosphate buffer saline (PBS). Preservation of the protein folding was tested by 1H NMR. With simple one-dimensional spectra (necessarily acquired during several minutes on a high-field spectrometer equipped with a cryocooled probehead), it was observed that a maximum of ten cryptophanes could be tethered on the transferrin. Beyond this value, the protein unfolds. Obviously there is a statistical distribution of the number of cryptophanes and of their locations on the protein. However the mass spectrometry indicated a narrow distribution of the number of grafted cryptophanes.
Finally we have chosen to use two constructs. In the first one, a 129Xe NMR-based biosensor made with a ratio of five cryptophanes per protein was built (biosensor B1). In the second one, a ratio of 2:2:1 was used for the cryptophane, fluorescent probe and protein, respectively (biosensor B2). Proton translational diffusion experiments completed the mass spectrometry to check that the cryptophanes were covalently linked and not trapped inside anfractuosities of the protein.
Figure 1 displays the xenon spectra obtained at 11.7 T and xxxxK with B1 at 1.3 ÂµM in water. The first observation is that the Xe@B1 line is unique and not broad. This constitutes a confirmation that the spacer between the cryptophane part and the protein is long and flexible enough. The detection threshold per time unit of the laser-polarized 129Xe NMR experiments with such constructs containing paramagnetic Fe3+ ions could be estimated. Given that the signal-to-noise ratio is 6.6 on the sub-spectrum obtained by repetitive selective excitation of the Xe@B1 signal (direct observation method [ref]) and that this experiment can be repeated at least 8 times with the gaseous xenon reservoir in the NMR tube on top of the solution simply by shaking, this leads to a detection threshold on the order of 100 nM. NON, A VIRER
Also, we have compared the 129Xe longitudinal relaxation rates encountered with similar concentrations of apo- and holo-transferrin. Obviously fast exchange conditions on the T1 timescale are encountered, and a calculation taking into account the fraction of encapsulated xenon has been done. The extracted values are xxx and xxx for the apo- and holo- forms, respectively.
Study of their interaction with biological cells
On a cell suspension, according to our procedure including shaking of the NMR tube as a way to introduce fresh hyperpolarized xenon into solution, it is difficult to envisage a direct MR contrast. Furthermore even if on the 129Xe NMR spectrum a tiny modification of the cryptophane environment is susceptible to give rise to a chemical shift variation of the Xe@biosensor signal,[refs] here the line broadening due to the presence of a high number of biological cells in suspension renders improbable the frequency separation between the signal of xenon caged in the biosensor free in solution (Xe@B_aq) and this of xenon in the biosensor in the cytoplasm (Xe@B_intracell). However Schröder and co-workers have shown that a chemical shift difference of up to xxxx ppm can occur between xenon in the cryptophane in the aqueous phase and xenon in the cryptophane in a lipidic phase.[ref]
These difficulties led us to imagine the following 'batch' procedure, which synopsis is represented in Table 1:
a) in a first step, 120 millions K562 cells have been incubated during one hour at 37Â°C with 400 ÂµL of a 5 ÂµM solution of B2 in PBS. Then the cells and the supernatant (after three washings) have been separated by centrifugation (details in Materials and Methods). This has given rise to two solutions: ca (cells a) and sa (supernatant a).
b) in parallel, 120 millions K562 have been treated with pronase, a mixture of proteolytic enzymes designed to suppress the transferrin receptors. Exactly the same procedure than in a) has then been applied: incubation during one hour at 37Â°C with 400 ÂµL of a solution of B2 in PBS (final concentration 5 ÂµM). The separation between the cells and the supernatant gave rise to solutions cb and sb, respectively. Pairwise comparison of the cell samples ca and cb through fluorescence microscopy and hyperpolarized 129Xe NMR could then be performed, as well as comparison between the supernatant samples sa and sb.refx As exchange of the transferrin biosensor between the intra- and extra-cellular compartments is driven by thermodynamics laws, xxxx
Figure 2 displays the fluorescence microscopy results. After incubation of the cells with B2, the internalization of the biosensor is clearly visible. The observation of fluorescent aggregates inside the cells could be due to the formation of vacuoles. Also the border of some cells appears green, signifying that a part of the biosensor lies in their plasmic membrane.
