Curious Results with Palladium- and Platinum ... - ACS Publications

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Curious Results with Palladium- and Platinum-Carrying Polymers in Mass Cytometry Bioassays and an Unexpected Application as a Dead Cell Stain Daniel Majonis, Olga Ornatsky, Robert Kinach, and Mitchell A. Winnik* Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, Canada M5S 3H6 S Supporting Information *

ABSTRACT: We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) and an orthogonal end-group, either a fluorescein molecule or a bismaleimide linker for antibody attachment. Polymer characterization by a combination of 1H NMR, UV/vis absorption measurements, and thermal gravimetric analysis (TGA) indicated that each chain of the fluorescein-terminated polymers contained one dye molecule. These polymer samples were loaded with three different types of lanthanide ions as well as palladium and platinum ions. The numbers of metal atoms per chain were determined by a combination of UV/vis and conventional ICP-MS measurements. The experiments with lanthanide ions demonstrated that a net anionic charge on the polymer is important for water solubility. These experiments also showed that at least one type of lanthanide ion (La3+) is capable of forming a bimetallic complex with pendant DTPA groups. Conditions were developed for loading these polymers with palladium and platinum ions. While these polymers could be conjugated to antibodies, the presence of Pd or Pt ions in the polymer interfered with the ability of the antibody to recognize its antigen. For example, a goat anti-mouse (secondary) antibody labeled with polymers that contain Pd or Pt no longer recognized a primary antibody in a sandwich assay. In mass cytometry assays, these Pd- or Pt-containing MCPs were very effective in recognizing dead cells and provide a new and robust assay for distinguishing live cells from dead cells.

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applications of this type of polymer.2,3 Using this approach, we have reported an 11-plex assay of the cell types present in human cord blood and a 20-plex assay of the biomarker distribution in leukemic cell lines and in patient samples obtained from the Leukemic Cell Bank of Quebec. While in principle there are more than 50 discrete isotopes of lanthanide (Ln) metals, which would make a 50-plex assay possible, in practice only 31 are readily available. When we began this research 5 years ago, the idea that 30plex immunoassays could be carried out seemed only a dream. Now such assays are routine, with instrumentation and reagents commercially available. With its success has come a growing need to increase the multiplicity of cell surface and intracellular biomarkers that can be detected at high throughput on individual cells. Thus, we need to turn our attention to water-soluble polymers that can bind other metals, with masses in the range of 100−220 amu that can be detected by mass cytometry. We have previously described the synthesis of well-defined metal-chelating polymers with a terminal maleimide group for attachment to antibodies via a Michael reaction with −SH groups generated by selective reduction of disulfide bonds in the hinge region of the antibody. These polymers carry a

INTRODUCTION Our research group is involved in a multidisciplinary project wherein we develop reagents for the simultaneous detection of multiple biomarkers on individual cells at high throughput by the new technique of mass cytometry.1 In mass cytometry, cells are injected (nebulized) stochastically into the plasma torch of an inductively coupled plasma mass spectrometer (ICP-MS), where they are vaporized, atomized, and ionized. The ion cloud generated in this way is analyzed by time-of-flight mass spectrometry, and the methodology allows one to take 20−30 mass spectra during the 200 μs transit time of the ion cloud through the instrument. ICP-MS is a powerful tool for the quantitative analysis of the metal content of samples, with single mass resolution and a large dynamic range. With current technology, one can analyze ca. 1000 cells per second. To carry out multiplexed immunoassays by mass cytometry, one labels antibodies with metal-chelating polymers in such a way that each distinct antibody, targeted to a specific cell biomarker, is labeled with a different metal isotope. Lanthanide isotopes are ideal for this purpose. They have low natural abundance and similar chemistry, and isotopically enriched lanthanides such as 151Eu and 153Eu are available commercially. This means that a common metal-chelating polymer, with appropriate end-functionality for attachment to an antibody, will serve to label many different antibodies for a multiplexed immunoassay. We have described the synthesis and some © 2011 American Chemical Society

Received: July 21, 2011 Revised: September 7, 2011 Published: September 28, 2011 3997

dx.doi.org/10.1021/bm201011t | Biomacromolecules 2011, 12, 3997−4010

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diethylenetriaminepentaacetic acid (DTPA) group on every repeat unit.2 DTPA is a strong chelating group for Ln ions. The synthesis began with the reversible addition−fragmentation chain transfer (RAFT) polymerization of an inexpensive monomer, tert-butyl acrylate (tBA), using di-1-phenylethyl trithiocarbonate as the chain-transfer agent (CTA). The purified polymer was treated with ethanolamine in a concentrated THF solution to destroy the RAFT trithiocarbonate moiety and to oxidize the polymeric thiols to a disulfide. The disulfide serves to protect the sulfur atoms during pendant group modification in a way that they can easily be converted to −SH groups after these transformations are complete. Among the advantages of this method is that the polymers are obtained with a narrow molar mass distribution, the mean degree of polymerization is easily established by spectroscopic means, and subsequent transformations of the polymer occur in high yield without affecting the polymer chain length. As shown in Scheme 1, the tert-butyl

as pendant groups to the polymer backbone. Another more serious challenge is that many of the conditions used to synthesize polyaminocarboxylates complexes of Pd(II) and Pt(II) are quite harsh and risk destroying the polymeric maleimide group used for attachment to an antibody. We have sought more gentle conditions for loading palladium and platinum into metal-chelating polymers. In the experiments reported here, we describe the synthesis of polymers containing EDTA, DTPA, TTHA, and DOTA pendant groups using a sample of the amino-polymer disulfide with DPn = 79 per phenyl end-group as a common precursor. We report conditions for coupling a fluorescein dye on the end of each chain via reduction of the disulfide to a pair of −SH group. The dye enables a rapid assay of the metal ion content of the polymers by a combination of UV/vis and ICP-MS measurements. This assay was used to examine the loading of lanthanide, palladium, and platinum ions into polymers with pendant EDTA, DTPA, TTHA, or DOTA ligands. On the basis of those results, we prepared samples of a Pd-loaded and a Pt-loaded EDTA-containing polymer and employed them in a model mass cytometric immunoassay. The results of the bioassay were unexpected. These polymers were uniquely effective in labeling the small fraction of dead cells in a cell population, rather than the target antigen of the labeled antibody.

Scheme 1. Synthesis of a Polymer with Pendant Primary Amines and a Polymeric Disulfide End-Group2 a

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EXPERIMENTAL SECTION

Materials. All reagents and solvents, including ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma, 99%), diethylenetriaminepentaacetic acid (DTPA) (98%, Aldrich), triethylenetetraminehexaacetic acid (TTHA) (98%, Aldrich), tetraazocyclododecanetetraacetic acid hexahydrate (DOTA·6H2O) (≥95%, Macrocyclics), DL-dithiothreitol (DTT) (99%, Aldrich), lanthanum(III) chloride hydrate (≥99.0%, Fluka), terbium(III) chloride hexahydrate (99.999%, Aldrich), ytterbium(III) chloride hexahydrate (99.9%, Aldrich), potassium tetrachloropalladate(II) (K2PdCl4, 99.99%, Aldrich), potassium tetrachloroplatinate(II) (K2PtCl4, 99.99%, Aldrich), and dichloro(ethylenediamine)platinum(II) (Pt(en)Cl2, Aldrich)), and other compounds were used without further purification unless otherwise noted. Water was purified through a Milli-Q water purification system (12 MΩ·cm). All buffers were prepared in our laboratory. The 4 and 15 mL 3 kDa MWCO Millipore Amicon spin filters were purchased from Fisher Science. The 0.5 mL Pall Nanosep 3K Omega filters and 0.45 μm MF filters were purchased from VWR. The Pall Acrodisc 13 mm syringe filters with 200 nm nylon membrane were purchased from VWR. 2,2′-(Ethylenedioxy)bis(ethylmaleimide) (prepared by Acanthus Research, Toronto, Canada), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, Acros Organics, 99+%, from Fisher Science, Canada), and N-(5-fluoresceinyl)maleimide (Sigma, 90+%) were stored in a desiccator inside a freezer at −20 °C. Before use, their temperatures were equilibrated in a desiccator kept at room temperature. Polymer Synthesis. Amino-Polymer Disulfide. A new batch of DPn = 79 amino-polymer disulfide was synthesized in an identical manner, and from the same batch of PAA-disulfide, as presented previously.2 Synthesis of P(EDTA). EDTA (ethylenediaminetetraacetic acid disodium salt dihydrate, 6.53 g, ca. 80 equiv to each polymeric amino group) and H2O (16 mL) were added to a 100 mL roundbottom flask. Next, aqueous NaOH (5 M) was added with stirring to create a solution of pH 8.75 (monitored with a pH meter). The resultant volume was 25 mL. DMTMM (500 mg, ca. 8 equiv to each polymeric amino group) was dissolved in water (10 mL) with sonication and added quickly with stirring to the first solution. This solution was given 5 min to prereact. Then a solution of the aminopolymer (50 mg) in water (10 mL) was added quickly with stirring. The reaction solution was stirred for 1 h, concentrated in 2× 15 mL 3 kDa MWCO Millipore Amicon spin filters, and washed with water

a

AMBN = 2,2′-azobis(2-methylbutyronitrile), THF = tetrahydrofuran, DCM = dichloromethane, and TFA = trifluoroacetic acid.

