Folate-Targeted PEG as a Potential Carrier for Carboplatin Analogs

The Hebrew University of Jerusalem, Jerusalem 91120, Israel, and Oncology Institute, Shaare Zedek Medical. Center; Jerusalem, Israel. Received Novembe...
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Bioconjugate Chem. 2003, 14, 563−574

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Folate-Targeted PEG as a Potential Carrier for Carboplatin Analogs. Synthesis and in Vitro Studies Olga Aronov,† Aviva T. Horowitz,‡ Alberto Gabizon,‡ 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, and Oncology Institute, Shaare Zedek Medical Center; Jerusalem, Israel. Received November 20, 2002; Revised Manuscript Received February 12, 2003

Like most low molecular weight drugs, carboplatin has a short blood circulation time, which reduces tumor uptake and intracellular DNA binding. Drugs conjugated to PEG carriers benefit from prolonged blood circulation, but suffer from reduced cell permeability. In this work we attempted to develop long-circulating PEGylated carboplatin analogues with improved cell permeation abilities, by conjugating the platinum moiety to folate-targeted PEG carriers capable of utilizing the folate receptormediated endocytosis (FRME). Two bifunctional FA-PEG conjugates, FA-PEG-Pt and FA-PEGFITC, were prepared, and their cell uptake, DNA binding, and cytotoxicity were studied by fluorescent microscopy, FACS, and platinum analysis. Folate-targeted PEG conjugates enter the cells efficiently by the FRME pathway but form relatively few DNA adducts and have higher IC50 values than carboplatin and their nontargeted analogues. Nontargeted PEG-Pt conjugates have a lower cellular uptake but produce higher levels of DNA binding and improved cytotoxicity. Carboplatin, used as a control, has the fastest cellular uptake, but after 16 h of postincubation a large percentage of the drug is excreted from the cells. The findings of this study suggest that folate-targeted conjugates such as FA-PEG-Pt, may not be an optimal prodrug for the carboplatin family compounds, because the conjugates or the active moieties are neutralized or blocked during the FRME process and do not manage to effectively reach the nuclear DNA.

INTRODUCTION

Carboplatin [Pt(CBDCA-O,O′)(NH3)2], where CBDCA is cyclobutane-1,1-dicarboxylate (Figure 1), is a second generation platinum anticancer drug, widely used for treatment of many types of cancer, including ovarian cancer, lung cancer, head and neck cancer. etc. (1-3). The antitumor effect of carboplatin is thought to be due to its interaction with DNA where the Pt(NH3)2 moiety binds covalently to two adjacent guanines on the same strand (4). It is those Pt-DNA adducts that are believed to lead to the eventual death of the cancer cells. Due to the electrostatic effect and the chelate effect of the dicarboxylate ligand, the substitution kinetics of the Pt(II) complex are slow, rendering it quite inert. Therefore, the hydrolysis and direct platination of free DNA by carboplatin have been reported to be very slow (5, 6). Interestingly, when the level of DNA platination obtained upon exposure of Chinese hamster ovary cells to carboplatin was compared with platination levels of cell-free Salmon sperm DNA by carboplatin, it was reported that the former is 10 times more efficient (7). This indicates that carboplatin is more efficient in platinating DNA when exposed to some as yet unknown intracellular activation mechanisms. Although carboplatin is highly efficient against many neoplasms, the drug has drawbacks that are related to its unfavorable pharmacokinetic properties. Like most * To whom correspondence should be addressed. Tel: +9722-6758702. Fax: +972-2-6757076. E-mail: [email protected]. † The Hebrew University of Jerusalem. ‡ Shaare Zedek Medical Center. § Affiliated with the David R. Bloom, Center for Pharmacy at The Hebrew University of Jerusalem, Israel.

Figure 1. Carboplatin.

low molecular weight drugs, carboplatin has a short blood circulation time (8). The main route for the clearance of carboplatin from the body is through urinary excretion where 50% of the administered carboplatin is cleared within 24 h (9). This is a disadvantage especially due to the fact that the high intracellular levels of carboplatin, needed for efficient DNA platination, are difficult to obtain when carboplatin is rapidly cleared from the plasma. The use of water-soluble polymers as macromolecular carriers for low-molecular weight conventional drugs is a very promising recent strategy in anticancer therapy. In comparison with a native drug, a macromolecular prodrug exhibits improved body distribution and prolonged blood circulation, due to the dominant pharmacokinetic properties of the macromolecular carrier (10). Poly(ethylene glycol) (PEG) is a water soluble amphiphilic polymer showing excellent biocompatibility and is frequently used in biomedical applications (11). Drugs modified with PEG carriers also benefit from lowered immunogenicity and better tumor uptake (12-14). However, as a result of the increase in molecular weight, cell uptake and consequently biological activity of prodrug complexes may be reduced. Cell internalization ability is especially important for stable prodrugs, such as carboplatin analogues. Among numerous PEG-containing prodrugs, which were synthesized and tested recently, there is only one report about an attempt to modify

10.1021/bc025642l CCC: $25.00 © 2003 American Chemical Society Published on Web 04/23/2003

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Figure 2. Folic acid.

