Targeted single-wall carbon nanotube-mediated Pt(IV) prodrug delivery using folate as a homing device

Article abstract of DOI:10.1021/ja803036e

Most low-molecular-weight platinum anticancer drugs have short blood circulation times that are reflected in their reduced tumor uptake and intracellular DNA binding. A platinum(IV) complex of the formula c,c,t-[Pt(NH₃)₂Cl₂(O₂CCH ₂CHC₂O₂H)(O₂CCH₂CH ₂CONH-PEG-FA)] (1), containing a folate derivative (FA) at an axial position, was prepared and characterized. Folic acid offers a means of targeting human cells that highly overexpress the folate receptor (FR). Compound 1 was attached to the surface of an amine-functionalized single-walled carbon nanotube (SWNT-PL-PEG-NH₂) through multiple amide linkages to use the SWNTs as a longboat delivery system for the platinum warhead, carrying it to the tumor cell and releasing cisplatin upon intracellular reduction of Pt(IV) to Pt(II). The ability of SWNT tethered 1 to destroy selectively FR(+) vs FR(-) cells demonstrated its ability to target tumor cells that overexpress the FR on their surface. That the SWNTs deliver the folate-bearing Pt(IV) cargos into FR(+) cancer cells by endocytosis was demonstrated by the localization of fluorophore-labeled SWNTs using fluorescence microscopy. Once inside the cell, cisplatin, formed upon reductive release from the longboat oars, enters the nucleus and reacts with its target nuclear DNA, as determined by platinum atomic absorption spectroscopy of cell extracts. Formation of the major cisplatin 1,2-intrastrand d(GpG) cross-links on the nuclear DNA was demonstrated by use of a monoclonal antibody specific for this adduct. The SWNT-tethered compound 1 is the first construct in which both the targeting and delivery moieties have been incorporated into the same molecule; it is also the first demonstration that intracellular reduction of a Pt(IV) prodrug leads to the cis-{Pt((NH ₃)₂} 1,2-intrastrand d(GpG) cross-link in nuclear DNA.

Full text of DOI:10.1021/ja803036e

Published on Web 07/29/2008
Targeted Single-Wall Carbon Nanotube-Mediated Pt(IV)
Prodrug Delivery Using Folate as a Homing Device
Shanta Dhar,† Zhuang Liu,‡ Ju¨rgen Thomale,§ Hongjie Dai,*,‡ and
Stephen J. Lippard*,†
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts
02139, Department of Chemistry and Laboratory for AdVanced Materials, Stanford UniVersity,
Stanford, California 94305, and Institute of Cell Biology, UniVersity of Duisburg-Essen,
45122 Essen, Germany
Received April 24, 2008; E-mail: lippard@mit.edu; hdai@stanford.edu
Abstract: Most low-molecular-weight platinum anticancer drugs have short blood circulation times that
are reflected in their reduced tumor uptake and intracellular DNA binding. A platinum(IV) complex of the
formula c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2CO2H)(O2CCH2CH2CONH-PEG-FA)] (1), containing a folate derivative
(FA) at an axial position, was prepared and characterized. Folic acid offers a means of targeting human
cells that highly overexpress the folate receptor (FR). Compound 1 was attached to the surface of an
amine-functionalized single-walled carbon nanotube (SWNT-PL-PEG-NH2) through multiple amide linkages
to use the SWNTs as a “longboat delivery system” for the platinum warhead, carrying it to the tumor cell
and releasing cisplatin upon intracellular reduction of Pt(IV) to Pt(II). The ability of SWNT tethered 1 to
destroy selectively FR(+) vs FR(-) cells demonstrated its ability to target tumor cells that overexpress the
FR on their surface. That the SWNTs deliver the folate-bearing Pt(IV) cargos into FR(+) cancer cells by
endocytosis was demonstrated by the localization of fluorophore-labeled SWNTs using fluorescence
microscopy. Once inside the cell, cisplatin, formed upon reductive release from the longboat oars, enters
the nucleus and reacts with its target nuclear DNA, as determined by platinum atomic absorption
spectroscopy of cell extracts. Formation of the major cisplatin 1,2-intrastrand d(GpG) cross-links on the
nuclear DNA was demonstrated by use of a monoclonal antibody specific for this adduct. The SWNT-
tethered compound 1 is the first construct in which both the targeting and delivery moieties have been
incorporated into the same molecule; it is also the first demonstration that intracellular reduction of a Pt(IV)
prodrug leads to the cis-{Pt((NH3)2} 1,2-intrastrand d(GpG) cross-link in nuclear DNA.
Introduction
ing blood circulation. Recently, single-walled carbon nanotubes
(SWNTs) have been investigated as carriers in living systems.11–13
In spite of the clinical success of cisplatin (cis-dichloro-
diammineplatinum(II) or cis-DDP),1–5 there are many occasions
when treatment must be discontinued. Drug resistance, acquired
and intrinsic, limits the effectiveness of cis-DDP and is manifest
as reduced cellular uptake, enhanced DNA repair, drug de-
activation, or a combination of these mechanisms.
