Gd(iii)–Pt(iv) theranostic contrast agents for tandem MR imaging and chemotherapy†

Pt(iv) prodrugs have emerged as versatile therapeutics for addressing issues regarding off-target toxicity and the chemoresistance of classic Pt(ii) drugs such as cisplatin and carboplatin. There is significant potential for Pt(iv) complexes to be used as theranostic agents, yet there are currently no reported examples of Gd(iii)–Pt(iv) agents for simultaneous MR imaging and chemotherapy. Here we report the synthesis, characterization, and in vitro efficacy of two Gd(iii)–Pt(iv) agents, GP1 and GP2. Both agents are water soluble and stable under extracellularly relevant conditions but are reduced under intracellular conditions. Both are cytotoxic in multiple cancer cell lines, cell permeable, and significantly enhance the T1-weighted MR contrast of multiple cell lines. Thus, GP1 and GP2 are promising agents for tandem MR imaging and chemotherapy and provide a versatile platform through which future Gd(iii)–Pt(iv) agents can be developed.

Minispec v 2.51 Rev.00/NT software (Bruker Biospin, Billerica, MA, USA) operating at 1.41 T (60MHz) and 37 o C. Measurements were made using an inversion recovery pulse sequence (T 1 _ir_mb) using the following parameters: 4 scans per point, 10 data points, monoexponential curve fitting, phase cycling, 10 ms first pulse separation, and a recycle delay and final pulse separation ≥ 5 T 1 . 10 L aliquots of each solution were taken for ICP-MS analysis to determine the concentration of Gd(III).
Relaxivity Measurements at 7 T. GP1 and GP2 and 1 were dissolved in 200 L PBS or 5 mM GSH and each solution was serially diluted to make solutions of varying concentration. 25 L of each solution were pipetted into flame sealed Pasteur pipettes. The pipette tips containing solution were scored, separated, and sealed with parafilm to make small capillaries containing solution. These capillaries were imaged using a Bruker PharmaScan 7 T MR imaging spectrometer (Bruker BioSpin, Billerica, MA, USA). T 1 relaxation times were measured using a rapid-acquisition rapid-echo (RARE-VTR) T 1 -map pulse sequence with static T E (10 ms) and variable T R (100, 200, 400, 500, 750, 1000, 2500, 7500, and 1000 ms) values. Imaging parameters were as follows: field of view (FOV) = 25 x 25 mm 2 , matrix size (MTX) = 256 x 256, number of axial slices = 5, slice thickness (SI) = 1.0 mm, and averages (NEX) = 4. T 1 analysis was carried out using the image sequence analysis tool in Paravision 5.0 pl2 software (Bruker, Billerica, MA, USA) with monoexponential curve-fitting of image intensities of selected regions of interest (ROIs) for each axial slice.
Stability of GP1 and GP2 in various aqueous media. Aliquots of GP1 and GP2 were prepared from a stock with known mass checked by ICP-MS. An aliquot of each was dissolved in H 2 O and analyzed by HPLC-MS using an Atlantis C18 column (4.6 × 250 mm, 5 μm) and the following method: MeCN held at 0% for 3 min followed by a 15 min ramp to 100% MeCN (GP1 retention time: 10.9 min, GP2 retention time: 11.6 min). Aliquots of both were additionally dissolved in PBS, MEM, RPMI-1640, pH 5 H 2 O, PBS with 10 units/mL porcine liver esterase, 5 mM glutathione, and 5 mM sodium ascorbate, incubated in a shaker at 37 o C, and were analyzed by HPLC-MS at different time points using the same method. At each time point, the area of the peak of GP1 or GP2 was determined by integration and compared to the area of the peaks in H 2 O. Difference in peak area was used as a means of determining the percentage of agent remaining in solution.
Cell Lines and Culture. A2780 cells were cultured using RPMI-1640 supplemented with 10% FBS. HeLa cells were cultured using phenol red free DMEM supplemented with 10% FBS. MCF-7 cells were cultured using phenol-red free MEM supplemented with 10% FBS. All three cell lines were grown in a humidified incubator at 37 o C and 5% CO 2 and were harvested using 0.25% TrypLE. Cells were grown for 24 hours after plating before each experiment. All solutions were filtered through 0.2 L sterile filters before use.
Viability Assays. A2780, HeLa, and MCF-7 cells were plated at a density of 3,000 cells per well (100 L) in an opaque white 96-well plate. Cells were dosed with 100 L of solutions of GP1, GP2, cisplatin, or carboplatin in MEM and incubated for 48 hours (GP1 or cisplatin) or 72 hours (GP2 or carboplatin). After incubation, 50 L CellTiter-Glo 2.0 (Promega, Madison, WI) was added to each well and the assay was carried out following the manufacturer's protocol.
Luminescence of the wells was measured using a Synergy H1 microplate reader (BioTek, Winooski, VT). Viability was determined by comparing luminescence readings of the cells treated with agent to untreated control cells.
Concentration-dependent Cell Uptake. A2780 and HeLa cells were plated at a density of 40,000 cells per well (500 L) in a 24-well plate. Cells were incubated with GP1, GP2, 1, cisplatin or carboplatin at varying concentrations in MEM (300 L) for 24 hours. After 24 hours, the media was aspirated and the cells were washed twice with 500 L of PBS, harvested, and centrifuged at 500 rpm for 5 minutes at 4 o C. The media was aspirated and the cells were resuspended in 200 L of media. A 50 L aliquot was taken for cell counting using a Guava PCA system using the Guava Viacount protocol provided by the manufacturer. An additional 100 L aliquot was used for ICP-MS analysis of Gd and Pt in the cells.
Cell fractionation experiments were performed using a cytosol/particulate rapid separation kit (BioVision, Milpitas, CA). Uptake experiments were performed in the same way, following the manufacturer's protocol after the first centrifugation. Gd and Pt content in each fraction was determined by ICP-MS and the total uptake for a given set of cells was taken to be the sum of the cytosol and particulate fractions.
Time-dependent Cellular Uptake. A2780 cells were plated at a density of 40,000 cells per well (500 L) in a 24-well plate. Cells were incubated with 300 L of 65 M GP1 or 62.5 M GP2 in MEM for variable amounts of time (0.5, 1, 2, 3, 6, 24 h). At each timepoint, the media was aspirated and the cells were washed twice with 500 L of PBS, harvested, and centrifuged at 500 rpm for 5 minutes at 4 o C. The media was aspirated and the cells were resuspended in 200 L of media. A 50 L aliquot was taken for cell counting using a Guava PCA system using the Guava Viacount protocol provided by the manufacturer. An additional 100 L aliquot was used for ICP-MS analysis of Gd and Pt in the cells.

