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Bioconjugate Chem. 2004, 15, 814−823
Nuclear Localization Signal-Targeted Poly(ethylene glycol) Conjugates as Potential Carriers and Nuclear Localizing Agents for Carboplatin Analogues Olga Aronov,† Aviva T. Horowitz,‡ Alberto Gabizon,‡ Miguel A. Fuertes,§ Jose´ Manuel Pe´rez,§ and Dan Gibson*,†,| Department of Medicinal Chemistry and Natural Products, School of Pharmacy, P.O. Box 12065, The Hebrew University of Jerusalem, Jerusalem 91120, Israel, Oncology Institute, Shaare Zedek Medical Center, Jerusalem, Israel, Departamento de Quimica Inorga´nica y Centro de Biologı´a Molecular (CSIC-UAM), Facultad de Ciencias, Universidad Autonoma de Madrid, 28049-Madrid, Spain, and The David R. Bloom Center for Pharmacy, The Hebrew University of Jerusalem, Israel. Received March 14, 2004; Revised Manuscript Received May 12, 2004
Carboplatin is a low-molecular-weight anticancer drug that acts by binding to the nuclear DNA of cells. Thus, efficient delivery of the platinum drugs to the nucleus of the cancer cells may enhance the cytotoxicity of the drug. Efficient drug delivery to the nucleus of cancer cells requires three levels of localization: targeting to the cancerous tissue, accumulation in the cancer cells, and intracellular localization in the nucleus. Nuclear localization signals (NLS) are short positively charged basic peptides that actively transport large proteins across the nuclear membrane. We have prepared conjugates in which the NLS is tethered to poly(ethyleneglycol)carboplatin conjugate (NLS-PEGPt) and compared their pharmacological properties to those of their untargeted analogues that do not possess the NLS (PEG-Pt). NLS-PEG-Pt conjugates are rapidly internalized into cancer cells and accumulate in the nucleus. Despite their rapid nuclear localization, they form less Pt-DNA adducts than the untargeted analogues, PEG-Pt, and are also less cytotoxic. These results support the hypothesis that carboplatin (unlike cisplatin) may require cytosolic activation prior to its binding to nuclear DNA.
INTRODUCTION
Cisplatin, carboplatin, and oxaliplatin (See Figure 1) are three FDA approved anticancer agents that are believed to owe their anticancer activity to their ability to covalently modify cellular DNA (1, 2). Cisplatin and carboplatin form the identical major adducts with DNA where the cis-[Pt(NH3)2]2+ moiety is covalently coordinated to the N7 atoms of two adjacent guanines on the same strand forming the 1,2-d(G*pG*) intrastrand crosslink (3). The 1,2 intrastrand cross-links bend the DNA toward the major groove, resulting in a local distortion of the double helix, and it is this distortion that is believed to trigger a series of cellular events that results in the eventual death of the cancer cell (4). The most common dose limiting effects for platinum based drugs are nephrotoxicity, ototoxicity, neurotoxicity, and myelosuppression. In addition, cancer cells are either inherently refractory to the platinum drugs or can acquire resistance to the drugs. There are several known resistance mechanisms to cisplatin: reduced cellular accumulation, increased production of intracellular platinophiles such as glutathione and metalothionein, enhanced DNA repair and increased tolerance to DNA platination, and decreased cellular apoptotic response (5-7). The first two mechanisms of resistance attempt to prevent the * Corresponding author. † School of Pharmacy, The Hebrew University of Jerusalem. ‡ Shaare Zedek Medical Center. § Universidad Autonoma de Madrid. | The David R. Bloom Center for Pharmacy, The Hebrew University of Jerusalem.
Figure 1. Platinum-based anticancer drugs: (a) cisplatin, (b) carboplatin, and (c) oxaliplatin.
drug from reaching the nuclear DNA, its critical binding site. Higher levels of Pt-DNA adducts are expected to result in improved cytotoxicity. Thus, rapid and efficient delivery of the platinum drugs to the nucleus of the cancer cells may overcome some of the resistance pathways and could enhance the cytotoxicity of the drugs. Efficient drug delivery to the nucleus of cancer cells requires three levels of localization: targeting to the cancerous tissue, accumulation in the cancer cells, and intracellular localization in the nucleus (8, 9). We recently described the preparation and pharmacological properties of a conjugate comprised of a poly(ethylene glycol) (PEG)1 carrier to which folic acid (FA) was attached on one end (to exploit the folic acid receptor mediated endocytotic
10.1021/bc0499331 CCC: $27.50 © 2004 American Chemical Society Published on Web 07/01/2004
NLS-Targeted PEG Conjugates