They internalize various cargos in cells, including fluoresceins,
plasmid DNA, proteins, and other substances that would not
otherwise be taken up, with no apparent side effects.14–16 Well-
functionalized nanotubes circulate in the blood with half-lives
on the order of a few hours. Nanotube uptake by the reticu-
loendothelial system (RES) is cleared slowly by the biliary
pathway, and the nanotubes end up in feces, without showing
obvious toxicity in vivo.17–19 Because of their extremely low
solubility, pristine SWNTs cannot circulate well in biological
systems. Their dispersion in biological fluids is achieved by
Nanocarriers6–10 for low-molecular-weight drugs offer a
promising strategy for improving body distribution and prolong-
† Massachusetts Institute of Technology.
‡ Stanford University.
§ University of Duisburg-Essen.
(1) Rosenberg, B.; VanCamp, L.; Trosko, J. E.; Mansour, V. H. Nature
1969, 222, 385–386.
(11) Kam, N. W. S.; Jessop, T. C.; Wender, P. A.; Dai, H. J. Am. Chem.
Soc. 2004, 126, 6850–6851.
(2) Jamieson, E. R.; Lippard, S. J. Chem. ReV. 1999, 99, 2467–2498.
(3) Wong, E.; Giandomenico, C. M. Chem. ReV. 1999, 99, 2451–2466.
(4) Galanski, M.; Jakupec, M. A.; Keppler, B. K. Curr. Med. Chem. 2005,
12, 2075–2094.
(12) Kam, N. W. S.; Liu, Z.; Dai, H. J. Am. Chem. Soc. 2005, 127, 12492–
12493.
(13) Liu, Z.; Cai, W.; He, L.; Nakayama, N.; Chen, K.; Sun, X.; Chen, X.;
Dai, H. Nat. Nanotechnol. 2007, 2, 47–52.
(5) Wang, D.; Lippard, S. J. Nat. ReV. Drug DiscoVery 2005, 4, 307–
320.
(14) Liu, Y.; Wu, D.-C.; Zhang, W.-D.; Jiang, X.; He, C.-B.; Chung, T. S.;
Goh, S. H.; Leong, K. W. Angew. Chem., Int. Ed. 2005, 44, 4782–
4785.
(6) Bogunia-Kubik, K.; Sugisaka, M. Biosystems 2002, 65, 123–138.
(7) Schmidt, J. J.; Montemagno, C. D. Drug DiscoVery Today 2002, 7,
500–503.
(15) Bottini, M.; Cerignoli, F.; Dawson, M. I.; Magrini, A.; Rosato, N.;
Mustelin, T. Biomacromolecules 2006, 7, 2259–2263.
(16) Kam, N. W. S.; Liu, Z.; Dai, H. Angew. Chem., Int. Ed. 2006, 45,
577–581.
(8) Roco, M. C. Curr. Opin. Biotechnol. 2003, 14, 337–346.
(9) Sahoo, S. K.; Labhasetwar, V. Drug DiscoVery Today 2003, 8, 1112–
1120.
(17) Deng, X.; Jia, G.; Wang, H.; Sun, H.; Wang, X.; Yang, S.; Wang, T.;
Liu, Y. Carbon 2007, 45, 1419–1424.
(10) Wilkinson, J. M. Med. DeVice Technol. 2003, 14, 29–31.
9
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Dhar et al.
Scheme 1. Targeted Prodrug-Delivery System
surface functionalization through oxidation, which leaves oxygen-
containing functional groups such as alcohols or carboxylic acid,
or by the attachment of solubilizing side-chains.20–25 Once
solubilized, SWNTs efficiently cross cell membranes, being
taken into the cytosol by endocytosis.26–28 As a result, various
molecules attached to the surface of the SWNT can be internalized.
Coupling of macromolecular carriers with a normally endocytosed
compound frequently improves its intracellular accumulation.
Recently we demonstrated that functionalized soluble SWNTs
can serve as “longboats” to carry Pt(IV) prodrugs into cells
through clathrin-dependent endocytosis.29 The SWNT longboats
produce platinum intracellular levels much higher than those
for the platinum unit administered in the traditional manner.
Efforts to produce potent platinum anticancer drugs have been
hindered by their inactivation in the body prior to reaching the
tumor. Longboat SWNTs provide an opportunity to shuttle
platinum compounds safely through this obstacle course and
into the cancer cell.
overexpressed by a wide variety of human tumors, including
ovarian, endometrial, breast, lung, renal, and colon.32,33 The
highest frequency of R-FR overexpression (>90%) occurs in
ovarian carcinomas.34,35 Expression of R-FR on tumor cell
surfaces has led to the exploitation of FA as an important ligand
for specific targeting by diagnostic or therapeutic cancer cell
agents.36–39
The SWNT longboats also have the potential to carry
additional passengers such as tumor-targeting components to
the cancer cell. Targeted drug-delivery systems promise to
expand the therapeutic window of platinum complexes, as
recently demonstrated for oxaliplatin uptake by colorectal cancer
cells via organic cation transporters.30 Traditional cancer therapy
relies on the premise that rapidly proliferating cancer cells are
more likely to be killed by a cytotoxic agent. In reality, however,
these agents have very little or no specificity, which leads to
systemic toxicity, causing undesirable side effects. Therefore,
targeted drug-delivery constructs are much desired. In general,
a tumor-targeting drug-delivery system consists of a cell surface
recognition moiety and a chemical warhead connected directly
or through a suitable linker to a delivery system such as a SWNT
(Scheme 1). The conjugate itself should be systemically
nontoxic, and the linker must be stable in blood circulation.