carboplatin by conjugation with PEG (15). Unfortunately, the lack of information about the cell uptake of the PEGPt conjugates and about DNA-platination levels precludes significant conclusions concerning the relationship between molecular weight, cell uptake, and biological activity of the complexes. There are many examples for exploiting existing endocytosis pathways for specific targeting and delivery of macromolecules (16). Coupling of macromolecular carriers with a normally endocytosed ligand frequently improves intracellular accumulation of the prodrug. Folic acid (FA) is one of the most popular targeting ligands, which were utilized for this strategy. There are many reports that macromolecules and other carriers, conjugated to FA (Figure 2), were successfully recognized by FA receptors and internalized into cells via folate receptor-mediated endocytosis (FRME) (17, 18). The folate receptor is a glycosyl-phosphatidylinositol-anchored glycoprotein (19), with high affinity for the FA vitamin (Kd ∼ 10-9-10-10 M).20 It is located in caveolae and participates in the cellular accumulation of folate through the process of potocytosis. In this process, a ligand-bound receptor is sequestered in caveolae, followed by internalization into postcaveolar plasma vesicles, released from the receptor via an intravesicular reduction in pH, and subsequently transported into cytoplasm (for polyglutamation) (21, 22). In addition, cell surface receptors for folic acid are generally overexpressed in human cancer cells (23, 24). The combination of these facts with low imunogenicity and the relatively simple chemistry of FA make it a very useful tool in specific drug targeting. In this study we prepared carboplatin-PEG conjugates (PEG-Pt) and their folic acid analogue (FA-PEG-Pt) and compared their cytotoxicities, cellular uptake, intracellular distribution and DNA platination levels. EXPERIMENTAL PROCEDURES

Materials. All reagents were purchased from SigmaAldrich Israel LTD, at least reagent grade and used without purification. Solvents for synthesis and for HPLC were synthesis and HPLC grade accordingly. 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. 2D-[1H,15N] HSQC Spectroscopy. The 2D spectra were recorded using a heteronuclear single quantum correlation (HSQC) with gradient selection and editing sequence according to Kay et al. (25). 15N spins were irradiated during the acquisition using the GARP-1 sequence. 15N chemical shifts were externally referenced to 1.5 M NH4Cl in 1 M HCl (δN ) 0 ppm, δH ) 7 ppm); 1 H chemical shifts were externally referenced to TSP (Me3Si(CD2)2CO2Na) (δH ) 0 ppm). Data were collected

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at 25 °C. A total of 64 transients and 128 increments were collected over 6.5 h. All data were processed using the VNMR software, using the sinebell and sinebell shift functions in both dimensions. Data were zero filled to 4096 data points. RP HPLC. Analytical monitoring of the products was conducted on a RP C-18 HPLC column (Merck 50833 Lichrosphere 250 × 4.0 mm ID, 5 µM), using a Spectra Series P100 Gradient Pump and a Thermo Separation Products variable wavelength UV100 detector. The gradient running was 5% A/95% B to 100% A over 30 min at a flow rate of 1 mL/min with detection at 220 nm, where A is 0.1% TFA in acetonitrile and B is 0.1% TFA in water. Mass Spectrometry. MALDI-TOF-MS of PEG conjugates was measured on Bruker REFLEX reflector timeof-flight instrument with SCOUT multiprobe (384) inlet and gridless delayed extraction ion source (Bruker, Breman, Germany). All spectra were acquired in a positive-ion reflectron mode. For acquisition of mass spectrometric data, 0.5 µL of sample (1:50 in water) was added to 0.5 µL of 2,5-dihydroxybenzoic acid (0.1% TFA/ acetonitrile 2:1) and dispensed onto the sample support. Angiotensin-2 and ACTH were used as external standards for MALDI-TOF MS spectra calibration. The spectra exhibited a bell-shaped distribution of 44-Da spaced lines. The shift in single mass peak of PEG conjugates spectra in comparison to the spectrum of the original PEG-3000 spectrum was detected. ESI-MS was measured on a ThermoQuest Finnigan LCQ- Duo in the positive ion mode. Elution was in a mixture of 49:49:2 water:methanol:acetic acid at a flow rate of 15 µL/min. UV Analysis. The contents of folate and fluorescein in PEG conjugates were determined by quantitative UV spectrometry of the conjugates in PBS (pH ) 7.4 solution). Using extinction coefficient values: 363 ) 6197 M-1cm-1 for FA (26) and 490 ) 67000 M-1 cm-1 for FITC (27). Atomic absorption (AA) measurements were performed on a Varian SpetraAA Zeeman 300 spectrometer. The platinum concentration was calculated according to a calibration curve of a known concentration of a K2PtCl4 stock solution (250 ng/mL Pt). Inductively coupled plasma mass spectrometry (ICPMS) measurements were performed on a Elan-DRC II, Perkim-Elmer Sciex spectrometer. The platinum concentration was calculated according to a known concentration of a K2PtCl4 standard solution (10 ng/mL Pt). Synthesis and Characterization of PEG Conjugates. 1,ω-Diaminopolyoxyethylene3000 (1). Ten grams (3.3 mmol) of dry poly(ethylene)glycol 3000 was dissolved in 100 mL of dry CH2Cl2. Seven milliliters of triethylamine was added, the reaction was cooled in an icewater bath, and 1 mL (13.3 mmol) of CH3SO2Cl was added dropwise with stirring. When all the CH3SO2Cl was added, the ice bath was removed, and the reaction was stirred overnight at room temperature. The reaction mixture was washed with 100 mL of 50 mM NaHCO3. The organic fraction was dried over MgSO4 and filtered, and the solvent was evaporated to give 1,ω-dimethanesulfonylpolyoxyethylene3000 that was immediately dissolved in 150 mL of a concentrated solution of aqueous ammonia and was left to stir for 48 h in a sealed flask. Then, the product was extracted twice with CH2Cl2, the organic fractions were combined, dried over MgSO4, and filtered, and the solvent was removed under reduced pressure. The product was crystallized from Et2O. The

