Materials and Measurements
The complexes cis
and 6-carboxy-2′,7′-dichlorofluorescein-3′,6′-diacetate succinimidyl ester49
were synthesized as previously described. SWNTs made by a high pressure CO (Hipco) method were purchased and used to construct amine-functionalized SWNTs via nonspecific interactions between the SWNT surface and an amine terminated PEGylated phospholipid (PL-PEG-NH2
Distilled water was purified by passage through a Millipore Milli-Q Biocel water purification system (18.2 MΩ) with a 0.22 μm filter. N-hydroxysuccinimide (NHS), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), paraformaldehyde, N,N-diisopropyl-ethylamine (DIPEA), hydroxyethyl starch (HEAS), and succinic anhydride were purchased from Aldrich. HATU [2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium] was purchased from Applied Biosystems. A monoclonal cisplatin 1,2-d(GpG) intrastrand cross-link specific antibody R-C18 was synthesized as previously reported.50
FITC labeled secondary antibody rabbit anti-(rat Ig) was obtained from Invitrogen. Specific adhesion slides for immunofluoresecence were purchased from Squarix Biotechnology, Marl, Germany. All other solvents and reagents were obtained from VWR International and used as received. 1
H NMR and 195
Pt NMR spectra were recorded on a Bruker AVANCE-400 NMR spectrometer with a Spectro Spin superconducting magnet in the Massachusetts Institute of Technology Department of Chemistry Instrumentation Facility (MIT DCIF). Atomic absorption spectroscopic measurements were taken on a Perkin Elmer AAnalyst 300 spectrometer. LC-MS analyses were performed on an Agilent 1100 series instrument. HPLC analyses were carried out using an Agilent 1200 series instrument. HRMS analyses were performed on a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer in the MIT DCIF. Electrochemical measurements were made at 25 °C on a 263 EG&G Princeton Applied Research electrochemical analyzer with electrochemical analysis software 270 for voltammetric work using a three electrode set-up comprising a glassy carbon working electrode, platinum wire auxiliary electrode, and a Ag/AgCl reference electrode. The electrochemical data were uncorrected for junction potentials. KCl was used as supporting electrolyte. Fluorescence imaging studies were performed with an Axiovert 200M inverted epifluorescence microscope (Zeiss, Thornwood, NY) equipped with an EM-CCD digital camera C9100 (Hamamatsu Hamamatsu City, Japan). An X-Cite 120 metal-halide lamp (EXFO, Quebec, Canada) was used as the light source. The microscope was operated with Volocity software (Improvision, Lexington, MA).
Synthesis of H2N(CH2)3O(CH2)2O(CH2)2O(CH2)3NHBOC (3)
A solution of 4,7,10-trioxa-1,13-tridecanediamine (7.5 g, 34.1 mmol) in 1,4-dioxane (100 mL) was treated with BOC-anhydride(3.7 g, 16.9 mL). The mixture was stirred at room temperature for 12 h. The solvent was removed and the resulting yellow oil was purified by silica gel flash chromatography (2–10% MeOH in CH2Cl2) to produce the oil 3 in 49% (5.5 g) yield. 1H NMR (CDCl3) δ 5.1 (s, 1H), 3.58–3.50 (m, 12H), 3.21 (d, J = 6.9 Hz, 2H), 2.79 (t, J = 8 Hz, 2H), 1.75–1.69 (m, 4H), 1.59 (s, 2H), 1.42 (s, 9H); 13C NMR (CDCl3) δ 155.0, 69.2, 68.9, 66.7, 48.0, 37.6, 30.4, 28.6, 27.3. HRMS-ESI: Calcd. = 321.2384 for [M+H]+, Found = 321.2372.