Upon internalization into the cancer cell, the conjugate should
be readily cleaved to generate the active agent.
Carboplatin, cis-[Pt(NH3)2(CBDCA-O,O′)], where CBDCA
is cyclobutane-1,1-dicarboxylate, is a second generation plati-
num anticancer drug widely used for cancer treatment.40
Carboplatin has a short blood circulation half-life, which reduces
tumor uptake and subsequent intracellular DNA binding. Recent
efforts to overcome these shortcomings include conjugation of
carboplatin, linked to FA by attachment to CBDCA at an
equatorial site, to a PEG carrier41 for folate receptor-mediated
endocytosis (FRME).42,43 The FA-PtII-PEG conjugates were
taken into the cytosol of tumor cells more rapidly than PEG-Pt
conjugates, but the latter afforded twice as many Pt-DNA
adducts. Apparently, the endocytosed FA-PtII-PEG conjugate
is sequestered in a compartment like the lysosome, where it is
separated from the cytosol by a membrane, thereby preventing
Pt from reaching its nuclear DNA target.
Platinum(IV) complexes have the potential to overcome these
problems. Their octahedral geometry introduces two axial
ligands, L1 and L2 in Scheme 1, one of which can be attached
to a folic acid or other ligand for cellular targeting. The other
axial site offers a position for conjugation to a nanocarrier for
efficient delivery. Platinum(IV) complexes are prodrugs because
they can be reduced in the cytoplasm to unveil an active
platinum(II) derivative with concomitant loss of the axial ligands
(Scheme 1).44,45
A variety of receptors have been identified as markers for
carcinomas. Among these is the folate receptor (R-FR), and its
substrate folic acid (FA) has the potential to target several types
of cancer cells because of its ability to react with this high-
affinity, membrane-anchored protein.31 Moreover, R-FR is
(18) Schipper, M. L.; Nakayama-Ratchford, N.; Davis, C. R.; Kam,
N. W. S.; Chu, P.; Liu, Z.; Sun, X.; Dai, H.; Gambhir, S. S. Nat.
Nanotechnol. 2008, 3, 216–221.
(31) Antony, A. C. Blood 1992, 79, 2807–2820.
(19) Liu, Z.; Davis, C.; Cai, W.; He, L.; Chen, X.; Dai, H. Proc. Natl.
Acad. Sci. U.S.A. 2008, 105, 1410–1415.
(32) Weitman, S. D.; Lark, R. H.; Coney, L. R.; Fort, D. W.; Frasca, V.;
Zurawski, V. R., Jr.; Kamen, B. A. Cancer Res. 1992, 52, 3396–3401.
(33) Ross, J. F.; Chaudhuri, P. K.; Ratnam, M. Cancer 1994, 73, 2432–
2443.
(20) Qu, L.; Martin, R. B.; Huang, W.; Fu, K.; Zweifel, D.; Lin, Y.; Sun,
Y.-P.; Bunker, C. E.; Harruff, B. A.; Gord, J. R.; Allard, L. F. J. Chem.
Phys. 2002, 117, 8089–8094.
(34) Garin-Chesa, P.; Campbell, I.; Saigo, P. E.; Lewis, J. L., Jr.; Old,
L. J.; Rettig, W. J. Am. J. Pathol. 1993, 142, 557–567.
(35) Parker, N.; Turk, M. J.; Westrick, E.; Lewis, J. D.; Low, P. S.; Leamon,
C. P. Anal. Biochem. 2005, 338, 284–293.
(21) Georgakilas, V.; Kordatos, K.; Prato, M.; Guldi, D. M.; Holzinger,
M.; Hirsch, A. J. Am. Chem. Soc. 2002, 124, 760–761.
(22) Hirsch, A. Angew. Chem., Int. Ed 2002, 41, 1853–1859.
(23) Holzinger, M.; Abraham, J.; Whelan, P.; Graupner, R.; Ley, L.;
Hennrich, F.; Kappes, M.; Hirsch, A. J. Am. Chem. Soc. 2003, 125,
8566–8580.
(36) Bae, Y.; Jang, W.-D.; Nishiyama, N.; Fukushima, S.; Kataoka, K. Mol.
BioSyst. 2005, 1, 242–250.
(37) Mueller, C.; Schubiger, P. A.; Schibli, R. Eur. J. Nucl. Med. Mol.
Imaging 2006, 33, 1162–1170.
(24) Hirsch, A.; Vostrowsky, O. Top. Curr. Chem. 2005, 245, 193–237.
(25) Prato, M.; Kostarelos, K.; Bianco, A. Acc. Chem. Res. 2008, 41, 60–
68.