Folate-Targeted PEG

yield was 80%. 1H NMR (CDCl3) δ(ppm): 3.68 (t, 2H, NH2CH2CH2), 3.6 (bs, ∼280H, PEG3000), 2.97 (t, 2H, NH2CH2). 1-FMOC,ω-aminopolyoxyethylene3000 (2). Three grams (1 mmol) of (1) was dissolved in 150 mL of a 10 mM NaHCO3 solution and diluted with an additional 150 mL of CH3CN, and 260 mg (1 mmol) of FMOC was dissolved in 50 mL of CH3CN and added dropwise with stirring to the reaction. The mixture was left to react for an additional 1 h at room temperature, and then the mixture was acidified with HCl. The CH3CN was evaporated, and the water was lyophilized. The mixture of products (unprotected, monoprotected, and bisprotected PEG-diamine) was desalted by dissolving the lyophilized sample in CH2Cl2 followed by filtering out of the NaCl. The CH2Cl2 was removed, and the mixture of products was dissolved in water and separated on Sephadex CM25 cation exchange column. The monoprotected fraction was eluted with a 25 mM NaCl solution, collected, acidified with HCl, and lyophilized. The final product was desalted again by dissolving it in CH2Cl2, filtering out the solids, and evaporation of the solvent. The product was crystallized from Et2O. The yield was 30%. 1H NMR (DMSO-d6) δ(ppm): 7.88 (d, 2H, fluor), 7.78 (d, 2H, fluor), 7.44-7.31 (dt, 4H, fluor), 4.28 (d, 2H, fluor-CH2O), 4.22 (t, 1H, H-(C9)), 3.6 (bs, ∼280H, PEG3000), 3.14 (q, 2H, FMOC-NHCH2), 2.95 (t, 2H, CH2NH2). FA-PEG-FMOC (3). Initially the active ester of the glutamic residue of the folic acid was prepared. FA-NHS Ester. Folic acid (300 mg, 0.67 mmol) was dissolved in 12 mL of dry DMSO to which 93 mg (0.45 mmol) of DCC and 77 mg (0.67 mmol) of NHS were added. The reaction was left overnight at room temperature, in the dark. The DCU precipitate (a side product of the reaction) was filtered out, and 100 mL of 30% acetone in Et2O was added with stirring. The yellow precipitate was collected on sintered glass and washed with acetone and ether and was used immediately in the next step of synthesis. NH2-PEG-FMOC (500 mg, 0.15 mmol) was coevaporated with three 50 mL portions of dry pyridine and finally dissolved in 100 mL of dry pyridine. Then FANHS ester (2) obtained from the activation of 300 mg (0.67 mmol) of folic acid (see previous paragraph) was added to the solution and the reaction was left overnight stirring in the dark. Upon completion of the reaction (confirmed by the ninhydrine test) the pyridine was evaporated and the remaining solid was dissolved in 20 mL of 100 mM NaHCO3. The reaction mixture was chromatographed on a Sephadex 25 column and lyophilized. The yield was 80%. 1H NMR (D2O) δ(ppm): 8.62 (s, 1H, folate-C(8)H), 7.85 (d, 2H, fluor), 7.78-7.65(m, 4H, fluor/folate(PABA)H2), 7.56-7.31 (dt, 4H, fluor), 6.81 (d, 2H, folate(PABA)H2), 4.51 (s, 2H, folate(NH-CH2)), 4.38 (d, 2H, fluor-CH2O), 4.32-4.22 (m, 2H, fluor(C9)-H/folate(Glu)R-CH)), 3.6 (bs, ∼280H, PEG3000), 3.14 (m, 4H, FMOC-NHCH2/folate-NHCH2) 2.21-1.95 (bm, 4H, folate(Glu)β,γ-CH2). HPLC Monitoring of CPG-Mediated Glutamic Acid Cleavage. The experiment was carried out according to a published protocol:26 0.1 mL of 0.5 mg/mL solution of FA-PEG-FMOC was incubated with 2 units of CPG in a 150 mM, pH 7.3 Tris buffer, at 30 °C. After 4 h of incubation, the decrease in total peak area of the conjugate (tR ) 18.7, detection wavelength λ ) 360) was 65%. An additional 1 unit of CPG and prolonged incubation (up to 24 h) did not change this result. Cbz-PEG-FMOC (4). NH2-PEG-FMOC (500 mg, 0.15 mmol) was dissolved in 50 mL of a 10 mM NaHCO3