ester groups were readily removed by treating a solution of the polymer in dichloromethane (DCM) with trifluoroacetic acid (TFA). The poly(acrylic acid) generated in this way was converted to the amino-polymer disulfide through a 4-(4,6-dimethoxy-1,3,5triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)mediated coupling step with t -BOC-ethylenediamine followed by deprotection with TFA in DCM in the presence of anisole. By 1H NMR, these reactions took place in quantitative yield. This chemistry is presented in Scheme 1. In our previous publication, we also described the quantitative addition of pendant DTPA groups via DMTMM coupling, reduction of the polymeric disulfide with DTT (dithiothreitol), and reaction of the polymeric thiol with a bismaleimide to yield a terminal maleimide group for attachment to antibodies. Palladium and platinum represent promising choices for use in mass cytometric bioassays. They are in the same periodic table group and thus have similar chemistry. Together they have 8 isotopes with at least 10% natural abundance that are commercially available in 95%+ isotopic purity. These 8 isotopes represent additional labels that can be used, in principle, to increase the multiplicity of immunoassays. In addition, there exists literature describing palladium(II) and platinum(II) complexes with polyaminocarboxylates such as EDTA, DTPA, and TTHA.4−8 One of the challenges we address in this paper is the extension of our synthetic methodology to attach EDTA (ethylenediaminetetraacetic acid), TTHA (triethylenetetraminehexaacetic acid), and DOTA (tetraazocyclododecanetetraacetic acid) 3998


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(50 mM, pH 8.50). P(EDTA)-disulfide (35 mg) was transferred to a 20 mL scintillation vial with stir bar and dissolved in 1050 μL of this DTT solution. The vial threads were wrapped with Teflon tape, the cap was secured, and the solution was stirred at 50 °C for 1 h. Immediately afterward the polymer solution was diluted with acetate buffer (10 mL, 50 mM, pH 3.0) and transferred to a 15 mL 3 kDa MWCO Millipore Amicon spin filter. The solution was spun through the filter, after which the polymer was washed (3 × 11 mL) with aqueous acetic acid (5 mM, pH = 3.5). Next, the resultant polymer solution (350 μL) was transferred to a 20 mL scintillation vial and diluted to a total volume of 1050 μL with phosphate buffer (200 mM, pH 7.00). A freshly prepared solution of N-(5-fluoresceinyl)maleimide (5 mg) in DMF (525 μL) was quickly added with mixing to the polymer solution, and the solution was stirred for 1 h at room temperature while protected from light. Directly after this, the solution was diluted with phosphate buffer (200 mM, pH 8.50), transferred to a new 15 mL 3 kDa MWCO Millipore Amicon spin filter, and washed with phosphate buffer (200 mM, pH 8.50) (4 × 11 mL) followed by water (3 × 11 mL). Finally, the aqueous solution was filtered through a Pall Acrodisc 13 mm syringe filter with 200 nm nylon membrane and freeze-dried to yield P(EDTA)−fluorescein. Yield = 32 mg (91%). SEC (aqueous, relative to poly(methacrylic acid) standards, RI): Mn = 20 700 Da, PDI = 1.20; strong corresponding peak observed in the UV/vis trace monitored at 494 nm. P(DTPA) N-(5-Fluoresceinyl)maleimide Reaction. Yield = 32 mg (91%). SEC (aqueous, relative to poly(methacrylic acid) standards, RI): Mn = 23 600 Da, PDI = 1.20; strong corresponding peak observed in the UV/vis trace monitored at 494 nm. P(TTHA) N-(5-Fluoresceinyl)maleimide Reaction. Yield = 33 mg (94%). SEC (aqueous, relative to poly(methacrylic acid) standards, RI): Mn = 25 100 Da, PDI = 1.19; strong corresponding peak observed in the UV/vis trace monitored at 494 nm. P(DOTA) N-(5-Fluoresceinyl)maleimide Reaction. Yield = 11.7 mg (75%). SEC (aqueous, relative to poly(methacrylic acid) standards, RI): Mn = 18 200 Da, PDI = 1.22; strong corresponding peak observed in the UV/vis trace monitored at 494 nm. Reduction of P(EDTA)-Disulfide with DTT Followed by Functionalization with a Bismaleimide Linker. A solution of DTT (dithiothreitol, 20 mM) was freshly prepared in phosphate buffer (50 mM, pH 8.50). P(EDTA) disulfide (12 mg) was transferred to a 20 mL scintillation vial with stir bar and dissolved in 360 μL of this DTT solution. The vial threads were wrapped with Teflon tape, the cap was secured, and the solution was stirred at 50 °C for 1 h. Immediately afterward, the polymer solution was diluted with acetate buffer (3.5 mL, 50 mM, pH 3.0) and transferred to a 4 mL 3 kDa MWCO Millipore Amicon spin filter. The solution was spun through the filter, after which the polymer was washed (3 × 4 mL) with aqueous acetic acid (5 mM, pH = 3.5). Next, the resultant polymer solution (150 μL) was transferred to a 2 dram vial and diluted to a total volume of 360 μL with phosphate buffer (200 mM, pH 7.00). A freshly prepared solution of 2,2′-(ethylenedioxy)bis(ethylmaleimide) (12 mg) in DMF (180 μL) was quickly added with mixing to the polymer solution, and the solution was stirred for 1 h at room temperature. Directly after this, the solution was diluted with water (2 mL), filtered through a Pall Acrodisc 13 mm syringe filter with 200 nm nylon membrane into a new 4 mL 3 kDa MWCO Millipore Amicon spin filter, and washed with water (3 × 4 mL), phosphate buffer (200 mM, pH 7.00) (1 × 4 mL), and again water (3 × 4 mL). Finally, the aqueous solution was filtered through a Pall Acrodisc 13 mm syringe filter with a 200 nm nylon membrane and freeze-dried to yield P(EDTA)−maleimide. Yield = 10.7 mg (89%). 1H NMR (D2O): δ (ppm, integrated peak areas reported based on C6H5 = 5H) 1.0−2.4 (broad, 3H per monomer, backbone, integration = 268), 2.7−4.0 (broad m, 4H ethylenediamine and 12H EDTA per monomer, integration = 1503), 6.88 (s, 2H vinylic maleimide, integration = 1.79), 7.15−7.45 (broad peaks, 5H phenyl, integration = 5.0). SEC (aqueous, relative to poly(methacrylic acid) standards, RI): Mn = 20 200 Da, PDI = 1.20. Metal-Loading Reactions. Lanthanides. P(EDTA)−fluorescein, P(DTPA)−fluorescein, and P(DOTA)−fluorescein were loaded with lanthanum, terbium, and/or ytterbium. Polymer (0.4 mg) was dissolved in 190 μL of ammonium acetate buffer (20 mM, pH 6.0)