(38) Mueller, C.; Schubiger, P. A.; Schibli, R. Bioconjugate Chem. 2006,
17, 797–806.
(26) Kam, N. W. S.; Dai, H. J. Am. Chem. Soc. 2005, 127, 6021–6026.
(27) Kam, N. W. S.; O’Connell, M.; Wisdom, J. A.; Dai, H. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 11600–11605.
(39) Zhang, Z.; Lee, S. H.; Feng, S.-S. Biomaterials 2007, 28, 1889–1899.
(40) Harrap, K. R. Cancer Res. 1995, 55, 2761–2768.
(41) Aronov, O.; Horowitz, A. T.; Gabizon, A.; Gibson, D. Bioconjugate
Chem. 2003, 14, 563–574.
(28) Gao, L.; Nie, L.; Wang, T.; Qin, Y.; Guo, Z.; Yang, D.; Yan, X.
ChemBioChem 2006, 7, 239–242.
(42) Wang, S.; Low, P. S. J. Controlled Release 1998, 53, 39–48.
(43) Sudimack, J.; Lee, R. J. AdV. Drug DeliVery ReV. 2000, 41, 147–162.
(44) Barnes, K. R.; Kutikov, A.; Lippard, S. J. Chem. Biol. 2004, 11, 557–
564.
(29) Feazell, R. P.; Nakayama-Ratchford, N.; Dai, H.; Lippard, S. J. J. Am.
Chem. Soc. 2007, 129, 8438–8439.
(30) Zhang, S.; Lovejoy, K.; Shima, J. E.; Lagpacan, L. L.; Shu, Y.; Lapuk,
A.; Gray, J. W.; Chen, X.; Lippard, S. J.; Giacomini, K. M. Cancer
Res. 2006, 66, 88847–88857.
(45) Mukhopadhyay, S.; Barnes, C. M.; Haskel, A.; Short, S. M.; Barnes,
K. R.; Lippard, S. J. Bioconjugate Chem. 2008, 19, 39–49.
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Targeted Pt(IV) Prodrug Delivery
A R T I C L E S
Figure 1. Folate receptor (FR)-mediated targeting and SWNT-mediated delivery of 1 by endocytosis and structure of 1.
solvents and reagents were obtained from VWR International and
used as received. 1H NMR and 195Pt NMR spectra were recorded
on a Bruker AVANCE-400 NMR spectrometer with a Spectro Spin
superconducting magnet in the Massachusetts Institute of Technol-
ogy 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 T
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 voltam-
metric work using a three-electrode setup comprising a glassy
carbon working electrode, a 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 200 M inverted epifluorescence microscope (Zeiss,
Thornwood, NY) equipped with an EM-CCD digital camera C9100
(Hamamatsu, 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).
The goal of the present study was to utilize the targeting
properties of folic acid with the delivery feature of SWNTs to
develop a Pt(IV)-SWNT longboat for targeting delivery of cis-
DDP upon reduction in the cell (Scheme 1). We therefore
designed a Pt(IV) complex c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2-
CO2H)(O2CCH2CH2CONH-PEG-FA)] (1, Figure 1), having
succinate as one of its axial ligands for conjugation with amine-
functionalized SWNTs.29 The second axial ligand contains a
folic acid derivative separated from the platinum center by a
PEG spacer. The use of PEG makes the compound more water
soluble and biocompatible.46
Experimental Section
Materials and Measurements. The complexes cis-[Pt(NH3)2-
Cl2],47 c,c,t-[Pt(NH3)2Cl2(OH)2],48 c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2-
CO2H)2],44 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).13 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-diiso-
propylethylamine (DIPEA), hydroxyethyl starch (HAES), and
succinic anhydride were purchased from Aldrich. 2-(1H-7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
methanaminium (HATU) 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
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 dimethylformamide (DMF) to which 0.31 g (1.52 mmol)
of dicyclohexylcarbodiimide (DCC) and 0.257 g (2.26 mmol) of
NHS were added. The reaction mixture was stirred for 14 h at room
temperature in the dark. The byproduct, 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
(46) Zalipsky, S. AdV. Drug DeliVery ReV. 1995, 16, 157–182.
(47) Dhara, S. C. Indian J. Chem. 1970, 8, 193–194.
(48) Hall, M. D.; Dillon, C. T.; Zhang, M.; Beale, P.; Cai, Z.; Lai, B.;
Stampfl, A. P. J.; Hambley, T. W. J. Biol. Inorg. Chem. 2003, 8, 726–
732.
(49) Woodroofe, C. C.; Won, A. C.; Lippard, S. J. Inorg. Chem. 2005, 44,
3112–3120.
(50) Liedert, B.; Pluim, D.; Schellens, J.; Thomale, J. Nucleic Acids Res.
2006, 34, e47.
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A R T I C L E S
Dhar et al.