pathway) and a carboplatin analogue to the other end (FA-PEG-Pt) (10). PEG was chosen as the carrier because attaching a low molecular-weight drug to a hydrophilic polymer such as PEG results in high aqueous solubility, slower clearance, reduced systemic toxicity, and efficient accumulation in tumors through enhanced permeability and retention (EPR) (11). PEG is a water soluble amphiphilic polymer showing excellent biocompatibility and is frequently used in biomedical applications. Several drugs such as doxorubicin, camptothecin and paclitaxel, have been conjugated to PEG carriers and these conjugates are in various stages of clinical trials (12). Folic acid has been used extensively to internalize covalently tethered cargos into cancer cells, via the FRME, since many cancer cell lines overexpress folic acid receptors (13, 14). When the cell uptake, cellular DNA binding and cytotoxicity of FA-PEG-Pt were compared to those of the PEG-Pt conjugate (without the FA), it was found that FA-PEG-Pt conjugates were taken into the cytosols of the cancer cells significantly more rapidly than the PEG-Pt conjugates. Yet incubation with the PEG-Pt conjugates afforded twice as many platinum cellular DNA adducts as did FA-PEG-Pt. This is not necessarily surprising since it has been reported that most endocytosed compounds end up in the lysosome or in some compartment that is separated from the cytosol by a membrane, preventing them from reaching their critical biological targets (15). Thus, there is a need to explore a nonendocytotic pathway for the efficient delivery of platinum complexes to the nuclei of cancer cells. The research following discovery that the HIV-1 Tat protein can cross cellular membranes, established that positively charged peptides such as Tat(49-57) and Antp(43-58) have membrane translocating properties that can be exploited for the delivery of oligonucleotides, proteins, imaging agents liposomes, etc. (16, 17). While the exact mechanism by which the basic peptides enter the cells remains to be elucidated, it seems that they do not enter the cells by endocytosis and it is the amphipathicity of the peptides that is responsible for the cellular translocation. Nuclear localization signals (NLS) are short positively charged basic peptides that actively transport large proteins across the nuclear membrane. They have been used to localize cargo molecules (to which they have been conjugated) from the cytosol to the cell nucleus (18). The cellular delivery and particularly intracellular localization of therapeutics are two of the foremost challenges facing the medicinal chemists. In this manuscript we report the design, synthesis, and preliminary pharmacological properties of novel PEG-NLS conjugates as carriers for carboplatin analogues. EXPERIMENTAL PROCEDURES
Materials. All reagents were purchased from SigmaAldrich Israel, Ltd., at least reagent-grade and were used 1 Abbreviations: CBDCA, cyclobutane-1,1-dicarboxylate; PEG,poly-(ethylene glycol); FA, folic acid; FRME, folate receptormediated endocytosis; FMOC, fluorenylmethyl chloroformate; Cbz-Cl, benzyl chloroformate; FITC, fluorescein isothiocyanate; DCC, dicyclohexylcarbodiimide; DCU, dicyclohexylurea; ECI, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride; DIEA diisopropylethyl-amine; AA, atomic absorption; CPG, carboxypepidase G; MALDI-TOFMS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HiFr, high folate receptor; Icp-Ms, inductively coupled plasma mass spectrometry; FACS, fluorescence-assisted cell sorting; MB, methylene blue; PEG3000,-(CH2CH2O)68-; NHS, N-hydroxysuccinimide.
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without purification. Solvents for synthesis and for HPLC were synthesis and HPLC-grade accordingly. Compound Preparation. NH2-GABA-Cys-Gly-GlyPro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly-CO-Resin (1). GABA-FMOC. A 1 g (9.7 mmol) sample of γ-aminobutyric acid (GABA) was dissolved in 10% NaHCO3. A 1.5 g (5.8 mmol) sample of FMOC-Cl was dissolved in 30 mL of acetonitrile and was added dropwise over 2 h to the GABA solution. The reaction mixture was allowed to stand for 1 additional hour, and then the acetonitrile was evaporated and the solution was acidified to pH 1 with HCl. The white precipitate was filtered, washed with two portions of water and one portion of ethyl acetate, and desiccated over P2O5 (yield 80%). NH2-Cys-Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-GlyCO-Resin. The peptide NH2-Cys-Gly-Gly-Pro-Lys-LysLys-Arg-Lys-Val-Gly-Gly-COOH was synthesized by the solid-phase FMOC method on a Rink amide resin with the carboxy terminus still attached to the resin and the amino acid side chains protected as follows: Lys (-NBOC), Arg(Pmc), and Cys(S-Trt). NH2-GABA-Cys-Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-ValGly-Gly-CO-Resin. A 50 mg (0.017 mmol) sample of the peptide on the resin was put in a Merrifield vessel, and 14 mg (0.043 mmol) of GABA-FMOC, 2 mL of NMP, 19 mg (0.043 mmol) of BOP-reagent and 50 µL of DIEA were added. The reaction was agitated overnight at room temperature, after which the reaction mixture was filtered and a sample from the resin was used for a Kaiser test (for primary amines). If the test was positive, the reaction was repeated until the test came negative, indicating complete coupling. The resin was washed with 2 mL portions of DMF, and then 2 mL of a 20% piperidine in DMF was added and the deprotection was allowed to proceed for 2 h at room temperature with constant shaking and subsequently the resin was washed with four 2 mL portions of DMF. FITC-NH-GABA-Cys-Gly-Gly-Pro-Lys-Lys-Lys-Arg-LysVal-Gly-Gly-CO-Resin (2). A 10 mg (0.043 mmol) sample of FITC, 2 mL of DMF, and 50 µL of TEA were shaken overnight with the resin at room temperature, the resin was filtered, and a small portion of the resin was tested for free amines using the Kaiser test. After a negative Kaiser test was obtained the resin was washed with four 2 mL portions of DMF. HO-NLS-Cys-GABA-FITC (3). A 150 mg sample of phenol, 0.05 mL of thioanisole, 0.05 mL of water, and 0.025 mL of dithioethane in 2 mL of TFA were added to the resin, and the reaction was left for 3 h at room temperature with constant shaking. The reaction mixture was filtered, 20 mL of diethyl ether was added, and the orange precipitate that was formed was isolated by centrifugation. The precipitate was washed with three 20 mL portions of diethyl ether and then dissolved in 2 mL of water and lyophilized to yield an orange powder (yield 60%). RP18 HPLC: Rt ) 10.2 min. Mass spectra (ESIMS): four peaks corresponding to four charge states of the compound were observed at m/z values of 423.4, 564.1, 845.3 and 1688.4, indicating a molecular mass of 1687.4 (calcd 1687.2) H2N-PEG-NH2 (4). The compound was prepared as described by us recently (10). Cbz-PEG-NH2 (5). A 500 mg sample of Na2CO3 and 3 g of H2N-PEG-NH2 (1 mmol) were dissolved in 150 mL of water, and 50 mL of acetonitrile was then added. A 170 mg (1 mmol) sample of benzyl chloroformate was dissolved in 50 mL of acetonitrile and was added very slowly at room temperature over approximately 3 h to the reaction mixture. After the addition of the benzyl
816 Bioconjugate Chem., Vol. 15, No. 4, 2004
chloroformate was completed, the acetonitrile was removed under reduced pressure, and the remaining aqueous solution was extracted with two 100 mL portions of dichloromethane. The organic fractions were combined, dried over MgSO4, and crystallized from 100 mL of diethyl ether. The crude product contained some starting material (H2N-PEG-NH2), the desired product (CbzPEG-NH2), and some Cbz-PEG-Cbz. The desired monocationic momoprotected PEG was purified by cation exchange chromatography on a Sephadex CM-50 resin with UV detection at 254 nm. The reaction mixture was dissolved in a minimal amount of water (5 mL) and loaded on the column, and the neutral Cbz-PEG-Cbz was removed from the column with deionized water. The desired product was eluted with 25 mM NaCl and showed a UV absorption at 254 nm and also gave a positive test for a primary amine with fluorescamine. The product was lyophilized and dissolved in 100 mL of dichloromethane, the NaCl was filtered off, the solvent was removed, and the product was crytalized from 50 mL of diethyl ether. The yield was 30%. RP18-HPLC: one peak Rt ) 16.2 min. 1H NMR (DMSO-d ) δ(ppm): 6 7.32(m, 5H, -CH-(ph)), 7.23(t, 1H, Cbz-NH-), (bs, ∼280H, -PEG3000-), 3.14(q, 2H, Cbz-NH-CH2-, 2.95(t, 2H, -CH2-NH2). FA-PEG-NH2 (6). The synthesis of FA-PEG-FMOC was described in ref 10. FA-PEG-FMOC was used as the starting material for the preparation of FA-PEG-NH2. The FMOC protecting group was removed from FA-PEGFMOC by piperidine treatment. FA-PEG-FMOC (400 mg) was dissolved in 50 mL of 25% aqueous piperidine. The white precipitate was removed by filtration, the solvents were evaporated to remove all traces of piperidine, and the product was dissolved in water and evaporated again. The process was repeated twice. RP18HPLC: Rt ) 14.34 min. 1H NMR (D2O) δ(ppm): 8.62(s, 1H, folate-C(8)H), 7.65(d, 2H, folate(PABA)H2), 6.81(d, 2H, folate(PABA)H2), 4.51(s, 2H, folate(NH-CH2)), 3.6(bs, ∼280H, -PEG3000-), 3.14(m, 2H, folate-NH-CH2), 2.95(t, 2H, -CH2-NH2), 2.21-1.95(bm, 4H, folate(Glu)β,γCH2) 4.22(m, 1H, folate(Glu)R-CH). FA-PEG-Maleimide (7). 300 mg (0.095 mmol) of FA-PEG-NH2 was dissolved in a 5 mL of a saturated solution of NaHCO3, and the solution was cooled in an ice bath. An 80 mg (0.52 mmol) sample of N-methoxycarbonylmaleimide was added with stirring, and the reaction mixture was left in the ice bath for 15 min and was stirred for an additional 30 min at room temperature. The product was isolated on a Sephadex 25 column and immediately lyophilized to yield a white powder. Yield, 90%. RP18-HPLC: Rt ) 16.51min. 1H NMR (D2O) δ(ppm): 8.62(s, 1H, folate-C(8)H), 7.65(d, 2H, folate(PABA)H2), 7.01(2H, -CH)CH-), 6.81(d, 2H, folate(PABA)H2), 4.51(s, 2H, folate(NH-CH2)), 4.22(m, 1H, folate(Glu)R-CH) 3.6(bs, ∼280H, -PEG3000-), 3.14(m, 4H, maleimide-NH-CH2-/folate-NH-CH2), 2.21-1.95(bm, 4H, folate(Glu)β,γ-CH2). NLS-FITC-PEG-FA (8). A 100 mg sample of FAPEG-maleimide (7) was dissolved in a 50 mM phosphate buffer (pH 5.8), and to the reaction mixture was added 70 mg of HO-NLS-Cys-GABA-FITC (3) (0.041 mmol) and the reaction mixture was stirred at room temperature for 45 min. The product was isolated by semipreparative RP-HPLC and was lyophylized. The yield was 50%. A 10 mg sample of (8) was dissolved in 10 mL of PBS buffer, and the concentrations of the fluorescein (490 ) 67 000) and folate (363 ) 6197) were determined. The ratio between the fluorescein and the folate was 1.0: 0.95, attesting to the purity of the product. RP18 HPLC:
Aronov et al.