(9 × 11 mL for each filter). Finally, the aqueous solution was freezedried to yield P(EDTA)-disulfide. Yield = 97 mg (95%). 1H NMR (D2O): δ (ppm, integrated peak areas reported based on C 6H5 = 5H) 1.0−2.4 (b, 3H per monomer, backbone, integration = 240), 2.7−4.2 (broad m, 4H ethylenediamine and 12H EDTA per monomer, integration = 1373), 7.15−7.45 (broad t, 5H phenyl, integration = 5.0). SEC (aqueous, relative to poly(methacrylic acid) standards, RI): Mn = 33 200 Da, PDI = 1.19. Synthesis of P(DTPA). DTPA (6.90 g, ca. 80 equiv to each polymeric amino group) and H2O (10 mL) were added to a 100 mL round-bottom flask. Next, aqueous NaOH (5 M) was added with stirring to create a solution of pH 8.5 (monitored with a pH meter). The resultant volume was 29 mL. DMTMM (500 mg, ca. 8 equiv to each polymeric amino group) was dissolved in water (10 mL) with sonication and added quickly with stirring to the first solution. This solution was given 5 min to prereact. Then a solution of the aminopolymer (50 mg) in water (10 mL) was added quickly with stirring. The reaction solution was stirred for 1 h, concentrated in 2× 15 mL 3 kDa MWCO Millipore Amicon spin filters, and washed with water (9 × 11 mL for each filter). Finally, the aqueous solution was freeze-dried to yield P(DTPA)-disulfide. Yield = 111 mg (87%). 1H NMR (D2O): δ (ppm, integrated peak areas reported based on C6H5 = 5H) 1.0−2.4 (b, 3H per monomer, backbone, integration = 233), 2.7−4.2 (broad m, 4H ethylenediamine and 18H DTPA per monomer, integration = 1845), 7.15−7.45 (broad t, 5H phenyl, integration = 5.0). SEC (aqueous, relative to poly(methacrylic acid) standards, RI): Mn = 37 200 Da, PDI = 1.18. Synthesis of P(TTHA). TTHA (5.0 g, ca. 80 equiv to each polymeric amino group) and H2O (2.9 mL) were added to a 100 mL roundbottom flask. Next, aqueous NaOH (5 M) was added with stirring to create a solution of pH 8.5 (monitored with a pH meter). The resultant volume was 16 mL. DMTMM (289 mg, ca. 8 equiv to each polymeric amino group) was dissolved in water (5.8 mL) with sonication and added quickly with stirring to the first solution. This solution was given 5 min to prereact. Then a solution of the aminopolymer (28.8 mg) in water (5.8 mL) was added quickly with stirring. The reaction solution was stirred for 1 h, concentrated in 2× 15 mL 3 kDa MWCO Millipore Amicon spin filters, and washed with water (9 × 11 mL for each filter). Finally, the aqueous solution was freezedried to yield P(TTHA)-disulfide. Yield = 78 mg (87%). 1H NMR (D2O): δ (ppm, integrated peak areas reported based on C 6H5 = 5H) 1.0−2.4 (b, 3H per monomer, backbone, integration = 230), 2.7−4.2 (broad m, 4H ethylenediamine and 24H TTHA per monomer, integration = 2272), 7.15−7.45 (broad t, 5H phenyl, integration = 5.0). SEC (aqueous, relative to poly(methacrylic acid) standards, RI): Mn = 39 200 Da, PDI = 1.19. Synthesis of P(DOTA). DOTA·6H2O (2.0 g, ca. 70 equiv to each polymeric amino group) and H2O (2.54 mL) were added to a 50 mL round-bottom flask. Next, aqueous NaOH (5 M) was added with stirring to create a solution of pH 8.5 (monitored with a pH meter). The resultant volume was 5 mL. DMTMM (123.4 mg, ca. 8 equiv to each polymeric amino group) was dissolved in water (2.54 mL) with sonication and added quickly with stirring to the first solution. This solution was given 5 min to prereact. Then a solution of the aminopolymer (12.7 mg) in water (2.54 mL) was added quickly with stirring. The reaction solution was stirred for 1 h, concentrated in a 15 mL 3 kDa MWCO Millipore Amicon spin filter, and washed with water (9 × 11 mL). Finally, the aqueous solution was freeze-dried to yield P(DOTA)-disulfide. Yield = 30.0 mg (91%). 1H NMR (D2O): δ (ppm, integrated peak areas reported based on C 6H5 = 5H) 1.0−2.4 (b, 3H per monomer, backbone, integration = 221), 2.7−4.2 (broad m, 4H ethylenediamine and 24H DOTA per monomer, integration = 2112), 7.15−7.45 (broad t, 5H phenyl, integration = 5.0). SEC (aqueous, relative to poly(methacrylic acid) standards, RI): Mn = 30 500 Da, PDI = 1.19. Reduction of P(EDTA)-Disulfide with DTT Followed by Reaction with N-(5-Fluoresceinyl)maleimide. We describe this reaction for P(EDTA) disulfide; however, the reaction was identical for P(DTPA) and P(TTHA) and scaled accordingly for P(DOTA). A solution of DTT (dithiothreitol, 20 mM) was freshly prepared in phosphate buffer 3999


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KNO3, 200 ppm of NaN3, and 25 mM pH 8.5 phosphate buffer was used. The system was calibrated with poly(methacrylic acid) standards. Samples were dissolved in eluent directly prior to injection. 1 H NMR. 1H NMR (400 MHz) spectra were recorded on a Varian Hg 400 or a Varian 400 spectrometer with a 45° pulse width and at a temperature of 25 °C. All water-soluble polymers were dissolved in D2O, with chemical shifts referenced to the HDO peak at 4.77 ppm. 9 Acquisition parameters included 512 transients and a delay time of 10 s.2 We assume an inherent ±5% error in the integration values from all 1 H NMR measurements. Thermogravimetric Analysis (TGA). TGA measurements were performed on a TA SDT Q600 instrument. The ceramic sample and reference cups were extensively cleaned with a Bunsen burner prior to every run and placed on the balance arms to cool. Once the cups cooled, the instrument’s balance was tared, sample (4−6 mg) was added to the sample cup, and the sample analysis was initialized. Data were collected with a custom heating profile of 10 °C/min ramp to 100 °C, isothermal for 4 h, 10 °C/min ramp to 600 °C, and isothermal for 5 h, except for the Na2CO3 control run (see text). All analyses were run under a stream of air. UV/vis Spectroscopy. UV/vis spectra of the fluorescein-labeled polymers were collected on a Perkin-Elmer Lambda 35 UV/vis spectrometer. Polymer sample (ca. 0.5 mg) was accurately weighed on a Mettler Toledo MX5 microbalance, transferred to a 20 mL scintillation vial with Teflon tape-wrapped threads, and then dissolved in a weighed amount of phosphate buffer (200 mM, pH 8.50). Data from TGA and 1 H NMR analyses were combined with the UV/vis measurement to accurately calculate the number of fluorescein molecules per chain or, alternatively, the effective extinction coefficient per chain. Inductively-Coupled Mass Spectroscopy. ICP-MS measurements were made on a commercial ELAN DRCPlus (PerkinElmer SCIEX) operated under normal plasma conditions. The sample uptake rate was ca. 500 μL/min, and sample size was 1500 μL. A MicroFlow PFA-ST concentric nebulizer (Elemental Scientific, Inc.) was used in all instances. Experiments were performed using an autosampler (Perkin-Elmer AS 91) modified for operation with Eppendorf 2 mL tubes. Lanthanide standards were prepared from 1000 μg/mL PE Pure single-element standard solutions (PerkinElmer, Shelton, CT) by sequential dilution with high-purity deionized water (DIW) produced using a Elix/Gradient (Millipore, Bedford, MA) water purification system. Palladium and platinum standards were prepared from 1000 μg/mL Aristar Plus single-element standard solutions (VWR International) by sequential dilution with 2% HCl. Polymer Metal Content. The metal content of a given sample was determined through a combination of UV/vis spectroscopy and solution inductively coupled plasma mass spectrometry (ICP-MS). Metal-loaded polymer (ca. 0.2 mg) was dissolved in phosphate buffer (200 mM, pH 8.00, 50 μL). A 2 μL aliquot was placed into a NanoDrop ND-1000 spectrophotometer, and the fluorescein absorbance was measured to yield polymer chain concentration. (In addition to chain concentration, this measurement is also an indication of polymer recovery or yield after the metal-loading reaction. This is done by comparing the found chain concentration to the concentration of an unloaded 0.4 mg aliquot of polymer dissolved in 100 μL of buffer.) Next, a 5 μL aliquot of the polymer solution was taken for ICP-MS analysis. Early experiments demonstrated that, with the exception of P(DOTA), the amount of ICP-MS signal for lanthanide-loaded polymers was independent of whether a digestion process was used. Thus, the 5 μL aliquot of polymer solution was directly diluted to 5000 μL with 2% HCl, after which a 5 μL aliquot of that solution was again diluted to 5000 μL with 2% HCl. In contrast, early experiments demonstrated that lanthanide-loaded P(DOTA) and all palladium- and platinum-loaded polymers do require a digestion process in order to observe full metal signal in ICP-MS. Therefore, the 5 μL aliquot of polymer solution was added to a 15 mL centrifuge tube, concentrated HNO3 (250 μL) and concentrated HCl (50 μL) were added, and the solution was heated in an 85 °C bath for 2 h.10 After cooling, the solution was diluted to 5000 μL with 2% HCl, after which a 5 μL aliquot of that solution was again diluted to 5000 μL with 2% HCl. ICP-MS signal was converted to ppb through the concurrent analysis