Figure 3 gives the corresponding 55-95 ppm region of the laser-polarized 129Xe NMR spectra, plus these of the supernatant samples. This region exclusively corresponds to the signal of the noble gas encapsulated in the cryptophane. The first remark is that no peak in this region appears for the cells previously treated with pronase (sample cb) although the xenon polarization level is similar to this of the other cell sample (Table 2). This is perfectly consistent with the observations of fluorescence microscopy. On the 129Xe NMR spectrum of sample ca, 2 peaks appear in this region: in addition to the peak at 67 ppm, a second peak appears near 80 ppm (raffiner les déplacements chimiques). A fast calculation taking into account the number of cells present in solution and the potential number of transferrin receptors per cell shows that the second peak cannot be due to xenon in the biosensor in the cell, whereas the first one remains the biosensor free in solution. Indeed a factor 0.001 for the current biosensor concentration should be found between the areas of these two peaks. Rather, as previously mentioned, the peak at 80 ppm can be representative of the biosensor in the cell membrane.
These two peaks have similar intensities. This indicates that a big part of the biosensor lies in a lipidic environment. Indeed whereas the left peak represents xenon in the cell membranes, the right peak should contain the contribution of xenon in the intra-cell aqueous compartment and in the free biosensor.
Assessment of the specificity of the biosensor
UTILITE DE LA PROCEDURE. In this batch experiment, the net difference between the 129Xe NMR spectra of xenon in the biosensor interacting with the cells after treatment or not by pronase (samples cb and ca) indicates that the transferrin receptors are required for a strong interaction of the biosensor with the cells via internalization, i.e. an active crossing occurs between the transferrin biosensor and the cells. The resemblance between the xenon spectra of the supernatant samples (sa and sb) proves xxxx. The appearance of a second peak on the selective xenon spectra witnessing a large part of the biosensor location in the membranes leads us however to address the question of the specificity of the interaction. Indeed addition of pronase may have led to a destructuration of the cell membrane. In order to study this specificity, we have built another biosensor by grafting the cryptophane precursor on a globular protein, the bovine serum albumin (BSA) and the fluorescent probe (at a ratio of 2 cryptophanes and 2 Rhodamine Green moieties per protein, leading to the biosensor B3). The same procedure than previously, cf. Table 1 (samples cc and sc) has been performed. As there is no receptor of this protein on the surface of the K562 cells, the 129Xe spectrum of the cc sample should be exempt of any peak in the 55-95 ppm region. Figure 4 displays the fluorescence microscopy image and the laser-polarized 129Xe NMR spectrum of the cc sample. Whereas the fluorescence image is less bright than this of the transferrin biosensor B1, surprisingly again two peaks appears. Even if the ratio between these peaks is more in favor of the cryptophane in the lipid part than for B1 (see Table 2), this indicates a lack of specificity of the biosensor B1. Given that there is no known affinity of BSA for any surface receptor of the K562 cells, a hypothesis is that the hydrophobicity of the cryptophane moieties is responsible for the anchoring of the biosensors in the cell membranes.
In order to assess this point, two other experiments have been performed. Firstly, cryptophane-A, the organo-soluble parent, has been introduced in the NMR tube containing B3 and the K562 cells after separation from the supernatant (i.e. sample cc). The 129Xe NMR spectrum displayed in Figure S1 of the Supporting Information shows that the area of the left peak, previously assigned to xenon in the cryptophane located in a lipidic environment, increases, in agreement with our hypothesis about the propensity of the cage-molecule to be inserted in the cell membrane. Secondly, two new transferrin-based constructs have been built. In the first one, the fluorescent probe has been grafted (again via the lysine sidechains) on the apo-transferrin. In the second one, the cryptophane precursor AND the fluorescent probe have been grafted on the protein, at a ratio 2:2 for one protein. These constructs have been introduced in solutions containing giant unilamellar vesicles. Comparison of the fluorescence microscopy images obtained in both cases is displayed in Figure S2 of the Supporting Information. Whereas xxxx, the location of the second construct in the lipid bilayer is clearly visible.