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solution and diluted with an additional 50 mL of acetonitrile. Cbz-Cl (51 mg, 0.3 mmol) was dissolved in 10 mL of CH3CN and added dropwise with stirring to the reaction, at room temperature. The mixture was left to react for an additional 1 h after the CH3CN, water, and excess Cbz-Cl was removed by evaporation. The product was desalted, as described above. The yield was 90%. 1H NMR (DMSO-d6) δ(ppm): 7.88 (d, 2H, fluor), 7.78 (d, 2H, fluor), 7.65 (t, 1H, FMOC-NH), 7.44-7.31 (m, 9H, Ph, fluor), 7.23 (t, 1H, Cbz-NH), 4.99 (s, 2H, PhCH2O), 4.73 (bs, ∼280H, PEG3000), 4.28 (d, 2H, fluor-CH2O), 4.22 (t, 1H, H-(C9)), 3.14 (q, 2H, Cbz-NHCH2), 2.95 (t, 2H, CH2NH2). FA-PEG-FITC (5). First, the FMOC protecting group was removed from FA-PEG-FMOC by piperidine treatment. FA-PEG-FMOC (400 mg) was dissolved in 50 mL of 25% aqueous piperidine. Then white precipitate was removed by filtration, and solvents were evaporated. To remove all traces of piperidine, the product was dissolved in water and evaporated again. The process was repeated twice. FA-PEG-NH2 (100 mg, 0.026 mmol) was dissolved in 5 mL of DMF, and 20 mg (0.05 mmol) of FITC was added in the dark to the reaction mixture which stood at room temperature. Ninhydrin test indicated that the reaction was complete after 20 h. Aminopropanol (0.01 mL) was added to deactivate the excess of FITC, and the solvent was removed by evaporation. The product was purified on a Sephadex-25 column and lyophilized. The yield was 90%. HPLC: tR ) 17.7; UV (FA/FITC): [FA]/[FITC] ) 0.94. Cbz-PEG-FITC (6). The FMOC protecting group was selectively removed from Cbz-PEG-FMOC (4) as described above, and the primary amine was coupled to FITC as described in the previous section. The yield was 90%. HPLC: tR ) 16.8; MALDI TOF: The shift in single mass peak (in comparison to original PEG) is 708 (calculated 691). Di-tert-butyl 2-(3-Phthalimidopropyl)malonate (7). Di-tert-butyl malonate (2.2 mL, 10 mmol) was dissolved in 100 mL of dry THF, and 1.8 g (8 mmol) of KOt-Bu was added to the reaction mixture with stirring and under dry N2. The reaction mixture was slightly heated until the solution became clear, and then 3-bromopropylphthalimide was added. The reaction was left to stand overnight with stirring and under reflux. The end of the reaction was confirmed by TLC [95% CHCl3/5% ethyl acetate; Rf (bromopropylphthalimide) ) 0.6, Rf(7) ) 0.4)]. The THF was removed by evaporation, and the product was redissolved in CH2Cl2 and washed with 5% aqueous acetic acid. The organic fractions were combined, dried over MgSO4, and filtered, and the solvent was evaporated. The final product was isolated by silica column chromatography (95% chloroform, 5% ethyl acetate) and crystallized from petroleum ether. The yield was 60%. 1H NMR (CDCl ) δ(ppm): 7.82 (d, 2H, Ph), 7.68 (t, 2H, 3 Ph), 3.69 (t, 2H, phthalimide-CH2), 3.17 (t, 1H, R-CH, malonate), 1.85 (m, 2H, CH2CH), 1.62 (m, 2H, CH2CH2CH2), 1.48 (s, 18H, t-Bu). 13C NMR (CDCl3) δ(ppm): 168.90 (C(O)-tBu), 168.31 (C(O), phthalimide), 133.87 (CH, phthalimide), 132.09 (C, phthalimide), 123.917 (CH, phthalimide), 81.81 (C quaternary, tBu), 53.41 (CH2NH), 39.29 (R-CH, malonate), 27.78 (CH3, tBu), 26.75 (NHCH2CH2), 25.69 (CH2CH). Di-tert-butyl 2-(3-Aminopropyl)malonate (8). 7 (4 g, 10 mmol) was dissolved in 100 mL of absolute ethanol, and 0.8 mL (25 mmol) of hydrazine monohydrate was added. The reaction was refluxed for 3 h; after that the solvent and excess of hydrazine monohydrate were