Rt ) 14.17 min. MALDI-TOF-MS showed a bell distribution with the center around 5412 amu). Cbz-PEG-Maleimide (9). A 300 mg (0.095 mmol) sample of Cbz-PEG-NH2 was dissolved in a 5 mL saturated solution of NaHCO3, and the solution was cooled in an ice bath. An 80 mg (0.52 mmol) sample of N-methoxycarbonylmaleimide was added with stirring, and the reaction mixture was left in the ice bath for 15 min and was stirred for an additional 30 min at room temperature. The product was isolated on a Sephadex 25 column and immediately lyophilized to yield a white powder. Yield, 90%. RP18 Rt ) 17.15 min. NLS-PEG-FITC (10). A 100 mg (0.031 mmol) sample of Cbz-PEG-maleimide was dissolved in a 50 mM phosphate buffer (pH 5.8), 70 mg (0.041 mmol) of NLSCys-GABA-FITC was added, and the reaction mixture was stirred for 45 min. The product was purified and isolated by semipreparative RP-HPLC and lyophilized to yield an orange powder. Yield, 60%. RP18 HPLC Rt ) 15.11 min. MALDI-TOF-MS a bell distribution centered around 5200 amu (Calc. 5203 amu). Di-tert-butyl 2-(3-succininylaminopropyl)malonate (11). The synthesis and characterization of compound 11 were detailed in ref 10. 1H NMR (DMSO-d ) δ(ppm): 7.84(bt, 1H, C(O)-NH-), 6 3.16(t, 1H, R-CH-), 2.99(q, 2H, NH-CH2-), 2.38(t, 2H, succ.-CH2-), 2.29(t, 2H, succ.-CH2-), 1.61(m, 2H, -CH2CH-), 1.41(bs, 20H, -CH2-CH2-CH2-/t-Bu). Cbz-PEG-Mal-(tBu) (12). The synthesis and characterization of compound 12 were detailed in ref 10. Maleimide-PEG-Malonic Acid, Disodium Salt (13). Initially the Cbz protective group of compound 12 was removed by hydrogenation, and then the primary amine was converted to the maleimide in the same way as was done for the preparation of compound 7. The hydrolysis of the tert-butyl protecting groups was performed by dissolving 500 mg of maleimide-PEG-Mal-tBu in 30 mL of 25% TFA in CH2Cl2; after 2 h of stirring the solvent was removed, and the product was taken up in 3 mL of 50 mM Na2CO3. The product was purified on a Sephadex 25 column and lyophilized yielding a white solid (yield 90%). 1 H NMR (D2O) δ(ppm): 6.88(s, 2H, -CH)CH-), 3.6(bs, ∼280H, -PEG3000-), 3.36(t, 4H, PEG-CH2-N), 3.14(t, 2H, -NH-CH2-), 2.92(t, 2H, R-CH-), 2.48(m, 4H, succ.-CH2-), 1.60(m, 2H, -CH2-CH-), 1.37(m, 2H, -CH2CH2-CH2-). cis-Diamminediaquaplatinum(II) (14). The cis-[Pt(NH3)2(D2O)]2+ was obtained by reacting cis-Pt(NH3)2Cl2 with 1.95 equiv of AgNO3 in D2O as previously described (10). 195Pt NMR (D2O) δ(ppm): -1580 maleimide-PEGPt (15). The platination, purification, and characterization of the Pt conjugate were performed by reacting a slight excess of compound 13 with compound 15, as previously described (10). RP18 HPLC: Rt ) 16.3 min 1H NMR (D O) δ(ppm): 6.88(s, 2H, -CH)CH-), 3.62 (bs, ∼280H, -PEG3000-), 3.36(t, 4H, PEG-CH2-NH-), 3.14(t, 2H, -NH-CH2-), 3.08(t, 2H, R-CH-), 2.48(m, 4H, succ.-CH2-), 2.21(m, 2H, -CH2-CH-), 1.49 (m, 2H, -CH2-CH2-CH2-). 195Pt NMR (D2O) δ(ppm): -1703. NLS-PEG-Pt (16). Compound 15 was coupled to the commercially prepared NLS peptide (including the terminal Cys) using the procedure described for the preparation of compound 8. The reaction was monitored by RPHPLC Rt(product) ) 14.8 min and Rt(reactant) ) 16.3 min. The reaction was over in 45 min, and the product was purified by semipreparative HPLC with a yield of 50%.
NLS-Targeted PEG Conjugates
RP18 HPLC: Rt ) 14.8 min. 195 Pt NMR (D2O) δ(ppm): -1701. FITC-PEG-Pt(17/18). Compounds 18 and 19 were prepared using the methodologies described above. 195Pt NMR (D O) δ(ppm): -1689(31), -1692(32). 2 FITC-Pt (19). Di-tert-butyl 2-(3-Aminopropyl)malonate was prepared as described (10). A 100 mg (0.37 mmol) sample of this compound was dissolved in 30 mL of pyridine and 100 mg (0.26 mmol) of FITC, and 50 µL of TEA was added with stirring. The reaction was left overnight, and subsequently the solvent was removed under reduced pressure. The residue was taken up by 50 mL of CH2Cl2, and the excess amine was removed by washing with two portions of 50 mL solutions of 10% acetic acid. After evaporation an orange solid is obtained. Yield 80%. Mass Spectrum, (LC ESIMS): single peak at 553.2 (calculated 552.7). The deprotection of the malonate and platination was performed as described above, and the product precipitated as an orange solid and was filtered and washed with ethanol and ether. Yield 90%. 195Pt NMR (D O) δ(ppm): -1708 ppm. 2 NMR Spectroscopy. NMR data were collected on a Varian Unity Inova 500 MHz spectrometer equipped with a 5 mm switchable probe for 1H, 13C, 195Pt NMR, and 500 MHz 1H{13C/X} 5 mm PFG tunable triple inverse detection probe for 2D-[1H,15N] HSQC. 195Pt NMR. The platinum chemical shifts were measured relative to the external reference signal of K2PtCl4, set at -1624 ppm. A line-broadening (lb) of 300 Hz was normally applied, and data were processed using the VNMR software. Inductively Coupled Plasma Mass Spectrometry (Icp-Ms). Icp-Ms measurements were performed on a Elan-DRC II, Perkin-Elmer Sciex spectrometer. The platinum concentration was calculated according to a known concentration of a K2PtCl4 standard solution (10 ng/mL Pt). Cell Culture and Biological Studies. Medium. Cells were cultured in FA-free RPMI medium (RPMI-1640, Biological Industries, Beyt Haemek, Israel), with 10% fetal bovine serum (GIBCO, Grand Island, NY), 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 passaged by treatment with a trypsin (0.05%)/ EDTA (0.02%) solution in calcium- and magnesium-free phosphate-buffered saline (PBS). Cell Lines. M109 is a murine lung carcinoma line of BALB/c mice (19). By culturing these cells in FA-free medium for several passages, we obtained a subline expressing a high amount of FA receptors, named M109 HiFR or M109FR, to emphasize the overexpression of folic acid receptors (20). Fluorescent Microscopy Studies. Cells were grown on eight-well glass slides (Nunk). Forty-eight hours prior to the experiment, 0.2 × 105 cells were seeded per each well of the slide. The cells were washed with FA, serumfree RPMI medium and incubated for 4 h at 37 °C with 0.2 mL of a 0.5 µM solution of fluorescein-labeled PEG’s in FA, serum-free RPMI medium. Free folate competition experiments were conducted under the same conditions, except that incubation solutions contained, in addition to fluorescein-labeled PEG, free folic acid at a concentration of 1 mM. After incubation, cells were washed three times with 0.5 mL of RPMI medium and fixed with 3% formaldehyde in PBS. The fluorescent images were