and transferred to a 2 mL centrifuge tube. A volume of LnCl3 solution (50 mM aqueous solution) containing the desired number of metal equivalents was added. The solution was incubated at 37 °C for 30 min or 3 h while protected from light. At this point, the reaction between P(DTPA) and 1.5 equiv of LaCl3 had completely precipitated as an orange solid (with colorless mother liquor) and could not be redispersed. Next, the solution was concentrated on a 0.5 mL Pall Nanosep 3K Omega spin filter and subsequently washed with tris-buffered saline (25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4) (3 × 300 μL) and water (3 × 300 μL). After the first tris-buffered saline wash, the reaction between P(EDTA) and 1.5 equiv of TbCl3 had precipitated as an orange solid (with colorless mother liquor) and would not redissolve with pipet mixing. After completion of the washes the resultant polymer solution was split into two 2 mL centrifuge tubes and dried on an Eppendorf Vacufuge Plus, and both ca. 0.2 mg aliquots were subsequently stored for further use at room temperature protected from light. Palladium. P(EDTA)−fluorescein, P(DTPA)−fluorescein, P(TTHA)− fluorescein, and P(DOTA)−fluorescein were loaded with palladium. Polymer (0.4 mg) was dissolved in 100 μL of water. A solution of K2PdCl4 (for P(EDTA) 0.305 mg [1.1 equiv per ligand] or 0.555 mg [2 equiv per ligand], for P(DTPA) 0.442 mg [2 equiv per ligand], for P(TTHA) 0.716 mg [4 equiv per ligand], and for P(DOTA) 0.449 mg [2 equiv per ligand]) in HCl (50 mM, 1500 μL) was added. The solution was incubated at room temperature for 2 h or overnight, while protected from light. Approximately 1 h after the addition of the palladium solution the polymer was observed to precipitate as a fluffy yellow precipitate. After the incubation period was complete, the precipitate was spun down by centrifugation (5 min at 11 000 RCF). The mother liquor was pipetted off, after which the precipitate was rinsed, vortexed, and spun down (5 min at 11 000 RCF) with 50 mM HCl (3 × 300 μL). To the precipitate was added phosphate buffer (200 mM, pH 7.00, 300 μL), and the mixture was vortexed until a clear solution formed. This solution was concentrated on a 0.5 mL Pall Nanosep 3K Omega spin filter and subsequently washed once more with phosphate buffer (200 mM, pH 7.00, 300 μL), followed by washes with water (2 × 300 μL). After completion of the washes the polymer solution was split into two 2 mL centrifuge tubes and dried on an Eppendorf Vacufuge Plus, and both ca. 0.2 mg aliquots were subsequently stored for further use at room temperature protected from light. Platinum. P(EDTA)−fluorescein was loaded with platinum. To Pt(en)Cl2 (0.220 mg [0.8 equiv per ligand] or 0.551 mg [2 equiv, per ligand]) in a 2 mL centrifuge tube with locking cap was added a solution of AgNO3 (0.220 or 0.549 mg, respectively [1.9 equiv per Pt(en)Cl2]) in H2O (100 μL). This mixture was incubated in a 60 °C oven for 3 h, with short sonication every half hour, during which the platinum reagent dissolved and AgCl precipitated. The mixture was then incubated at 4 °C for 1 h. Immediately, the platinum solution was filtered through a 0.5 mL Pall Nanosep 0.45 μm MF spin filter to remove AgCl. Polymer (0.4 mg) was dissolved in 100 μL of water and transferred to a 2 mL centrifuge tube, after which platinum solution (100 μL) was added. This solution was incubated at room temperature in the dark for 30 min (0.8 equiv of Pt) or 2 h (2 equiv of Pt), after which it was transferred to a 0.5 mL Pall Nanosep 3K Omega spin filter. The reaction centrifuge tube was rinsed with phosphate buffer (200 mM, pH 7.00, 100 μL), and the rinse was added to the spin filter. The solution was concentrated on the spin filter and subsequently washed with phosphate buffer (200 mM, pH 7.00, 2 × 300 μL) and water (2 × 300 μL). After completion of the washes the polymer solution was split into two 2 mL centrifuge tubes and dried on an Eppendorf Vacufuge Plus, and both ca. 0.2 mg aliquots were subsequently stored for further use at room temperature protected from light. Instrumentation and Characterization. Size Exclusion Chromatography. The nominal molecular weights and polydispersities of all anionic, water-soluble samples were measured with a Viscotek sizeexclusion chromatograph (SEC) equipped with a Viscotek VE3210 UV/vis detector, VE3580 refractive index detector, and Viscotek ViscoGEL G4000PWXL and G2500PWXL columns (kept at 30 °C). The flow rate was maintained at 1.0 mL/min using a Viscotek VE1122 solvent delivery system and VE7510 SEC degasser. An eluent of 0.2 M 4000


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Scheme 2. Synthesis of Metal-Chelating Polymersa

of separate 1 ppb metal standards. Finally, the number of metal atoms per chain was calculated by dividing the metal concentration of the original polymer solution, as calculated from the ICP-MS data, with the polymer chain concentration, as found by NanoDrop UV/vis. Antibody Labeling with Metal-Chelating Polymers. Metallabeled antibodies were prepared as follows. In advance, 0.4 mg of P(EDTA)−maleimide was loaded with palladium (2 equiv, 2 h method) or platinum (0.8 equiv, 0.5 h method), dried into a PCR tube on an Eppendorf Vacufuge Plus, and stored vacuum-packed in a −30 °C freezer. The day of the experiment, an antibody solution at 1 mg/mL in 150 mM sodium phosphate buffer, pH 7.2, and in the absence of bovine serum albumin (BSA) or gelatin was subjected to mild reduction by TCEP (tris(2-carboxyethyl)phosphine) to convert the disulfides in the Fc fragment to thiols. The reduction and subsequent Ab-polymer conjugation steps were performed in 0.5 mL 50K MWCO centrifugal devices (Millipore YM-50). Finally, the maleimide group of the metal-loaded metal-chelating polymer was bound to the thiol groups of the partially reduced Ab in tris-buffered saline (TBS, 25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4). The metal-tagged Ab was washed several times in EDTA-free TBS and stored at +4 °C. Mass Cytometry Bioassays. Goat anti-mouse (GAM) and CD45 antibodies were obtained from Pierce and Biolegend, respectively. KG1a and Ramos cells were obtained from ATCC (American Type Culture Collection, Manassas, VA). GAM was labeled with P(EDTA)− maleimide−Pd and P(EDTA)−maleimide−Pt as described above. To test GAM−P(EDTA)−maleimide−Pd, Ramos cells were incubated with a Rh intercalator11 to identify dead cells and then stained with primary CD45 followed by secondary GAM−P(EDTA)− maleimide−Pd. Washed cells were fixed in 3.7% formaldehyde and counterstained with an Ir intercalator1 for nucleated cell identification. To test GAM−P(EDTA)−maleimide−Pt, two separate aliquots of KG1a cells were prepared. The first aliquot consisted of live cells, and the cells of the second aliquot were fixed 3.7% formaldehyde. Separate aliquots of live and dead cells were stained with CD45 followed by secondary GAM−P(EDTA)−maleimide−Pt. As above, washed cells were fixed in 3.7% formaldehyde and counterstained with an Ir intercalator. Dead cell staining experiments were performed as follows. KG1a cells were killed by 3.7% formaldehyde or heat (5 min at 60 °C). Cells were counted with a hemacytometer and mixtures of live and dead cells were prepared by mixing given proportions of cells from live and dead aliquots of KG1a. These mixtures were first incubated with an Rh intercalator11 and were subsequently stained with a solution (50 μL per each cell pellet of 1 × 106 cells) of a palladium reagent. GAM− P(EDTA)−maleimide−Pd was used at 0.001 mg/mL, and P(EDTA)− fluorescein−Pd was used at 0.01 or 0.1 mg/mL. Finally, as above, washed cells were fixed in 3.7% formaldehyde and counterstained with an Ir intercalator. Labeled cells were analyzed by mass cytometry1 using a CyTOF instrument from DVS Sciences Inc., Markham, Ontario. Mass cytometry is a real-time analytical technique whereby cells or particles are individually introduced into an inductively coupled plasma flame, and each resultant ion cloud is analyzed multiple times by time-offlight mass spectrometry. Dual counting, the combination of digital counting and analogue modes of ion detection, allows a much wider range of ion signal (simultaneous detection of very small and very large signals). Data were collected in FCS 3.0 format and processed by FlowJo (Tree Star Inc., Ashland, OR) software. Standard Error Calculation. Standard errors were calculated using standard error propagation expressions assuming an uncertainty of ±5% in NMR integrations, an inherent ±5% error for each water/ DTPA value and sodium/DTPA value from the TGA measurements, ±2% precision on absorbance values determined with the Nanodrop instrument (manufacturer’s specification), and ±2% error in the metal ion intensities determined by ICP-MS.

a

DTT = dithiothreitol, PB = phosphate buffer, and DMF = dimethylformamide.

This dimeric polymer was obtained by quantitative pendant group transformation of a poly(tert-butyl acrylate) sample with DPn = 79 per phenyl end group prepared by RAFT polymerization, following the procedure reported previously.2 The disulfide group is stable to the various reactions carried out on this polymer. It serves as the source of end-functional thiol groups that can be generated by mild reduction and then further reacted with a bismaleimide linker for attachment to an antibody. Scheme 2 summarizes the polymer transformations described in this paper. In this work, our goal was to investigate commonly available polyaminocarboxylate ligands as carriers of palladium and platinum to create Pd- and Pt-loaded metal-chelating polymers. We have previously shown that the pendant primary amines of the amino-polymer disulfide could be quantitatively functionalized with DTPA groups using DMTMM as a coupling agent. Here we extend these experiments to include the available homologues EDTA, TTHA, and DOTA. In each case, we obtained quantitative ligand attachment, as monitored by 1H NMR. Analysis of the samples by aqueous size exclusion chromatography (SEC) showed no evidence for cross-linking or broadening of the molecular weight distribution. To gauge the success of loading the polymer with metal ions, we needed to be able to determine the molar concentration of polymer molecules as well as the concentration of polymerbound metal ions. One way to facilitate measurement of the polymer concentration was to attach a dye molecule of known molar extinction coefficient to the thiol group at the end of each chain. To proceed, we reduced the polymer disulfide with dithiolthreitol (DTT)2 and then reacted the liberated polymeric thiol with an excess of N-(5-fluoresceinyl)maleimide. Subsequent purification by the spin filter removed excess dye. The characterization of these dye-labeled polymers is described below. Polymer Chain Extinction Coefficients. The syntheses described above were designed to attach a single fluorescein

■

RESULTS AND DISCUSSION Polymer Synthesis. The starting point for the syntheses described here is amino-polymer disulfide shown in Scheme 2. 4001