Synthesis of 4
Folic acid (1 g, 2.26 mmol) was dissolved in 20 mL of dry DMF to which 0.31 g (1.52 mmol) of DCC and 0.257 g (2.26 mmol) NHS were added. The reaction mixture was stirred for 14 h at room temperature in the dark. The by-product, dicyclohexylurea, was filtered off and 100 mL of 30% acetone in diethyl ether was added with stirring. A yellow precipitate formed and was collected on a sintered glass crucible; after washing with acetone and ether several times, the material was used immediately for the next step in the synthesis. Folate-NHS ester (1.2 g, 2.2 mmol) was dissolved completely in 100 mL of dry pyridine and 3
(0.73 g, 2.27 mmol) was slowly added over 30 min. The mixture was stirred at room temperature in the dark for 20 h. After evaporating pyridine, the resulting compound was dissolved in 5 mL of TFA to remove the BOC group. Deprotection was carried out at room temperature for 5 h. TFA was removed under vacuum. The resulting compound was loaded onto a DEAE Sephadex A25 column packed with potassium tetraborate and the compound was eluted with 10–50 mM ammonium bicarbonate. Fractions were collected and lyophilized. Compound 4
was isolated in 72% (1.24 g) yield. 1
H NMR (DMSO-d6
) δ 8.67 (s, 1H), 7.86–7.64 (m, 6H), 6.64 (d, J
= 8 Hz, 2H), 4.5 (s, 2H), 4.33–4.26 (m, 1H), 3.6–3.3 (m, 14H), 3.07 (d, J
= 4 Hz, 2H), 2.88–2.82 (m, 2H), 2.49 (s, 2H), 2.36–2.15 (m, 2H), 1.76 (t, J
= 4 Hz, 2H), 1.61–0.99 (m, 4H); 13
C NMR (DMSO-d6
) δ 174.4, 172.1, 166.5, 160.8, 158.5, 153.3, 151.0, 148.3, 129.4, 128.3, 121.4, 111.2, 69.7, 69.6, 69.5, 68.0, 67.4, 46.2, 36.9, 35.6, 30.6, 29.3, 27.2. HRMS-ESI: Calcd. = 644.3151 for [M+H]+
, Found = 644.3140.
Synthesis of c,c,t-Pt(NH3)2Cl2(O2CCH2CH2CO2H)(O2CCH2CH2CONH-PEG-FA)] (1)
To a solution of c,c,t
] (0.2 g, 0.38 mmol) in DMF (10 mL) was added a DMF solution (0.5 mL) containing HATU (0.217 g, 0.57 mmol). This mixture was stirred for 10 min at room temperature. To the resulting solution was added a DMF solution containing 4
(0.193 g, 0.3 mmol) and DIPEA (0.056 g, 0.432 mmol). The mixture was stirred at room temperature for 24 h in the dark. The DMF was then removed under vacuum to afford a yellow oil. Diethyl ether was added to precipitate a yellow solid, 1
. Compound 1
was purified by reprecipitation, dissolving it first in MeOH and then adding diethyl ether. As a final purification, 1
was repeatedly washed with acetone and then isolated as a yellow solid in 51% yield (0.2 g). IR (KBr): νmax
3382, 2922, 1700, 1684, 1662, 1607, 1558, 1539, 1506, 1301, 1261, 1189, 1100, 846, 667, 558 cm−1
; NMR (DMSO- d6
H NMR: δ 8.6 [s, 1H(=NCH)], 7.9–7.7 [b, 2H(NH2
)], 7.7–7.5 [b, 2H(Ar)], 6.9 [b, 2H(NH)], 6.6 [b, 2H(Ar)], 6.5 [b, 6H(NH3
)], 4.44 [d, J
= 5.6 Hz 2H(NHCH2
)] 3.7–3.4 [m, 12H(OCH2
)], 3.15–2.91 [m, 4H(NHCH2
)], 2.65–2.1 [m, 10H(NCH2
)], 1.55 [t, J
= 6.4 Hz 6H(CH2
)] ppm (Figure S1, Supporting Information
Pt: δ = 1220.79 ppm. (Figure S2
); mp (decomposition), 167–170 °C. ESI-MS (−): Calcd. = 1158.28 for [M-H]−
, Found = 1158.10 (Figure S3
Synthesis of 5
Fluorescein isothiocyanate (FTTC, 0.25 g, 0.64 mmol) was dissolved in dry DMF (2 mL) and 3 (0.266 g, 0.832 mmol) was added. The reaction mixture was stirred at room temperature for 24 h. DMF was removed under vacuum. The residue was purified by silica flash chromatography (5–15% MeOH in CHCl3) to produce 5 in 87% (0.44 g) yield. 1H NMR (CD3OD) δ 7.98 (s, 1H), 7.05 (d, J = 8 Hz, 1H), 6.55 (t, J = 4 Hz, 1 H), 6.43 (s, 4H), 6.41 (t, J = 8 Hz, 2 H), 3.51 (s, 2H), 3.50–3.34 (m, 12H), 2.99 (d, J = 4 Hz, 2H), 1.81 (t, J = 4 Hz, 2H), 1.57 (t, J = 4 Hz, 2H), 1.29 (s, 9H); 13C NMR (CD3OD) δ 171.3, 161.5, 158.6, 154.3, 142.5, 130.5, 125.9, 113.8, 111.6, 103.7, 80.1, 79.7, 71.6, 71.5, 71.3, 71.2, 70.6, 70.0, 49.8, 48.5, 44.0, 38.8, 31.0, 29.9, 28.9. ESI-MS (−): Calcd. = 708.26 for [M-H]−, Found = 708.20.