Scheme 2. Synthetic Route to 1
Scheme 3. Synthetic Procedure for Synthesizing 2
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 12 h. After pyridine was evaporated,
the resulting compound was dissolved in 5 mL of trifluoroacetic
acid (TFA) to remove the BOC group. Deprotection was carried
out at room temperature for 4 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 53% (1.24
g) yield. 1H 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). 13C 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,
9
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Targeted Pt(IV) Prodrug Delivery
A R T I C L E S
Scheme 4. Synthetic Procedure for Synthesizing SWNT-1
Table 1. Comparison of Reduction Potentials for Pt(IV)
7.98 (s, 1H), 7.05 (d, J ) 8 Hz, 1H), 6.55 (t, J ) 4 Hz, 1H), 6.43
(s, 4H), 6.41 (t, J ) 8 Hz, 2H), 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% (0.28 g) yield. 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. Compound 6 (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 × 104 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 × 106 M-1 cm-1).
The number of amine groups on each nanotube was estimated to
be between 50 and 100.
Compounds
complexa
reduction potential (mV) at pH 7.4 vs NHE
ref
[Pt(en)(OCOCF3)2Cl2]
[Pt(dach)Cl4]
197
107
58
58
58
58
58
59
58
59
[Pt(a,cha)(OCOC3H7)2Cl2]
[Pt(en)Cl4]
47
37
[Pt(a,cha)(OCOCH3)2Cl2]
[Pt(en)(OCOCH3)2Cl2]
[Pt(ipa)(OH)2Cl2]
[Pt(en)(OH)2Cl2]
1
-53
-349
-533
-687
-698
this work
a en ) ethylenediamine; ipa ) isopropylamine; a ) amine; cha )
cyclohexylamine; dach ) diaminocyclohexane.
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)(O2CCH2-
CH2CONH-PEG-FA)] (1). To a solution of c,c,t-[Pt(NH3)2-
Cl2(O2CCH2CH2CO2H)2] (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, first dissolving it 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%
(0.2 g) yield. IR (KBr): νmax 3382, 2922, 1700, 1684, 1662, 1607,
1558, 1539, 1506, 1301, 1261, 1189, 1100, 846, 667, 558 cm-1
.
1H NMR (DMSO-d6): δ 8.6 [s, 1H (dNCH)], 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 and CH2)], 1.55 [t, J ) 6.4 Hz, 6H (CH2 and C(O)CH2)]
(Figure S1, Supporting Information). 195Pt NMR (DMSO-d6): δ )
1220.79 (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 (FITC, 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 82% (0.44 g) yield. 1H NMR (CD3OD): δ
Synthesis of SWNT-Pt(IV) Conjugates. 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
9
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A R T I C L E S
Dhar et al.
Figure 2. Survival of (a) FR(+) JAR, (b) FR(+) KB, and (c) FR(-) NTera-2 cells treated with 1 (9), SWNT-1 (2), or cisplatin (b).
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.
Cell Culture. a. Medium. 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.
b. Cell Lines. 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 3-4 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 concentra-
tions 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
was added NaH2PO4 (1.68 g), and the pH was adjusted to be in
the range 7.5-8.0 by adding NaOH.
Figure 3. Fluorescence of 2 in (a) FR(+) KB and (b) FR(-) NTera-2
cells.
stand at room temperature for 10 min. To this solution was added
0.8 mol equiv 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 of 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′-diacetate succinimidyl 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.
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 concentra-
tion 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 perme-
abilized 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
HPLC Monitoring of Carboxypeptidase-G (CPG)-Media-
ted 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
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Targeted Pt(IV) Prodrug Delivery
A R T I C L E S
Figure 4. Visualization of Pt-1,2-d(GpG) intrastrand cross-links in the nuclear DNA of KB cells after treatment with (b) SWNT-1 and (c) cis-DDP. (a)
Immunofluorescence of untreated KB cells. Nuclei were stained with Hoechst (blue), and Pt-1,2-d(GpG) in DNA were visualized using Mab R-C18 (green).
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 two washes using Millipore water. They were mounted
on microscope slides using the mounting solution for imaging.
Images were collected at 500 ms for the DAPI channel and 270
ms for the FITC channel.
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 precoated 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 three times. After blocking with
milk for 30 min 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, the cells were treated with a
PBS solution of Hoechst H33258 (250 µg/L) for 10 min at room
Determination of Platinum Concentrations from Cell Ex-
tracts. KB cells were grown in FA-free RPMI to >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 800g 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 11000g 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 20000g 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
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A R T I C L E S
Dhar et al.
temperature for nuclear staining. The cells were then washed three
times with PBS and mounted using the mounting solution for
imaging.
In order to demonstrate the folate receptor targeting properties
of 1, we employed a FITC-labeled conjugate of folic acid (FA-
PEG-FITC, 2). This compound allowed us to determine whether
a PEG-conjugated folic acid can interact with FR on the surface
of a tumor cell by using fluorescence microscopy.