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Table 1. H2O and Na+ Content and Adjusted Molecular Weights for P(EDTA)−Fluorescein, P(DTPA)−Fluorescein, P(TTHA)−Fluorescein, and P(DOTA)−Fluorescein Polymer Samples Calculated from TGA Analysis

dye to every polymer chain. The success of this approach needed to be tested. In addition, we needed to determine an effective molar extinction coefficient for the polymer-bound chromophore in order to use the UV/vis absorbance of fluorescein as a measure of polymer chain concentration. It is not difficult to prepare polymer solutions of known mass concentration, but the mass concentration must be converted to molar concentration. 1H NMR analysis at points along the synthesis up to and including P(ligand)-disulfide showed that the degree of polymerization was 79 for all four polymers and that every polymer repeat unit was functionalized with a ligand. This information allowed us to calculate Mn values corresponding to the fully protonated carboxylic acid form of the polymers. However, these “1H NMR molecular weights” need to be corrected for both the presence of sodium counterions associated with the pendant groups as well as any water that was not removed during sample lyophilization. Once these values are known, one can calculate adjusted molecular weights2 that include the contributions of sodium and residual moisture to convert mass concentration to molar concentration. This information is available from thermal gravimetric analysis (TGA) of the lyophilized polymer. Thermal Gravimetric Analysis. In our first publication,2 we described TGA experiments employing a linear temperature sweep. From this approach, there was no clear demarcation between the end of water loss and the start of polymer degradation. Here we employed a step-scan approach that separated the water-loss step from the thermal decomposition of the polymer. In this procedure, the samples were held at 100 °C for 4 h to drive off water without degrading the polymer. Then the temperature was increased rapidly and held at 600 °C for 5 h to destroy the polymer without degrading Na2CO3. Na2CO3 is the only nonvolatile product formed in the TGA analysis of the sodium salts of polyaminocarboxylates12 at temperatures less than 800 °C. Control experiments to test the validity of this approach are described in the Supporting Information. Step-scan TGA traces for P(EDTA)−fluorescein, P(DTPA)− fluorescein, P(TTHA)−fluorescein, and P(DOTA)−fluorescein are presented in Figure 1. All four polymer samples showed similar

sample P(EDTA)− fluorescein P(DTPA)− fluorescein P(TTHA)− fluorescein P(DOTA)− fluorescein

H2O per ligand unit

Na+ per ligand unit

2.3

1

H NMR mol wt (Da)b

adjusted mol wt (Da)c

1.7

30800 ± 12%

37200 ± 10%

2.9

2.2

38800 ± 12%

46700 ± 10%

3.2

2.5

48600 ± 12%

57600 ± 10%

2.5

1.6

39700 ± 12%

45900 ± 11%

a

The standard error calculation is described in the Experimental Section. bCalculated from DPn/phenyl end-group at the PtBA stage, attachment of a ligand to each pendant group, and assuming that the ligand groups are fully protonated (not partially neutralized by Na + ions).2 cAn apparent molecular weight that includes the mass contribution of attendant water molecules and sodium counterions.

data in Table 1, one can see that the number of H2O molecules and Na+ per ligand unit increased as the pendant ligand increased in size from EDTA through to TTHA. This is not unexpected as the larger ligands contain more carboxylic acids to attract water and to carry sodium counterions. In our previous report,2 the assumption of 150 °C as the water loss temperature for the linear temperature ramp in the TGA measurements yielded 2.2 H2O and 2.3 Na+ per DTPA unit. This is not very different from the more reliable results presented here. Polymer Chain Extinction Coefficients. Aliquots of all four fluorescein-end-labeled polymers were dissolved in phosphate buffer (pH 8.50) and examined by standard quantitative UV/ vis spectroscopy. All samples showed the visible absorption spectrum of the dianion form of fluorescein.13 This absorbance data were combined with the TGA data to calculate the number of fluorescein molecules per chain, as presented in Table 2. For Table 2. Fluorescein Labeling and Effective Molar Extinction Coefficients of P(EDTA)−Fluorescein, P(DTPA)− Fluorescein, P(TTHA)−Fluorescein, and P(DOTA)− Fluorescein Polymer Samples sample P(EDTA)− fluorescein P(DTPA)− fluorescein P(TTHA)− fluorescein P(DOTA)− fluorescein

fluorescein molecules per chain

effective extinction coeff (M−1 cm−1, at 494 nm)

0.99 ± 0.11

87200 ± 9800

0.96 ± 0.11

84100 ± 9400

1.14 ± 0.13

100000 ± 11000

1.13 ± 0.13

99600 ± 12000

this calculation a fluorescein extinction coefficient of 88 000 M−1 cm−1 was used.13 The results for P(EDTA) and P(DTPA) are exceptionally good, almost exactly one dye per polymer molecule. In contrast, the measured absorbance values for P(TTHA) and P(DOTA) are somewhat higher than expected, leading to calculated values of 1.14 and 1.13 (±0.13) dyes per polymer, respectively. These values are still within experimental error of 1 fluorescein molecule per chain. We used this data to calculate effective molar extinction coefficient values for each polymer. These values are presented in Table 2.

Figure 1. Step-scan TGA traces of P(EDTA)−fluorescein, P(DTPA)− fluorescein, P(TTHA)−fluorescein, and P(DOTA)−fluorescein, with temperature displayed on the right-hand y-axis. All four polymer samples display similar traces, with essentially flat baselines at the end of both isothermal (100 and 600 °C) periods.

traces, with essentially flat baselines at the end of both isothermal periods. The calculated amounts of water, sodium ions, and adjusted molecular weights are presented in Table 1. From the 4002


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solubility. Alric and co-workers observed a similar solubility problem with gold nanoparticles covered with DTPA−bisamide ligands. When fully loaded with gadolinium, the loss of anionic repulsion led to agglomeration. This problem was avoided by only loading Gd3+ into some of the ligands, leaving unloaded ligands to provide anionic repulsion.15 In the case of the DTPA−monoamide polymer, the extra carboxylic acid of the pendant group provides the net anionic charge per repeat unit to promote solubility in water. Precipitation was also expected with the DOTA polymer, as it too has three carboxylic acid groups per ligand; however, the polymer remained soluble. The data presented in Table 3 indicate that even with a 3 h metal-loading time, some DOTA units remain unloaded with a terbium ion, presumably in part due to the slow loading kinetics of DOTA−monoamide.16 Thus, the presence of unloaded DOTA units gave the polymer chain a net anionic charge, which we assume is important in maintaining water solubility. We also investigated the loading of lanthanum and ytterbium into the DTPA polymer. The results with ytterbium were quite similar to those with terbium. However, the lanthanum-loaded DTPA polymer precipitated during the loading step. Ternovaya and co-workers17 have shown it is possible to prepare binuclear lanthanide−DTPA complexes, depending on which lanthanides are used and the order of the lanthanide addition. Thus, in our reaction with 1.5 equiv of lanthanum chloride, an average of more than one La3+ was taken up by each DTPA group. This led to charge neutrality, with an attendant loss of water solubility. Alternatively, when we repeated the reaction with only 0.8 equiv of lanthanum, the polymer maintained water solubility throughout. From these results we have decided that in the future our standard procedure for loading any lanthanide metal will utilize 0.8 equiv. The data presented in Table 3 shows the degree of metal loading as well as the aqueous SEC data for all metal-loading experiments. Interestingly, the apparent Mn (aqueous SEC) of lanthanide-loaded polymers decreased significantly. This

Metal-Loading Experiments. The ability of the polymers described above to bind Pd2+ and Pt2+ ions was determined by a combination of ICP-MS measurements to determine the metal ion concentration in the solution and UV/vis measurements to determine polymer concentration via the absorbance of the fluorescein end-group. For the UV/vis measurements, control experiments were needed to establish an appropriate pH where the fluorescein chromophore would be in its dianionic form. This is necessary because the absorption spectrum of fluorescein depends on its level of deprotonation. The dye itself in water has pKa values of 2.2, 4.3, and 6.4,13 but these pKa values become raised when the fluorescein is located in a negatively charged microenvironment.14 The negative charge associated with the metal-laden polymers described herein may have a similar effect on the pKa values of the fluorescein endgroup. Control experiments were carried out on a sample of P(DTPA)−fluorescein loaded with terbium and then dissolved in buffers of different pH and analyzed by UV/vis. These spectra are presented in Figure S4 of the Supporting Information. At a pH of 8.0 or greater, fluorescein is fully in the dianionic form. Therefore, UV/vis measurements of chain concentration were carried out in a 200 mM, pH 8.00 phosphate buffer. In parallel, size exclusion chromatography (SEC) measurements were used to test for water solubility as well as polymer cross-linking by the formation of metal-ion bridges between chains. Lanthanides. We began our metal-loading experiments with lanthanide ions. P(EDTA)−fluorescein, P(DTPA)−fluorescein, and P(DOTA)−fluorescein were treated with 1.5 equiv of terbium chloride following the procedure described in ref 2. During the washing steps, the DTPA and DOTA polymers remained soluble, but the EDTA polymer irreversibly precipitated. The precipitation of the EDTA polymer demonstrates how net anionic charge promotes water solubility. Each EDTA−monoamide pendant group has only three carboxylic acids and will thus be neutral when loaded with a lanthanide(III) ion. This charge neutrality leads to a loss of water

Table 3. Metal Content, Polymer Yield, and Aqueous SEC Data of All Metal-Loading Reactions with P(EDTA)−Fluorescein, P(DTPA)−Fluorescein, P(TTHA)−Fluorescein, and P(DOTA)−Fluorescein sample P(EDTA) P(DTPA) P(TTHA) P(DOTA) P(EDTA) P(DTPA) P(DTPA) P(DTPA) P(DTPA) P(DOTA) P(DOTA) P(EDTA) P(EDTA) P(EDTA) P(DTPA) P(TTHA) P(DOTA) P(EDTA) P(EDTA)

reagent

TbCl3 LaCl3 LaCl3 TbCl3 YbCl3 TbCl3 TbCl3 K2PdCl4 K2PdCl4 K2PdCl4 K2PdCl4 K2PdCl4 K2PdCl4 Pt(en)(H2O)22+ Pt(en)(H2O)22+

metal equiv

1.5 1.5 0.8 1.5 1.5 1.5 1.5 1.1 2 2 2 4 2 0.8 2

time (h)