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evaporated under reduced pressure. The remaining solid was dissolved in methylene chloride and washed with 100 mL of 0.5 N NaOH. The aqueous phase was extracted with methylene chloride, the organic fractions were combined, dried over MgSO4, and filtered, and the solvent was removed to yield the product. The yield was 80%. 1H NMR (CDCl ) δ(ppm): 3.17 (t, 1H, R-CH), 2.71 (t, 3 2H, NH2CH2), 1.85 (m, 2H, CH2CH), 1.62 (m, 2H, CH2CH2CH2), 1.48 (s, 18H, t-Bu); 13C NMR (CDCl3) δ(ppm): 81.81 (C quaternary, tBu), 53.41 (CH2NH), 39.29 (R-CH, malonate), 27.78 (CH3, tBu), 26.75 (NHCH2CH2), 25.69 (CH2CH). ESI MS: 274 (calculated 274). Di-tert-butyl 2-(3-Succininylaminopropyl)malonate (9). 2 (2 g, 7.3 mmol) was dissolved in 100 mL of dry methylene chloride, and 1 g (10 mmol) of succinic anhydride was added with stirring to the reaction that was left overnight at room temperature. The end of reaction was confirmed by the ninhydrin test. Then, 50 mL of a 5% citric acid solution in water was added, and the reaction proceeded with stirring for an additional 60 min. The organic phase was separated and washed with an additional 50 mL portion of 5% citric acid solution. The organic fraction was dried under MgSO4 and filtered, and the solvent was evaporated. The product was crystallized from Et2O. The yield was 90%. 1H NMR (DMSO-d ) δ(ppm): 7.84 (bt, 1H, C(O)NH), 6 3.16 (t, 1H, R-CH), 2.99 (q, 2H, NHCH2), 2.38 (t, 2H, succCH2), 2.29 (t, 2H, succ-CH2), 1.61 (m, 2H, CH2CH), 1.41 (bs, 20H, CH2CH2CH2/t-Bu). 13C NMR (CDCl3) δ(ppm): 175.76 (COOH), 172.52 (C(O)NH), 168.90 (C(O)-tBu), 81.81 (C quaternary, tBu), 53.41 (CH2NH), 39.29 (R-CH, malonate), 30.75 (HOOCCH2), 29.91 (CH2C(O)NH), 27.78 (CH3, tBu), 26.75 (NHCH2CH2), 25.69 (CH2CH). ESI MS: 374.53 (calculated 373.44). FMOC-PEG-MAL(tBu)2 (10). NH2-PEG-FMOC (1.6 g, 0.5 mmol), di-tert-butyl 2-(3-succinylaminopropyl)malonate (9) (205 mg, 0.55 mmol), ECI (192 mg, 2 mmol), and DIEA (0.35 mL) were dissolved in 100 mL of dry methylene chloride at room temperature. The reaction was stirred for 3 h, and the completion of reaction was confirmed by the ninhydrin test. Then, the excess of ECI and DIEA was extracted with a 1% solution of citric acid. The organic fraction was dried over MgSO4 and filtered, and the solvent was evaporated. The product was crystallized from Et2O. The yield was 85%. %. 1H NMR (D2O) δ(ppm): 7.88 (d, 2H, fluor), 7.78 (d, 2H, fluor), 7.44-7.31 (dt, 4H, fluor), 4.28 (d, 2H, fluor-CH2O), 4.22 (t, 1H, H-(C9)), 3.6(bs, ∼280H, PEG3000), 3.34 (m, 4H, PEG-CH2NH), 3.26 (t, 1H,Mal-R-CH), 3.07 (t, 2H, NHCH2), 2.48 (m, 4H, succ-CH2), 1.61 (m, 2H, CH2CH), 1.46-1.38 (m+s, 20H, CH2CH2CH2/t-Bu). FA-PEG-MAL(tBu)2 (11). First, the FMOC was removed from FMOC-PEG-MAL(tBu)2 as described above. Then, it was coupled to FA, as described above. The yield was 80%. 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)), 4.22 (m, 1H, folate(Glu)R-CH), 3.6 (bs, ∼280H, PEG3000), 3.34 (m, 4H, PEG-CH2NH), 3.26 (t, 1H,Mal-R-CH), 3.07 (t, 2H, NHCH2), 2.48 (m, 4H, succ-CH2), 2.21-1.95 (bm, 4H, folate(Glu)β,γ-CH2), 1.61 (m, 2H, CH2CH), 1.46-1.38 (m+s, 20H, CH2CH2CH2/t-Bu). HPLC Monitoring of CPG-Mediated Cleavage. The experiment was carried according to a published protocol (26), as described in the FA-PEG-FMOC preparation procedure. Integration area of the conjugate (tR ) 19.1) was reduced to 35% of original size. Cbz-PEG-MAL(tBu)2 (12). First, the FMOC group was removed from FMOC-PEG-MAL(tBu)2 as described