Bioconjugate Chem., Vol. 15, No. 4, 2004 817
obtained with an LSM410 invert Laser Scan Confocal Microscope, at the wavelength I¨ ) 488 nm. Cell Growth Inhibition Studies. The cytotoxic effect of free carboplatin and two PEG-Pt conjugates (FA-PEG-Pt and PEG-Pt) was assayed colorimetrically by the MB staining method described previously (10). In our assays, M109HiFR cells in 200 µL aliquots were plated onto 96-well flat-bottom microtiter plates. Following 20 h in culture, 20 µL of the tested drug was added to each well. For each 10-fold increase in drug concentration, five drug concentration points were tested. Each test was performed in triplicate wells and in two parallel plates. The cells were treated continuously for 72 h. The drug concentration which caused a 50% inhibition of the control growth rate (IC50) was calculated by interpolation of the two closest values of the growth inhibition curve. Cell Lines and Culture Conditions for the Cytotoxicity Measurement with the Human Ovarian Cancer Cell Lines. Cultures of pairs of cisplatin sensitive and resistant ovarian cancer cell lines (A2780/A2780cisR, 41M/41McisR, and CH1/CH1cisR) have been described elsewhere (21). These pairs of cell lines were selected on the basis of encompassing all of the known major mechanisms of resistance to cisplatin: 41McisR being resistant primarily through reduced drug transport (22), CH1cisR through enhanced DNA repair/tolerance (23), and A2780cisR through a combination of decreased uptake, enhanced DNA repair/tolerance, and elevated GSH levels (24). Cell survival in compound-treated cultures was evaluated by the MTT method as previously reported (25). Platinum compounds were added to 96 microwell plates containing the cell cultures at final concentrations between 0 and 200 µM. After 24 h, cell survival was evaluated by measuring the absorbance at 520 nm, using a Whittaker microplate reader 2001. IC50 values (compound concentration that produces 50% of cell killing) were calculated from curves constructed by plotting cell survival (%) versus compound concentration (µM). Experiments were carried out in quadruplicate. Pt Accumulation Studies with Continuous Incubation. The cellular accumulation of the Pt compounds was assayed through measurement of the level of cellassociated Pt. Forty-eight hours prior to an assay, 2 × 106 cells were seeded per 60 mm dish, to obtain 5 × 106 cells/plate. At the day of the assay, plates were washed twice with RPMI medium and incubated for 4 h at 37 °C with 2 mL of FA-free RPMI medium, containing three different concentrations (100, 200, and 500 µM) of the platinum compound. Then the incubation solution was removed, and cells were washed with 2 mL of RPMI medium, released from plates with trypsin/EDTA treatment, washed three times with PBS, counted, and dried under reduced pressure. Cell-associated platinum was extracted with 0.1 mL of concentrated nitric acid for 15 min at 90 °C. Subsequently, solutions were diluted with water, and the Pt concentration was established using Icp-Ms. Pt Binding to Cellular DNA. Cells were centrifuged, and the DNA was extracted by the salting out procedure (26). Cells were re-suspended in 10 mL of Nuclear Lysis buffer (10 mM Tris at pH 8.2, 400 mM NaCl, 2 mM EDTA). The cell lysates were digested for 4 h at 37 °C with 0.5 mL of 10% SDS and 0.1 mL of protease K solution (Sigma, 20 µg/mL water solution). Then, 5.5 mL of a saturated NaCl solution was added to the lysates, and the mixture was shaken vigorously followed by centrifugation at 10 000 rpm for 15 min. The supernatant containing the DNA was transferred to another tube,
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Figure 2. The NLS conjugates that were prepared for this study.
containing 20 mL of absolute ethanol and mixed gently. DNA strands were removed, washed three times in water/ethanol mixture (1:1), and dissolved in 2 mL of distilled water. DNA concentration was established according to absorbance at 260 nm, and the samples were diluted with TE buffer (10 mM Tris pH 8, 1 mM EDTA) buffer. DNA purity was determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm. All samples showed the A260/A280 ratio of 1.7-1.9, which indicates that we obtained DNA with a high degree of purity. The platinum content in the DNA solutions was determined by Icp-Ms measurements. RESULTS
Synthesis. The conjugates that were prepared for this study, NLS-FITC-PEG-FA, NLS-FITC-PEG, NLSFITC, and NLS-PEG-Pt, are depicted in Figure 2. Since the NLS is the terminal moiety of all of these conjugates and since the NLS is prepared on a solid phase support, it seems like the synthesis of a NLS-PEG conjugates would best be performed by conjugating the PEG moiety to the peptide while the latter is still bound to the solid phase column. This would result in a single product that could be easily purified by size exclusion chromatography. All attempts to couple the PEG moiety to the peptide while still bound to the solid-phase resin were unsuccessful. Therefore another strategy for the synthesis of NLS-FITC-PEG-FA conjugate was devised and is shown in Scheme 1. The approach taken was to initially prepare the fluorophorated peptide (3), then the functionalized PEG (7), and in the final step to conjugate them to each other in solution to obtain the desired product (8). Often, conjugation of peptides is accomplished by coupling the primary amine of the peptide to a carboxylate group of the cargo thereby forming a stable amide linkage. There are two main reasons for not using the primary amine groups of the NLS peptide for coupling the peptide to the PEG: (a) since there are several primary amines on the peptide (four lysines and one terminal amine), from a chemical point of view, it will be impossible to obtain selectivity in coupling the peptide to a PEG-carboxylate and obtain only one product; (b)