0.5 0.5 0.5 0.5 0.5 0.5 3 2 2 16 16 16 2 0.5 2

polymer yield (%)

87 71 83 82 75 68 77 99 68 66 70 87 80

± ± ± ± ± ± ± ± ± ± ± ± ±

5 3 5 6 6 3 4 6 3 3 6 4 4

metal/chain

metal/ligand

Mn (aq SEC, Da)c

20 700 23 600 25 100 18 200 complete precipitation on metal loading complete precipitation on metal loading 60 ± 9 0.76 ± 0.11 18 100 77 ± 11 0.97 ± 0.13 16 600 71 ± 10 0.90 ± 0.13 17 800 64 ± 9 0.81 ± 0.12 10 600 69 ± 10 0.87 ± 0.13 10 800 52 ± 7 0.65 ± 0.09 18 100 79 ± 11 1.00 ± 0.14 19 200 76 ± 11 0.96 ± 0.14 19 100 119 ± 17 1.5 ± 0.2 18 200 157 ± 22 2.0 ± 0.3 22 400 115 ± 17 1.5 ± 0.2 17 100 47 ± 7 0.60 ± 0.09 19 500 90 ± 13 1.14 ± 0.16 18 500

PDI (aq SEC) 1.20 1.20 1.19 1.22

1.23 1.24 1.24 1.29 1.28 1.31 1.34 1.28 1.34 1.37 1.30 1.22 1.34

a

A full error propagation calculation was performed using the sources of error described in the Experimental Section. bData are organized by metal: unloaded, lanthanides, palladium, and platinum. cApparent molecular weights from aqueous SEC are relative to poly(methacrylic acid) standards. 4003


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indicates a smaller hydrodynamic volume in solution, likely due to the shielding effect of the cationic metal centers chelated by the polymer. Palladium. A number of considerations went into the design of the palladium-loading reaction. First, the reagent K2PdCl4 is a water-soluble salt easily produced from palladium sponge.18 This is an important consideration if this chemistry is to be extended to enriched and purified (stable) isotopes of palladium. Second, the solvent used for the metal-binding step was 47 mM HCl. Acidic conditions were chosen to prevent the polynuclear hydrolysis of K2PdCl4.8 We also tried to anticipate at this point reaction conditions that would be compatible with a terminal maleimide on the polymer, to be used in a subsequent step for attachment of the metal-containing polymer to antibodies. According to the literature, maleimide hydrolysis only occurs at high pH.19 In addition, we performed a control experiment wherein a sample of P(DTPA)−maleimide2 was dissolved in 47 mM HCl at 1 mg/mL concentration and incubated for 2 h, then washed in a 3 kDa spin filter with phosphate buffer (pH 7.00, 200 mM, 1 × 11 mL) and water (3 × 11 mL), and finally freeze-dried. Reanalysis by 1H NMR showed the same level of maleimide signal as before the treatment. The Pd2+ binding reaction was carried out on 0.4 mg of polymer in a total reaction volume of 1600 μL. This relatively dilute solution for the reaction was chosen to minimize the chance of polymer precipitation prior to metal chelation.20 Control experiments where 0.4 mg of each polymer was dissolved in 1600 μL of 50 mM HCl showed no visible precipitation over a period of weeks. Conveniently, however, the metal-loaded polymer itself precipitated out of solution ∼1 h after the addition of K2PdCl4. After rinsing several times with 50 mM HCl, the polymer was redissolved in phosphate buffer (pH 7.00), placed in a spin filter, and washed several times with phosphate buffer and water. In the literature, the mechanism of complex formation between polyaminocarboxylates and K2PdCl4 (or K2PtCl4) is normally described as a two-step process.4,6 In the first step, tertiary amino groups displace chloride ligands. For example, EDTA would form a bidentate complex with the metal center, whereas DTPA would form a tridentate complex. In the second step, the remaining chloride ligands are removed through (a) the intentional addition of Ag+ ions or (b) the reprecipitation of the complex in a solution that does not contain chloride ion. These methods allow carboxylic acid group(s) of the ligand to chelate the tetracoordinate, square-planar metal center as well.4,6 In this work, we expect that the polymers initially chelate palladium through tertiary amines. After dissolution in phosphate buffer, the spin filter washes with phosphate buffer and water will remove the remaining chloride ligands in a process analogous to reprecipitation in a solution that does not contain chloride ion. Owing to the ease of this palladium-loading reaction, all four polymers and a number of conditions were investigated, as summarized in Table 3. Perhaps the most interesting result is the behavior of the DTPA and TTHA polymers: in the presence of excess Pd2+, every DTPA unit chelates an average of 1.5 Pd, and every TTHA unit chelates an average of 2.0 Pd. Bimetallic complexes of palladium and TTHA have been previously reported.7 However, small-molecule DTPA complexes of palladium are usually prepared and purified as 1:1 complexes, where the metal is complexed by three tertiary amines and one carboxylic acid.8,5 In our experiments, it appears that each polymeric DTPA group chelates one

palladium with two of its tertiary amines and two of its carboxylic acids and in addition cooperatively chelates half of another metal center with a single tertiary amine and carboxylic acid. Scheme 3 shows one possible way this could occur. Scheme 3. Cooperative Chelation of Palladium by P(DTPA)a

a

Each DTPA group chelates an average of 1.5 palladium metal centers. Each metal center is square-planar, although not drawn as such. This is only one of several possible ways this cooperative chelation can be drawn.

It was our hope that DOTA, with its cyclic structure, would form a 1:1 complex with palladium that involved chelation only through the four tertiary amines. This result is desirable because palladium, being a soft metal, forms stronger bonds with amines than with carboxylic acids.4,6 While not directly comparable, palladium forms a 1:1 complex with 1,4,8,11-tetrathiacyclotetradecane, a cyclic thioether.21 To our knowledge, there are no reports of palladium or platinum complexes with DOTA. Unfortunately, when we reacted P(DOTA)−fluorescein with 2 equiv of K2PdCl4, we found 1.5 Pd per ligand. This shows that P(DOTA), like the other polymers, has a propensity to form complexes with mixed chelation by amino and carboxylic acid groups. While P(DTPA) and P(TTHA) are attractive options because they carry more palladium, we decided to use palladium-loaded P(EDTA) in the bioassay described below. The best loading conditions for P(EDTA) were 2 equiv of K2PdCl4 per EDTA and 2 h incubation. The recovered yield of polymer was somewhat lower than for longer reaction times, but reaction time is conveniently shorter. Platinum. Early experiments with platinum involved K2PtCl4 and a procedure analogous to that used for palladium. Upon treatment of the polymer with K2PtCl4 in the presence of aqueous HCl, some precipitation was observed. However, the precipitate did not fully dissolve in phosphate buffer. The precipitate disappeared after several phosphate buffer spin filter washes. Upon analysis of the product, we found ca. 50% polymer yield, a PDI of ≥1.70 by aqueous SEC, and only ca. 10 platinum atoms per chain. These results with K2PtCl4 showed poor metal loading as well as significant interchain crosslinking. This is not surprising; platinum is more kinetically inert than palladium, and it is this inertness which makes platinum complexes such as cisplatin suitable as cross-linking anticancer drugs.22 To overcome this problem, it was necessary to activate a platinum complex into a more reactive form. K2PtCl4 can be made more reactive to substitution by replacing chloride with more labile ligands. One not very attractive option is to prepare K2PtCl4 as a dilute solution in water and age it for a number of days. This will replace some of the chloride ligands with reactive aqua ligands. These species are an 4004


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Figure 2. Performance of GAM-Pd in a bioassay. (A) 191Ir vs 193Ir signal from the iridium DNA intercalator is plotted to select for cell events. (B) Selected cell events from (A) are identified as live or dead by plotting 103Rh vs 193Ir. High 103Rh signal shows a cell was dead, and low 103Rh signal shows the cell was live. (C) Cells with high 103Rh signal also have high palladium signal. (D) Cells with low 103Rh signal also have low palladium signal.

ill-defined mixture of starting material and mono- and diaqua complexes and upon aging can lead to polynuclear hydrolysis of the platinum species.23,24 Nevertheless, we attempted to load P(EDTA) with a solution of K2PtCl4 aged 36 h at room temperature. We found an apparent improvement in metal ion content (24 ± 3 Pt per chain), but also a 740 Da peak in the SEC chromatograph that was possibly a product of polynuclear hydrolysis. Another option was to treat K2PtCl4 with 4 equiv of KI to convert it to the more reactive K2PtI4 reagent.25 Unfortunately, our attempt with this strategy yielded no platinum per chain, as well as an exceedingly fine black precipitate, presumably Pt(0).26 According to the literature, it is possible to prepare the tetraaqua complex of platinum. We did not examine this approach, as the procedure is complicated, and the complex requires at least 0.5 M of perchloric acid to remain stable.27,28 A more accessible option is to prepare Pt(en)(H2O)22+, which is stable in water at acidic and neutral pH, and can be prepared in situ by treating Pt(en)Cl2 with 2 equiv of Ag+ at 60 °C for 2 h.29,30 The approach is also attractive for possible future experiments in which Pt(en)Cl2 could be prepared from isotopically enriched platinum sponge in two major steps: platinum sponge converted to K2PtCl4,18 and then the reaction of K2PtCl4 with 1 equiv of ethylenediamine.29 As a test of this approach, P(EDTA)−fluorescein was reacted with 2.0 or 0.8 equiv of Pt(en)(H2O)22+, with results presented in Table 3.