All these results seem to indicate that the cryptophane-based biosensors have a large propensity to interact with plasmic and intracellular membranes, due to the hydrophobic character of the xenon host molecule which is however not strong enough to unfold the protein.
MATERIALS AND METHODS
Synthesis of cryptophanol. Pure iodotrimethylsilane (65 ïL, 0.455 mmol) was added in one portion via syringe to a stirred solution of cryptophane-A (400 mg, 0.45 mmol) in CH2Cl2 (8 mL). The solution was strirred in the dark for 16 hours under an argon atmosphere at room temperature. CH2Cl2 (10 mL) was added to the solution and the solutrion was acidified with a HCl solution (1M, 6 mL). The organic layer was collected, washed with water and then dried over sodium sulfate. After evaporating the solvent under reduced pressure the residue was purified on a column chromatography (CH2Cl2/Et2O: 90/10) to give the cryptophanol as a white solid (144 mg, 37%). Spectroscopic data are identical to those previously reported for this compound.
Synthesis of cryptophane X. The protected PEG X (254 mg, 0.51 mmol, 1.5 eq.) was introduced in a three necks flask containing cesium carbonate (167 mg, 0.51 mmol), cryptophanol 1 (300 mg, 0.34 mmol) in freshly distilled DMF (12 mL). The mixture was stirred for 16 hours under an argon atmosphere at 60Â°C. CH2Cl2 (25 mL) and brine (10mL) ware added to the mixture. The aqueous layer was extracted twice with CH2Cl2 (10 mL). The combined layers are then washed with brine (5ï‚´ 10 mL) to remove DMF and dried over sodium sulfate. The solvent was removed under reduced pressure to give a residue, which was then purified on silica gel (a gradient of solvent Et2O/AcOEt was used: 100/0; 80/20; 50/50; 0/100). Evaporation of the solvent gave compound 3 as a white amorphous solid (282 mg, 69%). 1H NMR (500 MHz, CDCl3, 25Â°C) ï¤ 7.33-7.32 (m, 5H, Ar), 6.77 (s, 1H; Ar), 6.74 (s, 1H; Ar), 6.73 (s, 1H; Ar), 6.72 (s, 1H; Ar); 6.71 (s, 2H; Ar), 6.67 (s, 1H; Ar); 6.66 (s, 1H; Ar), 6.65 (s, 2H; Ar), 6.64 (s, 1H; Ar), 5.16 (s, 2H; OCH2Bn), 4.59-4.53 (m, 6H; Ha), 4.19-4.10 (m, 15H, OCH2, OCH2COO); 3.93 (m, 1H); 3.80-3.66 (m, 30 H; OCH2 and OCH3), 3.39-3.35 (m, 6H; He). 13C NMR (125.7 MHz, CDCl3, 25Â°C) ï¤ 170.32, 162.50, 149.85, 149.73, 149.61, 149.56, 148.96, 147.20, 146.72, 146.67, 146.62, 146.59, 135.45, 134.31, 134.26, 134.12, 134.04, 133.99, 132.72, 131.83, 131.57, 131.54, 131.47, 131.36, 128.60, 128.42, 128.39, 122.30, 121.39, 121.26, 120.54, 120.47, 120.33, 117.03, 114.64, 113.70, 113.59, 70.94, 70.86, 70.75, 70.65, 69.88, 69.60, 69.53, 69.38, 69.29, 69.20, 68.73, 68.64, 68.48, 56.09, 55.73, 55.66, 55.64, 55.59, 36.46, 36.30. HRMS ï›M+Naï+ calcd 1227.4949 found 1227.4935.