from a biological point of view, the lysines and arginines of the NLS are essential for attaining nuclear localization and should not be tampered with. Thus, a unique functional group that can be used for coupling to the PEG must be appended to the peptide. Thiols are known to react rapidly and selectively with maleimide groups, even in the presence of primary amine, and so we decided to add a thiol to the peptide and a maleimide to the PEG conjugate to allow selective and efficient coupling. The details of the synthetic approach are depicted in Scheme 1A. A cysteine was appended to the NLS sequence to provide a specific coupling moiety (1). A spacer of γ-aminobutyric acid (GABA) was added to the cysteine, and the fluorophore (FITC) was selectively coupled to the primary amine of the GABA on the solid-phase resin (while all the other amines were still protected). After deprotection and purification by RP-HPLC the peptide (3) was obtained and was characterized by electrospray ionization mass spectrometry and NMR spectroscopy. Thiols can be air oxidized to disulfides that are unreactive toward maleimide. High-performance liquid chromatography was used to check for the formation of intermolecular disulfide bond. Very little oxidation was observed but to be on the safe side, DTT was added to the peptide prior to coupling of the thiol to the maleimide. The second component of the final conjugate (see Scheme 1B) was a PEG3000 to which a maleimide group was appended on one end. The synthetic approach was to initially prepare the monoprotected Cbz-HN-PEGNH2 (5) as the universal starting material for all subsequent syntheses of the PEG conjugates. The unprotected free primary amine was coupled to the desired carboxylate, and subsequently, the Cbz protecting group was removed by hydrogenation and the primary amine at the other side of the PEG could be coupled to another carboxylate group, yielding a nonsymmetric bifunctionally modified PEG. In this specific case, the folic acid was activated by NHS as previously described (10), followed by the removal of the protecting group to yield H2NPEG-FA (6). Since the hydrodynamic properties of the PEG dominate the chromatographic behavior of the conjugates, it is difficult, if not impossible, to separate
NLS-Targeted PEG Conjugates
Bioconjugate Chem., Vol. 15, No. 4, 2004 819
Scheme 1
Scheme 2
the PEG conjugates from the unmodified PEGs. Therefore, a large excess of the low molecular weight moieties was used in all the conjugation reactions, to try and force the reaction to completion and to simplify the purifications. The preparation of a PEG having a terminal maleimide is depicted in Scheme 1B. The amino terminus of the FA-PEG-NH2 was converted to the maleimide using the N-methoxycarbonylmaleimide reagent, followed by purification by gel-permeation chromatography (GPC), and was characterized by 1H NMR spectroscopy. The double bond of the maleimide reacts specifically and rapidly with thiols yielding stable thioether bonds (Scheme 1C). The
reaction between the PEG-maleimide and the NLSCys-GABA-FITC was monitored by RP-HPLC and after 45 min the reaction was complete. The product, FAFITC-PEG-FA (8), was purified by HPLC and characterized by mass spectrometry. The synthesis of NLS-PEG-FITC (10) is depicted in Scheme 2. Initially the Cbz-PEG-NH2 (5) was synthesized, and subsequently the NH2 was converted to the maleimide with N-methoxycarbonylmaleimide (9). An excess of the flourophorated peptide (3) was reacted with the FA-PEG-maleimide, and the conjugate was purified by HPLC.
820 Bioconjugate Chem., Vol. 15, No. 4, 2004
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Scheme 3
The synthesis of the NLS-PEG-Pt is depicted in Scheme 3. Di-tert-butyl 2-(3-succininylaminopropyl)malonate (11) and 1-Cbz,ω-aminopolyoxyethylene3000, Cbz-PEG-NH2 (5), were prepared as previously described (10). and then were coupled using EDCI to yield compound (12). The Cbz was removed by hydrogenation, and the resulting primary amine was converted to a maleimide group as described to yield compound (13). The NLS with the appended cysteine (without the GABA and FITC) was prepared and purified as described. There are two synthetic pathways that could be taken at this point: to first couple the peptide to the PEG-di-tBumalonate (13a), remove the tert-butyl protecting group with acid, and neutralize the resulting diacid (13b), converting the maloante into the disodium salt (13c); and finally to platinate the ligand (conjugate). The second option is to first deprotect the malonate and convert it to the disodium salt (13c), then platinate and purify the platinum complex and finally to conjugate the NLS-PEG to the Pt-malonate. More often than not, the approach taken by the bioinorganic chemists and by the inorganic medicinal chemists is to initially complete the ligand synthesis and leave the platination for the last step. In this instance we chose the opposit approach, as depicted in Scheme 3. The rationale behind this choice stems from the fact that the reactive cis-[Pt(NH3)2(H2O)]2+ (14) could react with the thioether that connects the PEG-maleimide with the NLS-Cys and even possibly with the lysines and arginines of the peptide. On the other hand, the diamminemalonate coordination sphere of the platinum(II) renders the Pt(II) quite inert, and the reaction of the cysteine thiol with the maleimide is expected to be faster and more selective than its reaction with the inert Pt moiety. There are reports in the literature showing that carboplatin can react with the thioether of methionine (27), raising question as to the potential stability of the conjugate. We have checked the stability of the conjugate by HPLC and by preparing the 15N labeled Pt complex and measuring the two-dimensional NMR [1H,15N] HSQC spectrum. The cross-peak of the diamminemalonate at -78.8/4.15 was stable for weeks in solution, and no cross-peak was observed in the 15N chemical shift range around -40
Figure 3. Pt-PEG conjugates that were prepared for this study.