Results and Discussion
Synthesis and Chemical Characterization. The molecular
design of the Pt(IV) compound 1, containing both a folate
derivative and a succinate with a free carboxylic (Figure 1),
was inspired by the asymmetric Pt(IV) compound [Pt(NH3)-
Cl2(OEt)(O2CCH2CH2CO2H)] recently reported by us.29 The
folic acid derivative 4 (Scheme 2) was designed such that the
basic requirements for interaction with the folate receptor were
maintained. Folic acid contains two carboxylate groups that can
be used for conjugation to the Pt(IV) center. Modification of
the γ carboxylate group allows conjugation without significant
loss of binding affinity for the folate receptor. Folic acid was
therefore derivatized at the γ position with a polyethylene glycol
(PEG) spacer. Efficient FR binding can be achieved by
separating the folate from the Pt(IV) center by a PEG spacer.
The mono-BOC-protected PEG-amine was prepared by a
standard procedure using 4,7,10-trioxa-1,13-tridecanediamine.
The synthesis of the folate-γ-NHS ester, illustrated in Scheme
2, was carried out by the procedure reported earlier.51
The BOC-protected folate-PEG-amine was synthesized by
reacting mono-BOC-protected PEG-amine (3) with NHS-folate,
as indicated in Scheme 2. A ninhydrin assay was used to follow
the progress of the reaction. Folate-PEG-amine (4) was released
by a standard BOC deprotection method in the final step.
The isomeric purity of 4 is an important issue for binding
to the FR. We used a carboxypeptidase G2 (CPG)-digestion
assay52 to measure the percent of γ-isomer in the folate-
PEG-amine 4. CPG is a lysosomal, thiol-dependent protease
that hydrolyzes only γ-amides but not the R-isomer.53
Derivatization of the R-carboxyl group of folate prevents
hydrolysis by CPG, but derivatization of the γ-carboxyl has
little effect on the enzyme activity. A solution of 4 (155 µM)
in 150 mM Tris buffer (pH 7.3) shows two peaks (Figure
S6a) under HPLC conditions, both giving rise to an m/z peak
at 644.3 in their LC-MS spectra. The assignment of the
structures of the two isomers of folate-PEG-amine was carried
out by CPG hydrolysis. When a solution of 4 (155 µM) in
150 mM Tris buffer (pH 7.3) was tested as a substrate for
the CPG enzyme, the minor peak at 11.0 min was unchanged,
while the peak at 13.5 min revealed hydrolysis (Figure S6b).
This experiment showed that >90% of the FA residues in
conjugate 4 are γ-carboxyl-linked. HPLC chromatograms of
compound 4 before and after treatment with CPG are
available in Figures S6a and S6b, respectively.
The synthetic route to 2 is given in Scheme 3. The mono-BOC-
protected PEG amine (3) was coupled to FITC by a thiourea linkage
to afford 5, which was deprotected to give 6 containing a free
amine. Conjugation of 6 to folic acid by amide coupling was
performed by using the activated folate-γ-NHS ester in the presence
of HATU/DIPEA to yield the final compound 2.
In the next step we used noncovalently functionalized
SWNTs having phospholipid-tethered amines on their surface.
A polyethyleneglycol chain was introduced between the
amine and phospholipid moieties to improve the solubility
of the SWNTs and to keep functional groups away from the
nanotube surface. The diameters and lengths of the func-
tionalized SWNTs were 1-5 nm and 100-300 nm, respec-
tively. Compound 1 was tethered to SWNTs by EDC/NHS
amide coupling (Scheme 4).29 Platinum atomic absorption
spectroscopy (AAS) demonstrated that 1 can be easily
attached to the functionalized SWNTs, which can carry a
payload of around 82 Pt(IV) units per SWNT. This
Pt(IV)-SWNT construct can deliver a significant dose of
cisplatin selectively to FR(+) cancer cells, reducing side
effects that are often exhibited by the platinum anticancer
drugs, especially cis-DDP.
Redox Properties. Since reduction of Pt(IV) complexes
inside the cell to release an active Pt(II) species is one of
the key steps for their successful utilization in cancer therapy,
we determined the reduction potentials of 1 at two different
physiological pH values by cyclic voltammetry. An ideal
situation for Pt(IV) reduction demands that it be sufficiently
stable to travel through the body without decomposition until
it reaches a tumor cell and that it have the appropriate
reduction potential to be reduced only when inside the cell.
Premature reduction in the bloodstream is a serious problem
since it both sheds the axial targeting groups and produces a
reactive Pt(II) that can become deactivated in plasma and/or
produce detrimental side effects. Electrochemical studies of
1 at pH 7.4 and 6.0 revealed behavior characteristic of
irreversible loss of the axial ligands. At pH 7.4, a value close
to that in blood, the reduction potential of 1 is -0.698 V vs
NHE. At a pH value of 6.0, closer to that in endosomes and
lysosomes,54–56 there is a positive shift by 126 mV in the
reduction potential of 1 to -0.572 V vs NHE.
These potentials were obtained following extrapolation to
a scan rate of 0.0 mV s-1 to account for the irreversible
behavior of the reduction processes.57 Irreversible cyclic
voltammetric responses and the linear fits of potentials at
different scan rates are presented in Figures S7-S10.