The desired reaction mechanism is for the two tertiary amines of each EDTA group to displace the two aqua ligands, resulting in one platinum atom per ligand. However, reaction with 2.0 equiv for 2 h yielded 1.14 ± 0.16 atoms per ligand, which suggests that the polymer may form complexes with mixed chelation by amino and carboxylic acid groups. We also performed a loading reaction with 0.8 equiv of metal and a half hour reaction time to yield 47 ± 7 platinum atoms per chain. Mass Cytometry Experiments. For application in a mass cytometric bioassay, our approach is to prepare metal-chelating polymers with a maleimide group for conjugation to an antibody. To this end, we synthesized a sample of P(EDTA) with a maleimide end-group, as shown in Scheme 2. Next, aliquots of P(EDTA)−maleimide were loaded with palladium using 2 equiv per polymeric EDTA group and 2 h reaction time and with platinum using 0.8 equiv of metal and 0.5 h reaction time. We assumed that the degree of metal-loading for the maleimide-terminated polymer was comparable to that obtained above using the same conditions and the fluorescein-terminated polymer. In the next step, we treated two separate aliquots of goat anti-mouse (GAM) with TCEP to partially reduce disulfides in the hinge region of the antibody. The antibody thiol groups generated in this way were then covalently reacted with the maleimide end-groups of the two polymer samples to form two antibody−polymer conjugates: GAM-Pd and GAM-Pt. 4005


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Figure 3. Performance of GAM-Pt in a bioassay. (A and C) 191Ir vs 193Ir is plotted to select cell events for fixed and live cells, respectively. (B) Selected cell events from (A) are plotted for platinum signal ( 195Pt) against 193Ir. A large proportion have high platinum signal, and a smaller proportion do not. (D) Selected cell events from (C) are plotted for platinum signal. The live cells show low platinum signal.

palladium counts of ca. 102 or less as low palladium signal and counts above this value as high signal. These results show two problems with the performance of GAM-Pd. The first problem is that the live cells were not labeled with palladium. This indicates that, after conjugation of the metal-carrying polymer to GAM, GAM had lost its binding affinity to CD45. The second problem is that dead cells were labeled with palladium. This result suggests that some aspect of the antibody−polymer conjugate is capable of associating with dead cells. Platinum. The performance of GAM-Pt was tested in a similar assay. We began with two aliquots of live KG1a cells. The second aliquot was set aside. The cells of the first aliquot were fixed with formaldehyde. Next, the separate aliquots of fixed and live cells were separately stained with primary antibody CD45 followed by secondary GAM-Pt. Finally, as above, cells were fixed and incubated with an Ir intercalator. Results for the GAM-Pt bioassay are presented in Figure 3. In parts A and C the cell event plots (191Ir vs 193Ir) are presented for the fixed and live cells, respectively. The cell events from these plots are further analyzed in parts B and D, respectively, where the platinum signal (195Pt) is plotted against 193 Ir. In part B, ca. 3/4 of the fixed cells have high platinum signal, and the rest do not. In part D, the live cells have only a low platinum signal.

Palladium. The performance of GAM-Pd was tested in a bioassay with Ramos cells. Cells were first incubated with a Rh intercalator11 to identify dead cells. A dead (or fixed) cell characteristically has a permeable membrane, which allows the Rh intercalator to enter the cell and intercalate with the nuclear DNA. On the other hand, the Rh intercalator does not appreciably stain live cells because it is incapable of crossing an intact live cell membrane. Next, the cells were stained with a primary antibody, CD45, and then the cell-bound CD45 was in turn stained with GAM-Pd. Finally, cells were fixed and incubated with an iridium DNA intercalator. As all cells are fixed at this final stage, the Ir intercalator enters all cells and intercalates with the nuclear cell DNA. Results for the GAM-Pd bioassay are presented in Figure 2. In part A, 191Ir vs 193Ir is plotted to select for signals due to intact cells. Mass cytometry events with high 191Ir and 193Ir signal are identified as cell events. In contrast, events with low 191Ir and 193Ir signal are dismissed as debris and cell fragments. The selected cell events from part A are further analyzed in part B. In part B, live and dead cells are differentiated by plotting 103Rh vs 193Ir. The events with high 103Rh signal are dead cells, while those with low 103 Rh signal are live cells. In parts C and D, the palladium signal (106Pd vs 108Pd) is plotted for the high and low 103Rh populations from part B. In part C, we see that the cells with high 103Rh signal also have high palladium signal. In part D, we see that cells with low 103Rh signal also have low palladium signal. We consider 4006


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Figure 4. P(EDTA)−fluorescein−Pd dead cell staining experiment on 50% live cell mix. (A) 191Ir vs 193Ir is plotted to select for cell events. (B) Selected cell events from (A) are plotted for palladium signal ( 106Pd) against 193Ir to show two populations: dead cells with high palladium signal and live cells with low palladium signal. (C) Selected cell events from (A) are plotted for rhodium signal ( 103Rh) to identify dead and live cells. The populations of live and dead cells from (B) and (C) are in good agreement. (D) Rh-identified dead cells from (C) have correspondingly high palladium (106Pd vs 108Pd) signal. (E) Rh-identified live cells from (C) have correspondingly low palladium ( 106Pd vs 108Pd) signal, except for a small proportion of false positives.

ligands. Since carboxylic acid ligands are hard, they form a relatively weak bond with these metals; this bond is susceptible to hydrolysis and/or exchange with a more appropriate soft ligand. Platinum anticancer drugs usually have two carboxylic acid ligands (or one bidentate acid ligand) for this purpose. 22 Palladium, the relatively more reactive metal, can even undergo hydrolysis with soft

The results with GAM-Pt show the same problems as those with GAM-Pd. A notable difference, however, is that GAM-Pd appears to be more efficient than GAM-Pt at staining dead cells. Effect of Soft Metal Atoms. These curious results are most likely due to the soft nature of palladium and platinum ions. With P(EDTA), the palladium and platinum ions are chelated by a mixture of tertiary amino and carboxylic acid 4007


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Figure 5. Summary of the performance of GAM-Pd and P(EDTA)−fluorescein−Pd as dead cell stains. Live KG1a cells were mixed with dead KG1a cells at the % values shown on the X-axis. GAM-Pd was used at 0.001 mg/mL, and P(EDTA)−fluorescein−Pd was used at 0.1 mg/mL.

bidentate ligands, making some palladium complexes useful as catalysts for peptide cleavage. 31 As such, our idea is that the aforementioned problems are caused by metal centers along the polymer backbone. We suspect that these metal centers form mixed complexes with soft ligands along the antibody, and if this chemical modification occurred near the antigen-binding region, the antibody would lose its affinity.32 In a related fashion, the metal centers form mixed complexes with ligands encountered on the inside and/or outside of dead cells. Since we did not observe significant staining of live cells, it is clear that the formation of these bonds is encouraged by features specific only to dead cells. One notable feature of apoptotic and dead cells is a permeabilized cell membrane. A permeabilized cell membrane would allow the antibody− polymer conjugate to enter the cell, thus permitting the formation of mixed complexes with soft ligands found therein. Another notable feature connected to apoptosis and cell death is a change in the surface charge of the inner and outer cell membranes. In a live cell, the negatively charged lipid phosphatidylserine (PS) preferentially populates the inner membrane. A disruption of this asymmetry, which involves some PS flipping to the outer membrane, changes the surface charge of both inner and outer membranes. This change is connected to apoptosis33 and plays an important role in the electrostatic membrane association of proteins containing polycationic clusters or polybasic domains.34−36 In this work, soft ligands located in areas of the inner membrane with reduced PS may be more susceptible to the formation of mixed metal complexes. The palladium- and platinum-loaded metal-chelating polymers of this work are negatively charged, since each complex of Pd2+ or Pt2+ with (EDTA-monoamide)3− has a net negative charge. As such, it will be more electrostatically favorable for a polymer to approach an area with a reduced level of PS. Nothing would result if the polymer was loaded with hard lanthanide ions; however, the close contact between a palladium- or platinum-

loaded polymer and the inner cell membrane may encourage mixed metal complex formation. Palladium Polymers as an Apoptotic/Dead Cell Stain. The above results suggest an alternative application of palladiumcarrying polymers as a dead cell stain. To this end, mixtures of live and dead KG1a cells were prepared, incubated with the Rh intercalator to identify dead cells, and then stained with either GAM-Pd or P(EDTA)−fluorescein−Pd. Finally, as above, cells were fixed and incubated with an Ir intercalator to identify cell events. The result for the 50% live cell mix with P(EDTA)− fluorescein−Pd is presented in Figure 4. In part A, 191Ir vs 193Ir is plotted to select for cell events. In part B, these selected cell events are plotted for palladium signal (106Pd) vs 193Ir. This plot identifies two populations: a dead cell population characterized by high palladium signal, and a live cell population with low palladium signal. In part C, we present a similar plot in which rhodium signal (103Rh) of the Rh intercalator is used to identify dead cells. The populations of live and dead cells identified by Pd and Rh are in good agreement. In part D, the Rh-identified dead cells are shown to have correspondingly high palladium signal (106Pd vs 108Pd). In part E, the Rh-identified live cells are shown to have correspondingly low palladium signal (106Pd vs 108Pd), except for a small proportion of false positives. These false positives may be due to palladium binding to cell surface thiols of live cells. 37 Figure 5 summarizes the performance of GAM-Pd and P(EDTA)−fluorescein−Pd as dead cell stains. The x-axis shows the proportion of live cells added to each KG1a live/dead mix. Both Pd reagents performed well. When compared to the polymer-labeled GAM antibody, the polymer by itself required a higher concentration to yield an acceptable signal. It also showed a slightly higher number of cells with false positives.