Synthesis of cryptophane X and X. H2 gas was introduced to 25 mL flask containing compound 3 (560 mg, 0.465 mmol), CH2Cl2 (12 mL), Ethanol (2 mL), and Pd/C (70 mg, 0.0658 mmol, 0.14 eq). The mixture was stirred at room temperature for 5 h. After completion of the reaction the mixture was filtrated and the solid residue was washed with CH2Cl2 (2 ï‚´ 10 mL). The solvent was then removed under reduced pressure to give compound 4 (0.46 g; 89 %). 1H NMR (500 MHz, CDCl3, 25Â°C) ï¤ 6.77 (s, 1H; Ar), 6.74 (s, 1H; Ar), 6.73 (s, 3H; Ar), 6.725 (s, 1H; Ar), 6.72 (s, 2H; Ar), 6.69 (s, 1H; Ar), 6.66 (s, 1H; Ar), 6.65 (s, 3H; Ar); 4.59-4.53 (m, 6H; Ha); 4.21-4.12 (m, 15H; OCH2 and OCH2COO), 3.95 (m, 1H), 3.82 (t, 2H, 3J(H,H) = 5.0 Hz), 3.78-3.73 (m, 27 H, OCH2-OCH3), 3.41-3.36 (m, 6H, He). 13C NMR (125.7 MHz, CDCl3, 25Â°C) ï¤ 170.99, 149.83, 149.71, 149.63, 149.59, 149.55, 148.91, 147.21, 146.65, 146.60, 134.39, 134.26, 134.16, 134.09, 134.00, 132.75, 131.87, 131.59, 131.54, 131.40, 122.17, 121.28, 120.59, 120.54, 120.38, 117.12, 114.72, 113.72, 113.61, 71.59, 70.88, 70.76, 70.64, 70.37, 70.18, 69.85, 69.59, 69.53, 69.38, 69.31, 69.23, 69.13, 69.72, 56.16, 55.74, 55.66,j 55.64, 55.60, 36.22, 36.20, 36.15. HRMS ï›M+Naï+ calcd 1137.4460 found 1137.4452. Activation of the acid function was performed by adding in a three necks flask N,N'-disuccinimidyl carbonate (58 mg, 0.226 mmol, 2.1eq.) to a solution of 4 (0.12 g, 0.108 mmol) in acetonitrile (3 mL) and pyridine (0.1 mL) under an argon atmosphere. The mixture was stirred. The mixture was stirred for 6h at room temperature. Then CH2Cl2 (10 mL) and HCl 1M (3 mL) were added. The organic layer was separated and dried over sodium sulfate. The removal of the solvent gives 5, which was used without further purification.
Compound X: 11-tetrahydropyranyloxy-3,6,9-trioxaundecan-1-ol (5g, 18 mmol, 1eq.) in THF (60 mL) was added dropwise to a stirred solution of NaH 60% (1.8g, 45 mmol, 2.5 eq) in THF (50 mL) under an argon atmosphere. After complete addition the mixture was stirred for an additional 1 hour. A solution of bromoacetic acid (2.8 g, 20 mmol, 1.1 eq) in THF was then added dropwise. After addition the solution was heated overnight under reflux condition. The solution was allowed to reach room temperature and benzyl bromide (2.3 mL, 19.3 mmol, 1.1 eq.) was added in one portion. The solution was then heated overnight under reflux condition. After evaporating the solvent under reduced pressure, CH2Cl2 (250 mL) and water (100 mL) were added. The layers were separated and the aquous layer was extracted twice with CH2Cl2 (100 mL). The combined organic layers are then washed twice with water (50 mL) and dried over sodium sulfate. After evaporation of the solvent the crude product was purified by column chromatography (AcOEt) to give 5 as a colorless oil (5.75 g, 75 %). 1H NMR (500 MHz, CDCl3, 25Â°C) ï¤ 7.35-7.28 (m, 5H; Ar), 5.15 (s, 2H; OCH2Bn), 4.59 (m, 1H, OCHO), 4.17 (s, 2H, OCH2COO), 3.85-3.80 (m, 2H; OCH2), 3.71-3.69 (m, 2H, OCH2), 3.66-3.60 (m, 12H, OCH2), 3.59-3.54 (m, 1H, OCH2), 3.48-3.44 (m, 1H, CH2), 1.82-1.76 (m, 1H, CH2), 1.71-1.65 (m, 1H; CH2), 1.60-1.44 (m, 4H; CH2). 13C NMR (125.7 MHz, CDCl3, 25Â°C) ï¤ 170.26, 135.38, 128.53, 128.35, 128.33, 98.85, 70.88, 70.57, 70.55, 70.53, 70.50, 70.46, 68.61, 66.57, 66.41, 62.12, 30.49, 25.36, 19.41. HRMS ï›M+Naï+ calcd 449.2117 found 449.2142.