Facilitated reduction at pH 6.0 is driven thermodynamically
by protonation of the axial carboxylate groups as they depart
the platinum coordination sphere. The acidic environment
that builds up in and around cancer cells therefore facilitates
Pt(IV) reduction. The reduction potential of satraplatin, a
Pt(IV) compound currently in clinical trials, is -0.053 V vs
cis,trans,cis-[Pt(NH3)2(succinate)2Cl2] was used as the precur-
sor in the synthesis of the target Pt(IV) compound.44 Compound
1 was prepared by amide coupling of one of the succinate groups
of [Pt(NH3)Cl2(O2COCH2CH2)2] with the free amine group in
4 (Scheme 2). Standard amide coupling protocols using reagents
like EDC/NHS, PYBOP-HOBT, or DCC were unsuccessful.
After several attempts, we determined that HATU/DIPEA could
readily furnish 1 in 51% yield. The formation of 1 was
confirmed by the presence of the parent ion peak at 1158.1 in
the LC-MS (Figure S3).
(51) Lee, R. J.; Low, P. S. In Drug targeting: Strategies, principles, and
applications; Francis, G. E., Cristina, D., Eds.; Methods in Molecular
Medicine 25; Humana Press: Totowa, NJ, 2000; pp 69-76.
(52) Fan, J.; Pope, L. E.; Vitols, K. S.; Huennekens, F. M. Biochemistry
1991, 30, 4573–4580.
(54) Arunachalam, B.; Phan, U. T.; Geuze, H. J.; Cresswell, P. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 745–750.
(55) Collins, D. S.; Unanue, E. R.; Harding, C. V. J. Immunol. 1991, 147,
4054–4059.
(56) Kam, N. W. S.; Dai, H. Phys. Status Solidi B 2006, 243, 3561–3566.
(57) Nicholson, R. S.; Shain, I. Anal. Chem. 1964, 36, 706–723.
(53) Levy, C. C.; Goldman, P. J. Biol. Chem. 1967, 242, 2933–2938.
9
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Targeted Pt(IV) Prodrug Delivery
A R T I C L E S
NHE.58 The instability of satraplatin in blood has been a point
of concern for many years. Its half-life of only 35.8 min in
blood is a consequence of its relatively high reduction
potential at pH 7.4. Compound 1 is expected to be much
more stable in blood than satraplatin.
A comparison of the cathodic potentials of 1 at pH 7.4 with
those reported for several Pt(IV) compounds is presented in
Table 1. The cathodic reduction potential varies with the
electron-withdrawing ability and bulkiness of the axial ligands.59
The lowest reduction potential of 1 among the Pt(IV) complexes
listed in Table 1 might be a consequence of the bulky folate
ligand at an axial position.
Ability of 1 To Target and Destroy Folate Receptor-Po-
sitive Cancer Cells. The ability of SWNT-tethered 1 to destroy
cancer cells was studied by using the MTT assay. To prove
folate receptor-mediated targeting, we investigated FR-positive
[FR(+)] human choriocarcinoma (JAR) and human nasopha-
ryngeal carcinoma (KB) cell lines. FR-negative [FR(-)] tes-
ticular carcinoma cells (NTera-2) were used as a control (Figure
2, Table S1). Compound 1 and cis-DDP were examined as
additional controls. The IC50 values are 0.019 and 0.01 µM,
respectively, for SWNT-tethered 1 treatment of FR(+) JAR and
KB cells, significantly lower than those of cis-DDP or 1. The
IC50 values for cis-DDP and 1 with KB cells are 0.086 and
0.15 µM, respectively. The IC50 value of SWNT-1 increases
to 0.048 µM with FR(-) NTera-2 cells, demonstrating targeted
uptake by the FR(+) cells. In principle, comparison with JAR
or KB cells in which the folate receptor had been eliminated,
for example by RNAi, would have been more appropriate, but
such lines are at present not available to us. NTera-2 cells are,
in our experience, among the most sensitive to cisplatin,
however, so the comparison may be valid. The superior cell-
killing ability of SWNT-1 over 1 in FR(+) KB and JAR cells
indicates that the SWNT longboats can deliver their platinum
cargos containing a folate homing device selectively to folate
receptors overexpressed on the cancer cells. The Pt(IV)-SWNT
construct is >8 times more toxic to FR(+) cancer cells, a value
that would be significant in a clinical context.
One of the important concerns in the design of conjugated
systems is to ensure that the cytotoxic moiety, cis-DDP in
this case, releases from the carrier and can reach its ultimate
receptor, which is DNA in the cell nucleus in the case of
cis-DDP. The reduction potentials together with the IC50
values demonstrate that our SWNT-1 construct fulfills all
the criteria.
Folate-Targeted Cell Uptake Studies of FITC-Labeled Folate
Conjugate 2. The ability of a FITC-labeled conjugate of folic
acid (2) to target FR(+) cells was evaluated by fluorescence
microscopy. The results, summarized in Figure 3, clearly
indicate that 2 selectively accumulates in the FR(+) KB cells
(upper panel) when compared with uptake in FR(-) NTera-2
cells (lower panel). Compound 2 concentrates mainly in the
cytoplasm of KB cells after 2.5 h of incubation at 37 °C.