ppm, which is typical for the thioether binding. The observed stability is probably due to the polymeric moiety (PEG) that separates the Pt from the sterically hindered thioether and prevents facile intramolecular reactions. At pharmacological concentrations there is probably no need to worry about intermolecular interactions between two conjugates. To test how the Pt moiety and the properties of the PEG affect cellular uptake, three more fluorophorated platinum complexes (FITC-PEG6000-Pt, FITC-PEG3000Pt, and FTIC-Pt) (17, 18 and 19, respectively) were prepared as controls for the cell uptake and cytotoxicity studies of the conjugates. The molecules are depicted in Figure 3, and their synthesis was accomplished using the chemistry described above. Cell Accumulation and Intracellular Distribution. We initially designed and prepared the FITC-PEGNLS-FA conjugate in order to obtain: selectivity toward cancer cells, in which the FA receptors are overexpressed, uptake by FRME, and accumulation in the nucleus with the aid of the NLS. A 25 µM solution of compound (8) was incubated with M109FR cells for 2 h, and the results of the confocal microscopy are depicted in Figure 4A, clearly demonstrating that the conjugates have accumulated in the cell nucleus. Since this conjugate has two moieties (the NLS and the FA) capable of transferring the conjugate across the cell membrane, further
NLS-Targeted PEG Conjugates
Bioconjugate Chem., Vol. 15, No. 4, 2004 821 Table 1. Pharmacological Data on the NLS-PEG-Pt Conjugatea
carboplatin NLS-PEG-Pt a
IC50 µM
uptake ng Pt/106 cells
DNA-adducts ng Pt/mg DNA
33.15 ( 0.5 83.5 ( 2.1
6.7 ( 1.2 19 ( 1.7
10.75 ( 2.5 5.9 ( 1.8
Based on experiments with M109 cell line.
Figure 5. Cytotoxicity data for the interactions of carboplatin and compounds 17-19 (left to right) with human ovarian cancer cell lines. Each result is an average of three measurements with an average standard deviation of (3.
Figure 4. Confocal microscopy pictures showing the cellular localization of (a) NLS-FITC-PEG-FA with M109FR cells, (b) NLS-FITC-PEG with M109FR cells, (c) NLS-FITC with M109FR cells,(d) NLS-FITC-PEG-FA with M109FR cells in the presence of an excess of FA, (e) NLS-FITC-PEG-FA with wild-type M109 cells, and (f) FITC-PEG-FA with M109FR cells.
experiments were performed to establish the mechanism of cellular internalization. The FITC-PEG-NLS conjugate (10) was prepared and incubated with the M109FR cells, and again, nuclear localization was observed (Figure 4B), demonstrating that the peptide itself can translocate the conjugate across the cell membrane. Just to verify that it is indeed the NLS, and not the PEG, which is responsible for the cell internalization, the NLS was conjugated directly to the FITC (without the PEG), and again, nuclear localization was obtained (Figure 4C). To establish whether the NLS or the FA are the dominant factors in the cell internalization of compound (8) that contains both moieties, we studied the cell accumulation of this compound in the M109FR cell line when in competition with free FA. In the competition experiment with a large excess of free FA, the cellular accumulation was not affected (Figure 4D), indicating that the conjugate does not enter the cell via the FRME. In addition, we also studied the cellular accumulation in the native M109 cell line that is not enriched with FA receptors in order to factor out the contribution of the folic acid. The accumulation profile in M109 cells (Figure 4E) is practically identical to that observed in M109FR cells, reinforcing the results of the previous experiments. Incubation of the conjugate without the NLS (FITC-PEG-FA) with
the M109FR cells results in cytosolic accumulation of the conjugate (Figure 4f). Thus, both the experiments show clearly that the conjugate does not enter the cells via the FRME suggesting that it is the NLS that is responsible for both cellular accumulation and nuclear localization. Therefore, in the presence of NLS, the FA moiety does not contribute to selectivity via FR and pathway of uptake via FRME of the conjugates. NLS appears to be the prevailing moiety responsible for cell uptake of the conjugate. We also studied the cellular accumulation of Pt-PEG complexes with an FITC reporter group. Toward this end the molecules (17-19) in Figure 3 were prepared and purified, and their cellular accumulation was studied by confocal microscopy, FACS and ICPMS. The results indicate that the PEGless Pt-FITC conjugate (19) is accumulated within the M109 cells approximately twice as much as the Pt-PEG6000/3000-FITC conjugates (the FACS intensities were 71, 71, and 126 for compounds 17, 18, and 19, respectively). This indicates that PEGylation of the conjugate retards cell uptake. However, PEGylation may also retard efflux of the conjugate from the cell and enable more effective activation of carboplatin in the cytosol, as shown previously (10). Cytotoxicity and Intracellular DNA Adduct Levels. The pharmacological data for the studies performed in wild-type M109 cancer cells are listed in Table 1. Contrary to our expectation, the NLS-PEG-Pt conjugate was less cytotoxic than the Pt-PEG conjugate and produced less DNA-Pt adducts, despite the higher cellular accumulation and nuclear localization of the NLS conjugates observed by confocal microscopy. This indicates that NLS delivers carboplatin to the nucleus in a nonbioavailable form. The cytotoxicity of FITC-PEG3000-Pt, FITC-PEG6000Pt, and FTIC-Pt was measured in three human ovarian cell lines (A2780, CH1, and 41M), and the results are depicted in Figure 5. It is interesting to note that although FITC-Pt is accumulated within the cells twice as efficiently as the FITC-PEG-Pt conjugates, it is significantly less cytotoxic than the FITC-PEG-Pt conjugates. Also, it seems that the length of the PEG (3000 vs 6000) does not affect the cellular accumulation nor does it have an effect on the cytotoxicity.