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above and then the free amine coupled with Cbz, as described in above. The yield was 90%. 1H NMR (DMSOd6) δ(ppm): 7.84 (dt, 2H, CH2C(O)NH), 7.32 (m, 5H, CH(Ph)), 7.23 (t, 1H, Cbz-NH), 4.98 (s, 2H, Cbz), 3.6 (bs, ∼280H, PEG3000), 3.34 (m, 4H, PEG-CH2NH), 3.26 (t, 1H, Mal-R-CH), 3.07 (t, 2H, NHCH2), 2.48 (m, 4H, succ-CH2), 1.61 (m, 2H, CH2CH), 1.41 (bs, 20H, CH2CH2CH2/t-Bu). FA-PEG-MAL(Na)2 (13). First, the t-Bu protecting group was removed from FA-PEG-MAL(tBu)2, and then 500 mg (0.13 mmol) of FA-PEG-MAL(tBu)2 was dissolved in 30 mL of a 25% TFA solution in CH2Cl2. The reaction proceeded 2 h at room temperature, the CH2Cl2 and THF were removed by evaporation, and the product was washed with Et2O and dissolved in water. The pH of the solution was fixed at pH ) 7, by titration with 1 M solution of NaOH. 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(NHCH2)), 4.22 (m, 1H, folate(Glu)R-CH), 3.6 (bs, ∼280H, PEG3000), 3.34 (m, 4H, PEG-CH2NH), 3.07 (t, 2H, NHCH2), 2.92 (t, 1H, MalR-CH), 2.48 (m, 4H, succ-CH2), 2.21-1.95 (bm, 4H, folate(Glu)β,γ-CH2), 1.73 (m, 2H, CH2CH), 1.44 (m, 2H, CH2CH2CH2). RP-HPLC: tR ) 15.8. Cbz-PEG-MAL(Na)2 (14) was prepared as described in the procedure for the preparation of FA-PEG-MAL(Na)2. 1H NMR (D2O) δ(ppm): 7.32 (m, 5H, CH(Ph)), 4.98 (s, 2H, Cbz), 3.6 (bs, ∼280H, PEG3000), 3.36 (t, 4H, PEGCH2NH), 3.14(t, 2H, NHCH2), 2.92 (t, 2H, R-CH), 2.48 (m, 4H, succ-CH2), 1.73 (m, 2H, CH2CH), 1.44 (m, 2H, CH2CH2CH2). HPLC: tR ) 14.7. FA-PEG-Pt (15). Initially, cis-[Pt(NH3)2(D2O)2]2+(NO3)2 was synthesized. cis-PtI2(NH3)2 (40 mg, 0.082 mmol) and AgNO3 (28 mg, 0.162 mmol) were stirred vigorously overnight, in the dark in 2 mL of D2O. The yellow precipitate (AgI) was filtered out, and the filtrate was characterized by a single peak (δ ) -1580 ppm) in the 195Pt NMR spectrum. The solution of cis-[Pt(NH3)2(D2O)2]2+(NO3)2 was mixed with 152 mg (0.041 mmol) of FA-PEG-MAL(Na)2 and stirred at room-temperature overnight. The end of reaction was confirmed by 1H NMR. The product was purified on a Sephadex-25 column and lyophilized. 1H NMR (D2O) δ(ppm): 3.6 (bs, ∼280H, PEG3000), 3.36 (t, 4H, PEG-CH2NH), 3.14 (t, 2H, NHCH2), 2.92 (t, 2H, R-CH), 2.48 (m, 4H, succ-CH2), 2.26 (m, 2H, CH2CH), 1.48 (m, 2H, CH2CH2CH2). 195Pt NMR (D2O) δ(ppm): -1698; HPLC: tR ) 17.8; UV (FA): AA (Pt): [FA]/[Pt] ) 1.13. Cbz-PEG-Pt (16) was prepared as described in the procedure for the preparation of 15. 15N-Labeled Pt complexes were synthesized according to the published procedures for the synthesis of cis-DDP.27 1H NMR (D2O)δ(ppm): 7.32 (m, 5H, CH(Ph)), 4.98 (s, 2H, Cbz), 3.6 (bs, ∼280H, PEG3000), 3.36 (t, 4H, PEG-CH2NH), 3.14 (t, 2H, NHCH2), 2.92 (t, 2H, R-CH), 2.48 (m, 4H, succ-CH2), 2.26 (m, 2H, CH2CH), 1.48 (m, 2H, CH2CH2CH2). 195Pt NMR (D2O) δ(ppm): -1698; 2D-[1H,15N] HSQC (10% D2O) [4.15/-7.78] HPLC: tR ) 16.6; MALDI TOF: The shift in single mass peak (in comparison to original PEG) is 767 (calculated 764). 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 passed by treatment with a trypsin (0.05%)/EDTA

Folate-Targeted PEG

(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 (28). 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, to emphasize the overexpression of folic acid receptors (26). 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, serum-free 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 obtained with an LSM410 invert Laser Scan Confocal Microscope, at the wavelength λ ) 488 nm. Fluorescent Flow Cytometry Studies. Forty-eight hours prior to experiment, 2 × 106 cells were seeded per each 60 mm dish. Then cells were washed with RPMI medium and incubated for 4 h at 37 °C with 2 mL of 0.5 µM solution of fluorescein-labeled PEG’s in RPMI medium. Free folate competition experiments were conducted under the same conditions, except that the incubation solutions contained, in addition to fluorescein-labeled PEG’s, free folic acid at a concentration 1 mM. After incubation cells were washed three times with 0.5 mL of RPMI medium, released from plates with trypsin/EDTA treatment, centrifuged, and resuspended in 2 mL of PBS. The fluorescence scanning of the cells performed by FACScan (B&D Beckton-Dickinson) at the wavelength λ ) 488 nm. Cells Growth Inhibition Studies. The cytotoxic effect of free carboplatin and two PEG-Pt conjugates (FAPEG-Pt and PEG-Pt) was assayed colorimetrically by the MB staining method described previously (29). 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. Pt Uptake Studies with Continuous Incubation. The uptake of the Pt compounds was assayed through measurement of the level of cell-associated 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 ICPMS.

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Pt Uptake Studies with Postincubation. Six days prior to an assay, 1 × 106 cells were seeded per 162 cm2 flask, to obtain about 40 × 106 cells/flask. At the day of the assay, plates were washed twice with 15 mL FA, serumfree RPMI medium and incubated for 4 h at 37 °C with 15 mL of FA, serum-free RPMI medium, containing 200 µM of the platinum compound. Then, the incubation solution was removed and the cells were washed twice with 15 mL of FA, serum-free RPMI medium and were incubated in 15 mL of RPMI medium at 37 °C for an additional 16 h. After incubation cells washed with FA, serum-free RPMI medium and released from the flask by trypsin/EDTA treatment. Ten percent of cells were separated and taken to Pt uptake assay. Cells were counted, cell-associated platinum was extracted, and its content was established as described in previous section. The rest of the cell fraction (90%) was used for Pt-DNA binding measurement as detailed below. Pt-DNA Binding. Cells were centrifuged, and the DNA was extracted by the salting out procedure (30). Cells were resuspended in 10 mL of Nuclear Lysis buffer (10 mM Tris pH 8.2, 400 mM NaCl, 2mM 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 g ) 10000 rpm for 15 min. The supernatant containing the DNA was transferred to another tube, 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, 1mM 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 pure DNA. The platinum content in the DNA solutions was determined by ICPMS measurements. RESULTS