Compound X: A stirred solution of 5 (4.98 g, 11.3 mmol, 1 eq.) and pyridinium toluene sulfonate (0.95 g, 3.78 mmol, 0.33 eq) in ethanol was heated overnight at 45Â°C. The solvent was removed under reduced pressure and the oily residue purified by column chromatography (AcOEt). Evaporation of the solvent gives compound 6 as colorless oil (3.2 g; 83 %). Spectroscopic data are identical to those previously reported in the literature.
Compound X: para-chloro toluene sulfonate (1.5 g, 7.87 mmol, 1.3 eq) was added at 0Â°C in one portion to a stirred solution of compound 6 (2g, 5.84 mmol) in pyridine (6 mL). The solution was stirred at this temperature for an additional 2 hours. Water (10 mL) and diethyl ather (40 mL) were then added to the solution. The organic layer is collected and the aqueous layer is extracted twice with diethyl ether (2 ï‚´ 20 mL). The combined organic layer are then washed with brine (20 mL) and water (10 mL) and then dried over sodium sulfate. Evaporation of the solvent leaves a residue, which was purified by column chromatography (AcOEt). Different fractions are collected and the evaporation of the solvent gives compound 7 (2.32 g, 80 %) as colorless oil. Additional information 1H NMR (500 MHz, CDCl3, 25Â°C) ï¤ 7.77 (d, 3J(H,H)= 8.3 Hz, 2H; Ar), 7.33-7.29 (m, 7H; Ar), 5.16 (s, 2H; OCH2Bn), 4.17 (s, 2H; OCH2COO), 4.13 (t, 3J(H,H) = 4.9 Hz, 2H; OCH2), 3.71-3.54 (m, 14H, OCH2), 2.42 (s, 3H; CH3).
Synthesis of cryptophanol-A and of the linker. Derivative X has been prepared from cryptophanol X and a PEG molecule aimed at increasing sparingly the solubility of the final molecule in water and allowing its attachment to the protein (olo-transferrin). Herein, cryptophanol X whose synthesis has been independently reported by Darzac et al. and Spence et al. was prepared in a single step from cryptophane-A. [i] This new strategy allows the rapid synthesis for the mono-hydroxyled cryptophane and avoids the use of the multi-step synthesis described previously in the literature. Thus a removal of a single methyl group has been achieved by using iodotrimethylsilane in CH2Cl2 and provides cryptophanol in a 37% yield. During the purification procedure the unreacted cryptophane-A was recovered, which can be reused for subsequent reactions. This strategy affords an alternative way for preparing this important compound X, which has been used to build up a large range of different compounds. [ii] The yield and the ease of purification are strongly dependent of the quality of the highly reactive iodo-trimethylsilane used for the reaction and a monitoring of the reaction is usually necessary (the time of the reaction as well as the amount of the reactant are strongly dependent of the quality of the iodotrimethylsilane used). It is noteworthy that attempts to perform the reaction with iodo-trimethylsilane prepared in situ from chlorotrimethylsilane and sodium iodide in a mixture of dichloromethane and acetonitrile failed to achieve the mono-deprotection. Similarly attempts to use other reactants such as lithium diphenylphosphide in THF or sodium isopropylthiolate in DMF failed to achieve the demethylation reaction.