822 Bioconjugate Chem., Vol. 15, No. 4, 2004 DISCUSSION
A commonly accepted notion is that rapid nuclear localization should result in a higher level of formation of platinum-DNA adducts and hence superior cytotoxicity. Thus, over the years many attempts have been made to conjugate platinum complexes to DNA binding moieties such as intercalators (28-32) in order to increase the DNA binding affinity and specificity. The tethering of DNA binding agents to platinum complexes via stable covalent bonds yields in fact novel complexes that are bound to have properties that are quite different from those of the active moieties and thus they cannot be considered true delivery systems or prodrugs for the established drugs. One of the major concerns during the design of these systems is to ensure that the cytotoxic moiety, cis-[Pt(NH3)2]2+ in this case, separates from the carrier at the right time and location. This rationale dictated for our choice of a carboplatin analogue since the release of the cis-[Pt(NH3)2]2+ is expected to occur in a fashion similar to what happens with carboplatin itself. Yet, not only was the cytotoxicity of the NLS-PEG-Pt conjugate inferior to that of PEG-Pt but also the latter attained higher platination levels with cellular DNA despite its inferior cellular accumulation and intracellular localization. Clearly, the cytotoxic cis-[Pt(NH3)2]2+ moiety is not efficiently released from the carrier in the nuclear environment. The activation of carboplatin has been subject to a certain amount of debate since direct reaction of carboplatin with double stranded DNA results in very low levels of DNA platination (33). Fichtinger-Schepman observed that exposure of carboplatin to Chinese hamster ovary cells and subsequent reaction with Salmon sperm DNA resulted in much higher DNA platination levels when compared with platination levels of cell-free Salmon sperm DNA by carboplatin. In fact the platination was 10 times more efficient (34). This may indicate that carboplatin requires some intracellular activation by yet unknown mechanisms is order to be able to more efficiently platinate DNA. Sadler has shown that carboplatin can be chemically activated by thiols that react with the platinum complex, forcing the ring opening of the chelating malonate and rendering the Pt moiety more reactive (35). Sadler’s work was performed in vitro and describes one possible mechanism for the activation of carboplatin though other intracellular activation processes may be involved. When the platinum medicinal chemists design novel platinum anticancer agents, they normally tend to think of the cytosol of the cancer cell, with its wealth of platinophiles, as an obstacle that the platinum drug has to be overcome en route to the nuclear DNA. Yet it may be worthwhile to make a distinction between the more reactive diaminedichloroplatinum(II) complexes such as cisplatin, trans-platinum complexes with iminoether, aliphatic amines, planar amines, and nonplanar cyclic amine ligand and the inert compounds such as carboplatin, oxaliplatin, and nedaplatin, for which intracellular activation may be necessary for their activity. The results of this study suggest that the activation process, whatever it may be, probably takes place in the cytosol rather than in the nucleus. Thus, NLS based delivery is probably not suitable for carboplatin, oxaliplatin, and nedaplatin or any other platinum complex that requires cytosolic activation. We are in the process of testing the NLS-PEG conjugates as delivery systems for other anticancer drugs that bind to nuclear DNA. The PEG itself, at the lengths tested, impedes the cellular uptake of the platinum complexes yet it enhances
Aronov et al.
the cytotoxicity of the conjugates relative to those without the PEG. This may be due to the fact that while carboplatin or FITC-Pt are taken into the cell efficiently they might also be effluxed more easily than the PEGylated analogues (10), and since carboplatin analogues probably require relative long cytosolic residence times for activation, the PEG conjugates may just provide this necessary time frame for activation. It is noteworthy that the FITC-Pt conjugate is significantly less cytotoxic than carboplatin, indicating that the intracellular interactions of the two compounds are not identical. Since attempts to study the intracellular distribution of platinum compounds often entail functionalizing them with fluorophores that significantly alter the properties of the drug, the results reported here indicate that great care must be taken in the interpretation of such experiments. ACKNOWLEDGMENT
This work was supported partially by the European Cooperation in the Field of Scientic and Technical Research Network (COSTD20/003/00 Action: “Biochemistry, Structural and Cellular Biology of Non-Classical Antitumor Platinum Compouds” (J.M.P. and D.G.). The work done at Dr. Gabizon’s laboratory was supported in part by a grant from the Israel Cancer Association. A specific and institutional grant from Fundacio´n Ramo´n Areces is also acknowledged (J.M.P.). D.G. acknowledges the support from “The Grass Center for Drug Design and Synthesis of Novel Therapeutics”. LITERATURE CITED (1) Lokich, J. (2001) What is the “best” platinum: cisplatin, carboplatin, or oxaliplatin? Cancer Invest. 19, 756-760. (2) Lokich, J., and Anderson, N. (1998) Carboplatin versus cisplatin in solid tumors: an analysis of the literature. Ann. Oncol. 9, 13-21. (3) Cohen, S. M., and Lippard, S. J. (2001) Cisplatin: from DNA damage to cancer chemotherapy. Prog. Nucleic Acid Res. Mol. Biol. 67, 93-130. (4) Brabec, V. (2002) DNA modifications by antitumor platinum and ruthenium compounds: their recognition and repair. Prog. Nucleic Acid Res. Mo.l Biol. 71, 1-68. (5) Fuertes, M. A., Alonso, C., and Perez, J. M. (2003) Biochemical modulation of Cisplatin mechanisms of action: enhancement of antitumor activity and circumvention of drug resistance. Chem. Rev. 103, 645-662. (6) Perez, R. P. (1998) Cellular and molecular determinants of cisplatin resistance. Eur. J. Cancer 34, 1535-1542. (7) Kelland, L. R. (1994) The molecular basis of cisplatin sensitivity/resistance. Eur. J. Cancer 30A, 725-727. (1) (8) Widder, K. J., Senyei, A. E., and Ranney, D. F. (1979) Magnetically responsive microspheres and other carriers for the biophysical targeting of antitumor agents. Adv. Pharmacol. Chemother. 16, 213-271. (9) Langer, R. (1998) Drug delivery and targeting. Nature 392, 5-10. (10) Aronov, O., Horowitz, A. T., Gabizon, A., and Gibson, D. (2003) Folate-targeted PEG as a potential carrier for carboplatin analogues. Synthesis and in vitro studies. Bioconjugate Chem. 14, 563-574. (11) Takakura, Y., and Hashida, M. (1996) Macromolecular carrier systems for targeted drug delivery: pharmacokinetic considerations on biodistribution. Pharm. Res. 13, 820-831. (12) Zalipsky, S. (1995) Chemistry of Polyethylene-Glycol Conjugates with Biologically-Active Molecules. Adv. Drug Delivery Rev. 16, 157-182. (13) Wang, S., and Low, P. S. (1998) Folate-mediated targeting of antineoplastic drugs imaging agents, and nucleic acids to cancer cells. J. Controlled Release 53, 39-48. (14) Sudimack, J., and Lee, R. J. (2000) Targeted drug delivery via the folate receptor. Adv. Drug Delivery Rev. 41, 147-162.
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