Synthesis of 6
To a solution of 5 (0.4 g, 0.56 mmol) in CH2Cl2 (2 mL) was added TFA (5 mL) at 0 °C. The mixture was stirred for 4 h at room temperature. The solvent was evaporated, the oily residue was dissolved in MeOH, and 6 was precipitated as a yellow solid by addition of excess diethyl ether. The solid was washed with diethyl ether several times and dried in vacuo. Compound 6 was isolated in 82% yield (0.28 g). 1H NMR (CD3OD) δ 8.14 (s, 1H), 7.72 (dd, J= 1.92 and 8.3 Hz, 1H), 7.09 (d, J= 8 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 2 Hz, 2H), 6.68–6.66 (m, 2H), 3.55 (m, 2H), 3.49–3.41 (m, 12H), 2.89 (t, J= 6.6 Hz, 2H), 1.79–1.69 (m, 4H); 13C NMR (CD3OD) δ 181.6, 173.7, 160.4, 159.1, 158.7, 132.7, 132.5, 132.0, 131.5, 130.1, 124.1, 104.4, 80.2, 71.4, 71.3, 71.2, 70.4, 67.0, 44.3, 39.9, 30.0, 28.4. ESI-MS (+): Calcd. = 610.22 for [M+H]+, Found = 610.1.
Synthesis of 2
(60 mg, 0.098 mmol) was added to a solution of folate-NHS ester (54 mg, 0.098 mmol) in pyridine (1 mL), and the mixture was stirred for 24 h at room temperature in the dark. After evaporation of pyridine, the residue obtained was purified by reverse-phase HPLC (Figure S4
) using a C-18 column to produce 2
in 21% (23 mg) yield. ESI-MS (−): Calcd. = 1031.34 for [M-H]−
, Found = 1031.5 (Figure S5
). HRMS-ESI: Calcd. = 1055.3328 for [M+Na]+
, Found = 1055.3343.
Synthesis of SWNT-PL-PEG-NH2
SWNTs functionalized with phospholipid (PL) polyethylene glycol (PEG)-linked amines (SWNT-PL-PEG-NH2
) were prepared by a known procedure.13
Briefly, raw Hipco SWNTs were sonicated in PL-PEG-NH2
for 1 h followed by centrifugation (2.4×l04
g, 6 h) to remove catalysts and large aggregates. Excess free PL-PEG-NH2
was removed from the supernatant by ultrafiltration using 100 kDa Millipore filters. The resulting solution contained SWNTs with an average length of about 200 nm as determined by atomic force microscopy. The molar concentration of the functionalized SWNTs was computed by measuring the absorption at 808 nm (ε = 7.9×l06
). The number of amine groups on each nanotube was estimated to be between 50 and 100.