The PEG function used for the attachment of the biosensor molecule to the protein has been prepared in three steps from a mono-protected PEG derivative X previously described in the literature. [iii] An activated acid function has been introduced at one extremity in order to allow the coupling reaction with the protein and a tosyl group has been introduced at the other extremity to allow the coupling reaction between the phenol moiety of the cryptophane and the PEG molecule. Firstly the introduction of the carboxylic function was achieved by reacting the PEG X with bromomethylacetic acid in THF in presence of strong base. Secondly the protection of the acid derivative with benzyl bromide was carried out to allow the purification by column chromatography of compound X with 75 % yield. Deprotection of the tetrahydropyranyl moiety was then achieved with pyridinium p-toluene sulfonate in Ethanol to give rise to compound X with 83 % after purification. [iv] In turn the free alcohol function was used to introduce a tosyl group to provide derivative X with 80 % yield. [v]
Synthesis of cryptophane-PEG-succinimide. The efficient coupling reaction between cryptophanol-A and derivative X has been carried out in DMF in presence of cesium carbonate and gave rise to monofunctionalized cryptophane X with a 69 %. The formation of the desired product can be easily detected by 1H NMR spectroscopy. Indeed the presence in the 1H NMR spectrum of two diastereotopic protons located at 3.9 ppm appears characteristic of the coupling reaction since this signal stands for the two protons of the PEG derivative directly attached to the cryptophane molecule. The deprotection of the carboxylic acid was obtained by hydrogenation at atmospheric pressure in presence of a Pd/C catalyst in a mixture of dichloromethane and ethanol. A filtration of the solution after the reaction allows the obtention of the clean derivative X with 89 % yield. The activation of the acid function was finally achieved with Nhydroxysuucinimide in the presence of dicyclohexylcarbodiimide. After an appropriate treatment the cryptophane-PEG-succinimide was used without further purification for the coupling reaction with the protein.
b. Preparation of the biosensors
Biosensor B1. The human transferrin and Bovin Serum Albumin were purchased from Sigma Aldrich. Each protein was incubated with the activated cryptophanes during one hour in Phosphate Buffer Saline (PBS) medium pH 7.4 at room temperature. The ratio of cryptophanes versus protein used for NMR experiments was 5:1 and 2:1 for fluorescence imaging. The unreacted dye was removed from the protein solution using a gel Sephadex G25 medium chromatography. The elution was made with PBS, pH 7.4.
Rhodamine Green -transferrin. Rhodamine green succinimide ester was purchased from Molecular Probe. The protein was incubated with the fluorescent dye in bicarbonate medium pH=8,4 during 1h, at room temperature under agitation. The ratio of 2 was used. The free dye was removed from the protein solution using a Sephadex G25 medium gel chromatography. The elution was made with PBS. The labeling efficiency was controlled using an absorption spectrometer.
c. Characterization of the constructs
The folding of the labeled proteins was controlled with 1H NMR on a 700 MHz Bruker spectrometer equipped with a HCN cryoprobe. Solutions at 1 mM in 500 ÂµL H2O:D2O 90:10 were analyzed in 5 mm tubes.
Cell culture. Human erythroleukaemic K562 cells were purchased from ATCC and grown in IMDM medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum, 1% of l-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. The cells were maintained in exponential growth in a 5% CO2 incubator at 37Â°C. Prior to the experiment, the cells were washed tree times in PBS and viable cells were counted using trypan blew exclusion. The chosen amount of cells is re-suspended to a final volume of 1,5 L.
Fluorescent labeling. Cells were incubated with 200 nM of fluorescent probe during 1h. The cells were washed 3 times with PBS at 4Â°C in order to block the membrane dynamic. Then the cells were fixed on the glass substrate using PFA 5% during 30 minutes at 4Â°C. The observation was made using an inverted microscope.
Preparation of the samples. 120 millions of cells were washed three times with PBS. For the pronase sample, the cells were treated using a 2 mg/mL solution of pronase during 30 minutes at 37Â°C. (à verifier) Then the cells were washed 3 times with PBS and incubated with 5 ÂµM of the biosensor during 1h at 37Â°C. At the end, the cells were washed 3 times at 4Â°C in order to block plasma membrane dynamics, and re-suspended in a final volume of 1,5mL.
e. 129Xe NMR experiments
Laser-polarized xenon. Xenon 86%-enriched in isotope 129 was purchased from CornetNet, France. The hyperpolarized gas was prepared via the spin-exchange method, using our batch apparatus previously described.[ref EPJD, PINS]. The amount of gas produced with this set-up using a titanium:sapphire laser, on the order of 1 mL (in ~10 minutes), was sufficient to fill the NMR tube with a pressure of ~1 bar and an average polarization of 40%. The transfer of xenon between the cold finger designed to separate xenon from nitrogen after optical pumping and the NMR tube of interest was made through a vacuum line in the fringe field of the NMR magnet in order to preserve polarization.