Synthesis and Characterization of FITC-Labeled PEG Conjugates. FITC-labeled PEG conjugates were synthesized in order to evaluate, by fluorescence-sensitive techniques, whether the conjugated folate alters the PEGs accumulation in HiFR cells. The general strategy of the synthesis is shown on Scheme 1. The synthesis of monoprotected PEG diamine (2) provided us a universal starting material for all subsequent syntheses. Through stable amide bonds, desirable functions can be easily attached to either end of the polymer. Because of the difficulty of separating modified and unmodified PEGs, we used an excess of the low molecular weight ligands in the conjugation reactions, to increase the yield and to simplify the purifications. The next step was conjugation of FA to the unprotected primary amine. Folic acid has two carboxy groups, and in order to couple only one PEG-NH2 to one folic acid, care must be taken to activate only one ester group per folic acid molecule prior to coupling. Thus, complete solubilization of the folic acid is imperative prior to the addition of 1 equiv of the coupling reagents (NHS and DCC). Folic acid suffers from low solubility in most organic solvents, and the only solvent in which folic acid is completely soluble is DMSO. Unfortunately, when the coupling reaction was carried out in DMSO, it was

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Scheme 1. Synthesis of FITC-Labeled PEG Conjugates

Scheme 2. Preparation of Activated Folate

impossible to isolate the product from the reaction mixture. Thus, we decided to separate the ester activation and the coupling step, and we first prepared and isolated the N-hydroxysuccinimide activated FA derivative (Scheme 2), which precipitates readily from DMSO, when a mixture of acetone/ether mixture is added. The activated ester was immediately reacted with a pyridine solution of the monoprotected PEG diamine 2. The excess of unreacted FA was easily remove by GPC (gel permeation chromatography). Since it was reported that only the folic acid conjugates that were attached through the γ carboxyl group possess biological activity (17), it was important to measure the percentage of the γ-isomer in the FA-PEG conjugates. For this purpose we used the carboxypeptidase G HPLC assay (26), that is based on the ability of CPG to cleave the glutamic residue of FA only if its γ-carboxyl is unconjugated. In view of the fact that 65% of the FA-PEG conjugate was enzymatically cleaved by CPG we assume that γ-isomer fraction is 65%. To prepare a nontargeted control conjugate, we blocked the free amine with a Cbz protective group, instead of folate (4). Cbz was chosen because of its relatively low weight and high stability in physiological conditions. After removal of the FMOC-protecting group, it was easy to conjugate the free amine with FITC (5, 6). Synthesis of Pt-PEG Conjugates. To prepare PEG conjugates that can release the Pt from the carrier once inside the cells (analogues of carboplatin), the Pt moiety

was attached to the PEG through a modified dicarboxylate ligand. Malonic acid derivatives form bidentate chelates with Pt(II), through the binding of the two carboxylic groups. To enable easy conjugation of the dicarboxylate ligand to the PEG-amine carriers, we modified the protected malonate with short side chain, having free carboxylic acid at the end (Scheme 3). Compound 8 is a versatile synthon that allows for easy coupling of the protected malonate to any primary or secondary amine. The subsequent stages of the synthesis, including the platination steps, are depicted in Scheme 4. The free amine of the monoprotected PEG-diamine 2 was coupled to the side-chain carboxylate of 7 using ECI as the coupling agent. Then amine group of 10 was deprotected and conjugated to either folate (11) or Cbz (12), as described in the previous section. According to the CPG assay the fraction of the γ-isomer in FA-PEG-MAL(tBu)2 was ∼65%. After deprotection, the dicarboxylate was platinated using a standard procedure, in which an excess of the reactive diaqua Pt complex {Pt(NH3)2(H2O)2]2+} was reacted with the carrier. Monitoring of the platination reaction was carried out by proton NMR. The chemical shifts of the two methylene groups adjacent to the malonate change significantly upon platination going from 1.73 and 1.44 ppm in the unplatinated carrier to 2.26 and 1.48 ppm, respectively, in the platinated con-

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Scheme 3. Synthesis of Functionalized Malonic Moiety

Scheme 4. Synthesis of PEG-dicarboxylato-Platinum Conjugates

Figure 3. 1H NMR of platinated (A) and unplatinated (B) PEGdicarboxylate ligand.

jugate (see Figure 3). Total disappearance of the peaks related to unplatinated ligand indicated that the reaction

was completed. The excess of the unreacted diaqua Pt complex was removed by gel permeation chromatography. 195Pt NMR spectroscopy was used to characterize the Pt complex. 195Pt NMR chemical shifts are sensitive to the nature of the first coordination sphere of Pt(II) and coupled with the fact that the chemical shift scale of 195Pt NMR is quite large (thousands of ppm), this technique allows us to establish the nature of the complex. The 195Pt chemical shift of the diaqua Pt complex in water is δ ) -1580 ppm and the chemical shift of carboplatin is δ ) -1710 ppm. Thus, the single peak at δ ) -1699 ppm that was obtained for two products (15, 16) indicates that the desired platinum conjugate was obtained. Cell Uptake Studies of Fluorescent Labeled PEGs. All biological experiments were carried out with the M109HiFR cell line, enriched with folate binding receptors. The cell uptake of FITC-PEGs was evaluated by two fluorescent sensitive techniques: FACS and direct fluorescent microscopy. FACS enables us to compare the

570 Bioconjugate Chem., Vol. 14, No. 3, 2003

Figure 4. Flow cytometry profiles of fluorescence from cellassociated fluorescein. M109 HiFR cells were incubated with 50 µM PEG-fluorescein conjugates for 4 h at 37 °C. Upper panel: cells treated with FA-PEG-FITC in folate-free RPMI (A) or 1 mM folate containing RPMI (B). Lower panel: cells treated with PEG-FITC in folate-free RPMI (C) or 1 mM folate containing RPMI (D).