The conjugate 2, having two moieties, FITC and folic acid,
linked by a PEG linker was able to internalize by FRME,
indicating that the folic acid moiety in 1, which contains a
Pt(IV) center connected to folic acid by a PEG spacer, is
suitable for the FRME pathway.
We also investigated the intracellular localization of SWNT-
tethered 1 by fluorescence microscopy. We cotethered a
fluorescein-based fluorophore to our SWNT-Pt(IV) conjugate,
and the results indicated the presence of fluorescent SWNTs in
the endosomes, supporting the conclusion that uptake of SWNTs
occurs through endocytosis (Figure S11).
Intracellular DNA Adduct Levels. To learn whether or not,
once inside cells, platinum is released from SWNT or folate
receptors, a platinum localization experiment was performed.
Cytosolic and nuclear extracts were collected from KB cells
incubated with 1 µM SWNT-1 for 3 h, and platinum
concentrations were determined by AAS. The nuclear fraction
showed a considerable amount of platinum (86 ng of Pt/mg
of protein). The level of platinum in the cytosol was
comparatively less (11 ng of Pt/mg of protein). We did not
observe any detectable amount of platinum in the cytosol or
nucleus of cells treated with untethered 1 under similar
experimental conditions. Not only was the cytotoxicity of
the SWNT-1 conjugate superior to that of 1, but the latter
also attained higher platination levels with nuclear DNA,
clearly demonstrating that the cis-DDP moiety is efficiently
released from the SWNT and receptor in the cancer cell
environment.
Formation of Pt-1,2-d(GpG) Intrastrand Cross-Links. The
major reaction product of cis-DDP with nuclear DNA is the
1,2-d(GpG) intrastrand cross-link, which occurs in >75% of
all adducts. We employed monoclonal antibody R-C18,50
which is specific for cis-DDP intrastrand 1,2-d(GpG) cross-
links, to probe whether cis-DDP released from SWNT-1
forms this adduct with nuclear DNA. In KB cells, after a
12 h treatment with SWNT-1, the presence of the 1,2-
d(GpG) intrastrand cross-links was detectable by antibody-
derived green fluorescence in the nuclei of the cells (Figure
4). The appearance of 1,2-d(GpG) intrastrand cross-links was
also carried out using cis-DDP as a control (Figure 4). To
our best knowledge, this is the first demonstration of 1,2-
d(GpG) intrastrand cross-link formation with an active Pt(II)
species formed upon reduction of a Pt(IV) precursor with
nuclear DNA in a cell.
Conclusions
In this study, we introduce a route for preparing Pt(IV)
complexes containing both a cell receptor targeting moiety
and a delivery system. In particular, we established that 1,
containing a folic acid derivative as one of its axial ligands,
specifically targets folate receptor-enriched tumor cells.
Compound 1 has significantly enhanced cell-killing properties
when conjugated to SWNTs, which facilitate its delivery via
endocytosis. SWNT-tethered 1 is more active by a factor of
8.6 compared to cisplatin and, upon reduction in the cellular
environment, it forms the major cisplatin 1,2-d(GpG) intras-
trand cross-links with nuclear DNA. The internalization
studies showed high and specific binding to the FR for the
SWNT-1 conjugate. Our data provide strong evidence that
platinum-SWNT constructs containing a folate component
will be valuable for targeted delivery of platinum anticancer
agents. The modular nature of the synthetic approach and
the ability of the longboats to carry 2 orders of magnitude
greater platinum payloads per molecule than mononuclear
complexes such as cisplatin or carboplatin should be valuable
in future research.
(58) Choi, S.; Filotto, C.; Bisanzo, M.; Delaney, S.; Lagasee, D.; Whitworth,
J. L.; Jusko, A.; Li, C.; Wood, N. A.; Willingham, J.; Schwenker, A.;
Spaulding, K. Inorg. Chem. 1998, 37, 2500–2504.
(59) Ellis, L. T.; Er, H. M.; Hambley, T. W. Aust. J. Chem. 1995, 48,
793–806.
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1
Supporting Information Available: H, 195Pt, and ESI-MS
spectral data of 1 (Figures S1-S3), HPLC analysis (Figure S4)
and ESI-MS (Figure S5) for 2, CPG hydrolysis of 4 (Figure
S6), cyclic voltammograms of 1 (Figures S7-S10), a fluores-
cence microscope image of fluorophore-tethered SWNT-1
(Figure S11), and a table of IC50 values (Table S1). This material
is available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by the National
Cancer Institute under grants CA034992 at MIT and NCI-NIH-
CCNE-TR at Stanford and by the Deutsche Forschungsgemeinschaft
under grant TH 251/5-1 to J.T. S.D. is thankful to the Anna Fuller
Fund in molecular oncology for a postdoctoral fellowship. We thank
Dr. Elisa Tomat for preparing 6-carboxy-2′,7′-dichlorofluorescein-
3′,6′-diacetate succinimidyl ester. We thank Dr. Katie R. Barnes
and Ms. Caroline Saouma for initial studies in our laboratory toward
the goals of this article.
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11476 J. AM. CHEM. SOC. VOL. 130, NO. 34, 2008