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SUMMARY Metal-chelating polymers with four different polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) were synthesized from a common amino-polymer precursor. These polymers 4008


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were characterized by 1H NMR measurements to determine degree of polymerization and ligand functionalization and then were end-labeled with N-(5-fluoresceinyl)maleimide. The fluorescein-terminated polymers were characterized by a combination of UV/vis and TGA to find the number of dye molecules per chain. For all four samples we found 1 dye molecule per chain, within experimental error. Different metalloading conditions were investigated for these polymers, using chloride salts of three different lanthanide isotopes as well as palladium and platinum salts. Aqueous SEC measurements were used to monitor polymer solubility and to establish the absence of significant metal-ion-induced chain cross-linking. The number of metal atoms per polymer chain was determined by a combination of UV/vis and conventional ICP-MS measurements. Metal-loading experiments with La3+, Tb3+terbium, and Yb3+ yielded important results. First, we found that a net anionic charge is important for maintaining water solubility. Second, we found that La3+ is capable of forming bimetallic complexes with DTPA,17 which led to a loss of solubility. This result can be avoided by loading the polymers with less than 1 equiv of metal ion per DTPA group. Conditions were developed for gently loading these polymers with palladium and platinum ions. Palladium was loaded through the use of K2PdCl4 in 47 mM HCl, while platinum was loaded through the use of an activated species, Pt(en)(H 2O)22+, in water. We were able to load all four polymers with palladium and loaded the EDTA polymer with platinum. We imagine that in these samples metal binding involved interaction of the Pd and Pt ions with both the tertiary amines and carboxylic acids of the chelating group. When we employed P(EDTA)−maleimide loaded with palladium or platinum in mass cytometric bioassays, we found curious results. The secondary antibody−polymer conjugate lost its affinity for the target primary antibody and instead bound selectively to dead cells. We believe this occurs because the soft metal atoms are not sufficiently sequestered by their ligands, and thus these palladium or platinum atoms form new bonds with soft ligands found inside and/or outside dead cells. This was a disappointing result in terms of our initial objective of creating new metal-containing polymers to increase the multiplexing capabilities of immunoassays based on mass cytometry. Nevertheless, we discovered that these polymers, particularly the palladium-loaded polymer and antibody−polymer conjugate, act as effective and sensitive stains for dead cells. Future work will be directed toward ligand systems capable of fully sequestering these soft metal atoms for applications in assays by mass cytometry.

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ACKNOWLEDGMENTS The authors thank NSERC Canada, DVS Sciences, Amgen, and the Province of Ontario for their support of this research. We also thank Mr. I. Herrera for helpful discussions.

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REFERENCES

(1) Bandura, D. R.; Baranov, V. I.; Ornatsky, O. I.; Antonov, A.; Kinach, R.; Lou, X.; Pavlov, S.; Vorobiev, S.; Dick, J. E.; Tanner, S. D. Anal. Chem. 2009, 81, 6813−6822. (2) Majonis, D.; Herrera, I.; Ornatsky, O.; Schulze, M.; Lou, X.; Soleimani, M.; Nitz, M.; Winnik, M. A. Anal. Chem. 2010, 82, 8961− 8969. (3) Lou, X.; Zhang, G.; Herrera, I.; Kinach, R.; Ornatsky, O.; Baranov, V.; Nitz, M.; Winnik, M. A. Angew. Chem., Int. Ed. 2007, 46, 6111−6114. (4) Busch, D. H.; Bailar, J. C. J. Am. Chem. Soc. 1956, 78, 716−719. (5) Grevtsev, A. M.; Zheligovskaya, N. N.; Popov, L. V.; Spitsyn, V. I. Russ. Chem. Bull. 1977, 26, 867−869. (6) Grevtsev, A. M.; Zheligovskaya, N. N.; Popov, L. V.; Spitsyn, V. I. Russ. Chem. Bull. 1978, 27, 213−215. (7) Napoli, A. Talanta 1984, 31, 153−154. (8) Anderegg, G.; Malik, S. C. Helv. Chim. Acta 1976, 59, 1498− 1511. (9) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512−7515. (10) Ashoka, S.; Peake, B. M.; Bremner, G.; Hageman, K. J.; Reid, M. R. Anal. Chim. Acta 2009, 653, 191−199. (11) Tanner, S. D.; Ornatsky, O.; Bandura, D. R.; Baranov, V. I. Spectrochim. Acta, Part B 2007, 62, 188−195. (12) Esteban, M. F. G.; Vizcaino, M. C. P.; Gonzalez-Vilchez, F. Thermochim. Acta 1983, 62, 257−265. (13) Klonis, N.; Sawyer, W. H. J. Fluoresc. 1996, 6, 147−157. (14) Agi, Y.; Walt, D. R. J. Polym. Sci., Part A: Polym. Chem. 1997, 35, 2105−2110. (15) Alric, C.; Taleb, J.; Duc, G. L.; Mandon, C.; Billotey, C.; MeurHerland, A. L.; Brochard, T.; Vocanson, F.; Janier, M.; Perriat, P.; Roux, S.; Tillement, O. J. Am. Chem. Soc. 2008, 130, 5908−5915. (16) Keire, D. A.; Kobayashi, M. Bioconjugate Chem. 1999, 10, 454− 463. (17) Ternovaya, T. V.; Kostromina, N. A.; Kundrya, V. T. Zh. Neorg. Khim. 1975, 20, 2957−63. (18) Livingstone, S. E. Synth. React. Inorg. Met.-Org. Chem. 1971, 1, 1−7. (19) Hermanson, G. Bioconjugate Techniques, 2nd ed.; Academic Press: Amsterdam, 2008. (20) Battaglia, G.; Cigala, R. M.; Crea, F.; Sammartano, S. J. Chem. Eng. Data 2008, 53, 363−367. (21) Bell, M. N.; Blake, A. J.; Gould, R. O.; Holder, A. J.; Hyde, T. I.; Lavery, A. J.; Reid, G.; Schroder, M. J. Inclusion Phenom. Macrocyclic Chem. 1987, 5, 169−172. (22) Jones, C. Medicinal Applications of Coordination Chemistry; Royal Society of Chemistry: Cambridge, 2007. (23) Sanders, C. I.; Martin, D. S. J. Am. Chem. Soc. 1961, 83, 807− 810. (24) Lederer, M.; Leipzig-Pagani, E. Anal. Chim. Acta 1997, 350, 203−208. (25) Ziegler, C. J.; Silverman, A. P.; Lippard, S. J. J. Biol. Inorg. Chem. 2000, 5, 774−783. (26) Oksanen, A.; Kivekäs, R.; Lumme, P.; Laitalainen, T. Acta. Crystallogr., Sect. C: Cryst. Struct. Commun. 1991, 47, 719−722. (27) Elding, L. I. Inorg. Chim. Acta 1976, 20, 65−69. (28) Groening, O.; Elding, L. I. Inorg. Chem. 1989, 28, 3366−3372. (29) Summa, N.; Schiessl, W.; Puchta, R.; van Eikema Hommes, N.; van Eldik, R. Inorg. Chem. 2006, 45, 2948−2959. (30) Djinovic, V. M.; Galanski, M.; Arion, V. B.; Keppler, B. K. Dalton Trans. 2010, 39, 3633−3643. (31) Ašanin, D. P.; Rajković, S.; Molnar-Gabor, D.; Djuran, M. I. Monatsh. Chem. 2004, 135, 1445−1453.

ASSOCIATED CONTENT

S Supporting Information *

Control experiments for the TGA step-scan approach, a description of the aqueous SEC measurements for metalloaded polymers, and UV/vis spectra of P(DTPA)−fluorescein− Tb at different pH. This material is available free of charge via the Internet at http://pubs.acs.org.

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(32) Paik, C. H.; Murphy, P. R.; Eckelman, W. C.; Volkert, W. A.; Reba, R. C. J. Nucl. Med. 1983, 24, 932−936. (33) Kay, J. G.; Grinstein, S. Sensors 2011, 11, 1744−1755. (34) McLaughlin, S.; Murray, D. Nature 2005, 438, 605−611. (35) Heo, W. D.; Inoue, T.; Park, W. S.; Kim, M. L.; Park, B. O.; Wandless, T. J.; Meyer, T. Science 2006, 314, 1458−1461. (36) Yeung, T. Science 2006, 313, 347−351. (37) Sahaf, B.; Heydari, K.; Herzenberg, L. A.; Herzenberg, L. A. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 4001−4005.

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