NMR experiments. Each acquisition of an NMR spectrum was preceded by vigorous shaking (to artificially introduce the hyperpolarized gas into solution) and a wait time of ~10 s to enable the elimination of bubbles. The experiments were run on a 500 MHz Avance II Bruker spectrometer equipped with a 129Xe/1H micro-imaging probehead accepting tubes with a maximal outer diameter of 8 mm. Selective excitation was performed using a 1% truncated Gaussian pulse of 500 Âµs. PROCESSING
In this article we have demonstrated the proof-of-concept of the 129Xe NMR-based biosensing approach on a real in vitro system. The presence of paramagnetic Fe3+ ions on the biosensor itself and in the K562 cells did not impede the observation of xenon caged in cryptophanes grafted on this biosensor. It is worth noting that the 129Xe NMR spectra were obtained in one shot by repeated selective excitations in the spectral region 55-95 ppm. At the concentrations of protein used (~5 ÂµM) the observed xenon relaxation time remains long, which enables to fully use the reservoir of polarization constituted by dissolved xenon. Moreover, given the large reservoir of gaseous hyperpolarized xenon on top on the solution, if required we could have benefited from additional spectra to increase the signal-to-noise ratio simply by shaking the NMR tube between two acquisition series.
In the presence of K562 cells expressing many transferrin receptors, the transferrin biosensor is endocyted, as proved by fluorescence microscopy experiments. The laser-polarized 129Xe NMR spectra of the biosensor B1 alone and in the presence of the cells are largely different. However the lack of specificity of this biosensor for the transferrin receptors is undeniable. We have shown that it is due to the presence of the strong hydrophobic character of the cryptophane. Without leading to protein unfolding, it tends to attract the biosensor to the cell membrane.
This shows that the current approaches aiming at rendering soluble the cryptophanes in water through introduction of a hydrophilic ligand far from the cavity can have some drawbacks, as was already observed with a cryptophane biosensor designed to recognize a complementary RNA strand in solution.[ref ChemPhysChem2007]. The amphiphilicity created by the presence of the cage-molecule and the aliphatic spacer on one side and the oligonucleotide on the other side led to the formation of self-organized systems of the micelle or vesicle type. A solution would be to place hydrophilic groups closer to the aromatic rings of the cryptophanes, however paying attention to maintaining the xenon in-out exchange.[ref cryptophan-6-ol]
Figure 1. Structure of the biosensor B1 and laser-polarized 129Xe NMR spectrum of the noble gas in this biosensor in PBS. In the frame, a zoom of the 55-95 ppm frequency range, corresponding to the Xe@cryptophane region, is displayed.
Figure 2. Fluorescence images of K562 cells incubated by the B2 biosensor, without (left) and with (right) previous pronase treatment.
Figure 3. 129Xe NMR spectra obtained by selective excitation in the xenon@cryptophane. Upper left: with K562 cells after incubation with the biosensor during 1h at 37Â°C; upper right: spectrum of the corresponding supernatant; lower left: spectrum with K562 treated with pronase and then incubated with the biosensor during 1h at 37Â°C; lower right: spectrum of the corresponding supernatant.
Figure 4. Fluorescence images of K562 cells incubated by the B3 biosensor and 129Xe NMR spectrum obtained by selective excitation in the xenon@cryptophane spectral region.
Scheme 1. Synthesis of cryptophanol-A from cryptophane-A and synthesis of the linker.
Scheme 2. Synthesis of cryptophane-PEG-succinimide.