MR Imaging of Cell Pellets at 7 T. A2780 and HeLa cells were grown to ~60% confluency
in T-75 flasks. A2780 cells were dosed with 5 mL of 30 M GP1, 60 M GP2, 100 M 1, 7.5 M, or vehicle (MEM) and HeLa cells were dosed with 5 mL of 60 M GP1, 250 M GP2, 100 M 1, 15 M, or vehicle (MEM). Cells were incubated for 6 hours. After incubation, the media was aspirated and the cells were washed twice with 5 mL PBS, harvested, and centrifuged at 500 rpm for 5 min at 4 o C. The cells were resuspended in 1 mL of media and 950 L of the suspension was added to flame-sealed Pasteur pipettes while the rest was used for cell counting and ICP-MS. The pipettes were centrifuged at 200 rpm for 5 minutes and were separated to form small capillaries containing the cell pellets. The capillaries were sealed with parafilm and imaged using a Bruker PharmaScan 7 T MR imaging spectrometer following the same imaging protocol previously described.

Cell Counting with a Guava EasyCyte Mini Personal Cell Analyzer (PCA) System.
Aliquots of cell suspensions were mixed with the Guava ViaCount Reagent and allowed to stain for 5 minutes. The samples were vortexed for 20 seconds and cell count was determined via manual analysis using a Guava EasyCyte Mini PCA and ViaCount software. 1000 events were acquired for each sample and dilutions were performed to assure the cell count was in the optimal range for instrument performance (10-100 cells/L). Performance of the instrument was assessed daily using Guava-Check Beads and the manufacturer's protocol and Daily Check software.

Quantification of Gadolinium and Platinum with Inductively Coupled Plasma Mass
Spectrometry. Quantification of Gd and Pt was accomplished using ICP-MS of acid digested samples. Specifically, samples were digested in concentrated trace nitric acid (> 69%, Thermo Fisher Scientific, Waltham, MA, USA) and trace hydrochloric acid (> 34%, Thermo Fisher Scientific, Waltham, MA, USA) and placed at 65 °C for at least 4 hours to allow for complete sample digestion. Ultra-pure H 2 O (18.2 MΩ•cm) was then added to produce a final solution of 2.0% nitric acid and 2.0% hydrochloric acid (v/v) in a total sample volume of 10 mL. Quantitative standards were made using a 10,000 µg/mL Gd elemental standard and a 1,000 ug/mL Pt elemental standard (Inorganic Ventures, Christiansburg, VA, USA) which were used to create a 200 ng/g mixed element standard and a 2 ng/g mixed element standard in 2.0% nitric acid and 2.0% hydrochloric acid (v/v) in a total sample volume of 50 mL. A solution of 2.0% nitric acid and 2.0% hydrochloric acid (v/v) was used as the calibration blank.
ICP-MS was performed on a computer-controlled (QTEGRA software) Thermo iCapQ ICP-MS (Thermo Fisher Scientific, Waltham, MA, USA) operating in STD mode and equipped with a ESI SC-2DX PrepFAST autosampler (Omaha, NE, USA). Internal standard was added inline using the prepFAST system and consisted of 1 ng/mL of a mixed element solution containing Bi, In, 6 Li, Sc, Tb, Y (IV-ICPMS-71D from Inorganic Ventures). Online dilution was also carried out by the prepFAST system and used to generate a calibration curve consisting of 200, 100, 50, 20, 10, 2 ppb Gd and Pt and a calibration curve consisting of 2000, 1000, 500, 200, 100, and 20 ppt Gd and Pt. Each sample was acquired using 1 survey run (10 sweeps) and 3 main (peak jumping) runs (40 sweeps). The isotopes selected for analysis were 194,195 Pt,56,57 Gd,and 115 In, 159 Tb, 209 Bi (chosen as internal standards for data interpolation and machine stability). Instrument performance is optimized daily through autotuning followed by verification via a performance report (passing manufacturer specifications).       In all cases, ≥94% of Gd localized in the cytosol. The majority of Pt localized in the cytosol, but significant amounts were also found in particulate fractions. The differences in subcellular localization between Gd and Pt supports that GP1 and GP2 dissociate intracellularly.