Synthesis of SWNT-Pt(IV) Conjugates, SWNT-Pt(IV)
The synthesis of SWNT-Pt(IV) conjugates was carried out by using standard amide coupling reactions. In a typical reaction, a 1.0 mM aqueous solution of NHS (20 μL) was added to an equal volume of an aqueous 1.0 mM solution of EDC and the resulting solution was allowed to stand at room temperature for 10 min. To this solution was added 0.8 molar equivalents of compound 1 in ddH2O (40 μL). After 10 min, a solution of SWNT-PL-PEG-NH2 was added; the mole ratio of amine-to-Pt was 0.5. The solution was heated to 50 °C for 2 h and then stirred for 12 h at room temperature. The solution was dialyzed against deionized water in a 3500 MW cutoff dialysis cassette for 10 h, changing the water at 5 h. The concentration SWNT-Pt(IV) was subsequently determined by platinum AAS.
Synthesis of SWNT-Pt(IV)-Fl Conjugates
Following the procedure mentioned above, SWNT-Pt(IV) conjugates with an amine-to-Pt ratio of 2:1 were prepared. These conjugates were then allowed to react with a 0.4 mM aqueous solution of 6-carboxy-2′,7′-dichlorofluorescein-3′,6′-diacetatesuccinimidyl ester for 12 h at room temperature. The resulting solution was dialyzed against water using a 3500 MW cutoff dialysis cassette for 12 h and the platinum concentration was determined by AAS.
HPLC Monitoring of Carboxypeptidase-G (CPG) Mediated Cleavage of 4
Carboxypeptidase-G2 (CPG) digestion was followed by using reverse-phase HPLC. An analytical (VYDAC) C18 column was used at a flow rate of 1 mL/min monitoring absorbance at a wavelength of 285 nm. A solution of 4 (155 μM) was prepared in 150 mM Tris buffer (pH 7.3). An aliquot of this solution was injected into the HPLC system to obtain the time zero peak intensity. To this solution was added carboxypeptidase G (CPG, Sigma, 1 unit) and the resulting solution was incubated at 30 °C for 24 h. The solution was then analyzed by HPLC after 24 h.
Electrochemical Studies of Platinum(IV) Compounds
The platinum(IV) complex 1 was dissolved to a final concentration of 2.0 mM in 0.1 M aqueous KCl buffered with phosphate to either pH 6.0 or 7.4. Cylic voltammetric (CV) measurements were carried out at varying scan rates of 50–300 mV s−1. The solvent was degassed by several freeze-pump-thaw cycles and measurements were taken under an atmosphere of argon.
Cells were cultured in FA-free RPMI medium (RPMI-1640, Invitrogen), with 10% fetal bovine serum, 2 mM glutamine, 50 units/mL penicillin, and 50 μg/mL streptomycin. The concentration of FA in serum-containing FA-free medium is only 3 nM, as opposed to 2.26 μM (1 mg/L) under normal culture conditions. Cells were routinely passed by treatment with trypsin (0.05%)/EDTA.
Human nasopharyngeal epidermoid carcinoma (KB), choriocarcinoma (JAR), and human testicular cancer (NTera-2) cells were cultured in FA-free RPMI for several passages. Cells were passed every 3 to 4 days and reseeded from frozen stocks after reaching passage number 20.
MTT Cell Proliferation Assay
KB, JAR, or NTera-2 cells were seeded in 96-well tissue culture plates and maintained overnight in RPMI medium. Cells were then treated with various concentrations of 1, SWNT-1, or cis-DDP at different concentrations. The plates were incubated for 72 h at 37 °C. The cells were then treated with 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL in PBS) and incubated for 5 h. The medium was removed, the cells were lysed by adding 100 μL of DMSO, and the absorbance of the purple formazan was recorded at 550 nm using a Spectra MAX 340PC plate reader.
Cell Fixing Solution
Paraformaldehyde (4.0 g) and NaOH (0.4 g) were dissolved in 100 mL of distilled water. To this solution NaH2PO4 (1.68 g) was added and the pH was adjusted to be in the range 7.5 to 8.0 by adding NaOH.
Fluorescence Sample Mounting Media
For sample mounting, a solution containing 20 mM Tris (pH 8.0), 0.5% N-propyl gallate, and 50–90% glycerol was used.