Table 1. Synopsis of the protocols used in our batch procedure, designed toâ€¦
Table 2. Relative areas of the peaks of xenon caged in cryptophaneâ€¦
REFERENCES (Word Style "TF_References_Section").
1. Spence, M. M.; Rubin, S. M.; Dimitrov, I. E.; Ruiz, E. J.; Wemmer, D. E.; Pines, A.; Qin Yao, S.; Tian, F.; Schultz, P. G., Functionalized xenon as a biosensor. Proc. Natl. Acad. Sci. USA 2001, 98, 10654-10657.
2. Brotin, T.; Dutasta, J.-P., Cryptophanes and Their Complexes--Present and Future. Chem. Rev. 2009, 109, 88-130.
3. Lerouge, F.; Melnyk, O.; Durand, J.-O.; Raehm, L.; Berthault, P.; Huber, G.; Desvaux, H.; Constantinesco, A.; Choquet, P.; Detour, J.; SmaÃ¯hi, M., Towards thrombosis-targeted Zeolite nanoparticles for laser-polarized 129Xe MRI. J. Mater. Chem. 2009, 19, 379-386.
4. Berthault, P.; Bogaert-Buchmann, A.; Desvaux, H.; Huber, G.; Boulard, Y., Sensitivity and multiplexing capabilities of MRI based on polarized 129Xe biosensors. J. Am. Chem. Soc. 2008, 130, 16456-16457.
5. Spence, M.; Ruiz, E.; Rubin, S.; Lowery, T.; Winssinger, N.; Schultz, P.; Wemmer, D.; Pines, A., Development of a Functionalized Xenon Biosensor. J. Am. Chem. Soc. 2004, 126, 15287-15294.
6. Roy, V.; Brotin, T.; Dutasta, J.-P.; Charles, M.-H.; Delair, T.; Mallet, F.; Huber, G.; Desvaux, H.; Boulard, Y.; Berthault, P., A cryptophane biosensor for detection of specific nucleotide targets through xenon-NMR. ChemPhysChem 2007, 8, 2082-2085.
7. Wei, Q.; Seward, G. K.; Hill, P. A.; Patton, B.; Dimitrov, I. E.; Kuzma, N. N.; Dmochowski, I. J., Designing 129Xe NMR Biosensors for Matrix Metalloproteinase Detection. J. Am. Chem. Soc. 2006, 128, 13274-13283.
8. Chambers, J. M.; Hill, P. A.; Aaron, J. A.; Han, Z.; Christianson, D. W.; Kuzma, N. N.; Dmochowski, I. J., Cryptophane Xenon-129 Nuclear Magnetic Resonance Biosensors Targeting Human Carbonic Anhydrase. J. Am. Chem. Soc. 2009, 131, (30), 563-569.
9. Schlundt, A.; Kilian, W.; Beyermann, M.; Sticht, J.; Günther, S.; Höpner, S.; Falk, K.; Roetzschke, O.; Mitschang, L.; Freund, C., A Xenon-129 biosensor for monitoring MHC-peptide interactions. Angew. Chem. Int. Ed. 2009, 48, 1-5.
10. Du, X.-L.; Wang, K.; Ke, Y.; Yuan, L.; Li, R.-C.; Zhong, C. Y.; Ping, H. K.; Qian, Z. M., Apotransferrin Is Internalized and Distributed in the Same Way as Holotransferrin in K562 Cells. J. Cell. Physiol. 2004, 201, 45-54.
11. Lowery, T. J.; Garcia, S.; Chavez, L.; Ruiz, E. J.; Wu, T.; Brotin, T.; Dutasta, J.-P.; King, D. S.; Schultz, P. G.; Pines, A.; Wemmer, D. E., Optimization of xenon biosensors for detection of protein interactions. ChemBioChem 2006, 7, 65-73.
refx. Despite a xenon polarization not fully reproducible, quantitative estimation of the xenon NMR signals has been rendered possible through normalization of the integral of the peak of xenon free in solution, considering that the same amount of xenon was each time introduced in the NMR tube (checked by weighting of the tubes before and after the experiment).