Fluorescence Microscopy Experiments on KB and NTera-2 Cells
KB or Ntera-2 cells were seeded on microscope coverslips (1 cm) at a confluence of 1600 cells per slip and incubated overnight at 37 °C in FA-free RPMI. The medium was changed and SWNT-Pt(IV)-Fl or 2 was added to a final fluorophore concentration of 1.0 μM. The cells were incubated for 2.5 h at 37 °C. RPMI was then removed and the cells were incubated with fixing solution for 1 h at room temperature followed by three washes with phosphate buffered saline (PBS, pH 7.4). Cells were then permeabilized with 0.1% Triton-X 100 in PBS for 10 min followed by five washes using PBS. Cells were treated with a PBS solution of Hoechst (H33258) (250 μg/L) for 10 min at room temperature for nuclear staining. The cells were then washed three times with PBS followed by 2 washes using Millipore water. They were mounted on microscope slides using the mounting solution for imaging. Images were collected at 500 msec for the DAPI channel and 270 msec for the FITC channel.
Determination of Platinum Concentrations from Cell Extracts
KB cells were grown in FA-free RPMI to greater than 95% confluence in 175 cm2 flasks. These cells were then treated either with 1 μM 1 or SWNT-1 and subsequently incubated for 3 h at 37 °C. Cells were washed with PBS three times and released by trypsinization into PBS. Solutions containing cells were then centrifuged at 800 × g for 10 min and the cell pellet obtained was resuspended in 100 μL of ice-cold lysis buffer (1.0 mM DTT, 1.0 mM PMSF, 10 mM KCl, 10 mM MgCl2, pH 7.5) for 15 min. This process was repeated and finally the pellets were resuspended in 40 μL of ice-cold lysis buffer. The cell membranes were lysed by 10 strokes of a 28 ga. syringe. The resulting suspension was centrifuged at 11,000 × g for 20 min and the cytosolic fraction of the cells was collected as supernatant. The pellet was resuspended in 40 μL of extraction buffer (1.0 mM DTT, 1.0 mM PMSF, 1.5 mM MgCl2, 0.2 M EDTA, 0.42 M NaCl, 25% glycerol, pH 7.9) and lysed with 10 strokes of a 28 ga. syringe. The lysate was shaken at 1000 rpm for 45 min at 4 °C and then centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant was collected as the nuclear fraction. Platinum concentrations in all the fractions were determined by AAS. The protein concentration in each fraction was determined by using bicinchoninic acid (BCA) assay. Total platinum concentrations were expressed as nanograms of Pt per microgram of protein.
Immunofluorescence for Platinum 1,2-d(GpG) Intrastrand Cross-link Detection
KB cells were cultured in a six well plate in folate free RPMI and treated with SWNT-Pt(IV) or cis
-DDP at a concentration of 1 μM for 12 h. The cells were released by trypsinization and washed with medium followed by PBS. The cells were then resuspended in HAES-sterile-PBS at a density of 1×106
per mL. A 10 μL portion of the cell solution was placed onto a pre-coated slide (ImmunoSelect, Squarix) and air dried. The cells were then fixed using methanol at −20 °C for 45 min. Samples were washed two times with PBS. Alkali denaturation of nuclear DNA was then performed by using a solution of 60% 70 mM NaOH/140 mM NaCl-40% MeOH at 0 °C for 5 min. After two washes with PBS, removal of cellular proteins was carried out by digestion with pepsin (30 μg/mL) for 10 min at 37 °C followed by treatment with proteinase K (6 μg/mL) for 10 min at 37 °C in a humid chamber. The cells were washed with PBS-0.2% glycin. After blocking with milk (5% in PBS) for 30 min at room temperature, cells were incubated with anti-Pt-1,2-d(GpG) antibody R-C1850
(0.2 μg/mL) at 4 °C for 12 h in a humid chamber. The cells were then washed with PBS for three times. After blocking for 30 min with milk at room temperature, cells were incubated with FITC-labeled rabbit anti-(rat Ig) secondary antibody at 37 °C for 1 h. After three washes with PBS, cells were treated with a PBS solution of Hoechst (H33258) (250 μg/L) for 10 min at room temperature for nuclear staining. The cells were then washed three times with PBS and mounted using the mounting solution for imaging.