uptake of folate targeted PEG and untargeted PEG in low folate and free folate competition conditions. The results summarized in Figure 4 clearly indicate that the uptake of folate-targeted PEG increases significantly in folate free medium (profile A) when compared with the uptake otf the untargeted PEG (profile C). The competition with free folate reduces the uptake of folatetargeted PEG (profile B), but does not influence the uptake of nontargeted PEG (profile D). The FACS results were confirmed by direct fluorescence microscopy (Figure 5). Both targeted (image B) and nontargeted (image A) PEGs concentrated mainly in cytoplasm after 4 h of incubation, but there is a major increase in fluorescence intensity with the folate-targeted PEG (image B). As in the FACS experiment, free folate competition reduces the cell uptake of folate-PEG (image C). These results indicate that folate targeted PEG accumulates in M109-HiFR cells through the folate receptor endocytosis pathway. Cells Growth Inhibition Study. To estimate the impact of folate targeting on the cytotoxicity of PEG-Pt conjugates, growth inhibition tests of M109 HiFR cell line were carried out. In this test we compared targeted PEG-Pt complex to the untargeted one, and also we used free carboplatin as a general reference, since both complexes are structural analogues of carboplatin. The IC50 values are presented in Table 1. Surprisingly the folate-targeted complex was the least cytotoxic, among the three molecules tested. There are two possible explanations for this phenomenon: (a) for

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some reason the PEG-Pt conjugates are not internalized by the FRME as is the case for the PEG-FITC conjugates; (b) folate-targeted PEG-Pt complexes do enter the cells through folate endocytosis, but this pathway prevents interaction with DNA and thereby reduces the cytotoxic potency of the PEG-Pt conjugates. To clarify which one of two hypotheses is right, Pt uptake and DNA-Pt adducts measurements were performed. Platinum Cell Uptake. We measured Pt content in M109 HiFR cells incubated with PEG-Pt conjugates or with free carboplatin. After a 4 h incubation at three different concentrations of each compound, Pt concentrations was measured by ICPMS technique. The results of the experiment are shown in Figure 6. According to these results, the conjugated folate significantly elevates the uptake of PEG-Pt complex in HiFR cells. This indicates that FRME participates in the tumor cell uptake of folate-targeted PEG-Pt conjugate as in the case of folate PEG-FITC. Another observation from Figure 6 is that, unlike PEG-Pt or carboplatin, the uptake of FA-PEG-Pt does not increase proportionally with the Pt-compound concentration. This is likely to result from saturation of the FR-mediated uptake. In contrast nontargeted PEG-Pt and carboplatin do not enter the cell through FR endocytosis pathway, and therefore their uptake increases proportionally to their concentration in incubation solution. So, folate-containing PEG-Pt conjugates enter cells through the FR endocytosis pathway. Their efficient internalization should result in higher DNA platination levels. Pt-DNA Adducts. Since carboplatin and its analogues exert their cytotoxicity by formation of adducts with DNA, one of the most important measures for the potency of carboplatin analogues is their ability to form Pt-DNA adducts. To allow more efficient platination of the DNA by the Pt complexes, we prolonged the incubation period till 20 h (7). Cells were incubated with the drugs (PEG-Pt, FA-PEG-Pt, and carboplatin) for only 4 h, the medium was changed and the experiment proceeded for an additional 16 h in the drug free medium. When the postincubation ended, 10% of cells were taken for Pt cell uptake studies and the remaining cell fraction was used for DNA extraction (30). The results of DNA platination levels presented in Table 1 indicate that folate targeted FA-PEG-Pt produces less DNA adducts than the nontargeted PEG-Pt complex. This may explain its decreased cytotoxicity. More surprising was the fact that carboplatin yielded a very poor DNA platination level in comparison with PEG-Pt despite the fact that it displayed the highest Pt accumulation levels after 4 h of incubation. The results of the Pt uptake experiments with and without postincubation (Table 1) point to massive efflux of carboplatin from the cells, during the postincubation period in contrast with the stable levels for the PEG conjugates. The elevated efflux of carboplatin during postincubation period provides at least a partial explanation for the phenomenon of reduced DNA-platination potency of carboplatin. In fact, a collateral finding of this study is that nontargeted PEG-Pt causes more effective DNA platination than carboplatin itself. Pt-DNA adducts expressed as a fraction of whole cell drug uptake, were more than twice as high for PEG-Pt treated cells, than for carboplatin (Table 1).

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Figure 5. Cellular accumulation of fluorescein-labeled PEG conjugates in M109 HiFR cells. Cells were incubated with 50 µM substrates for 4 h at 37 °C followed by fixation. Panels A and B show fluorescence images of M109 HiFR cells incubated in folate-free RPMI medium with PEG-FITC (A) or FA-PEG-FITC (B). Panel C shows fluorescence images of M109 HiFR cells incubated with 50 µM FA-PEG-FITC in RPMI medium containing 1 mM of free FA. Panel D shows zoomed image of panel B. Table 1. Biological Monitoring of PEG-Pt Compounds Pt-DNA adducts ng Pt/mg DNAc

cell uptakeb (ng Pt/106 cells) platinum compound

IC50a, µM

4 h incubation

20 h incubation

PEG-Pt FA-PEG-Pt Carboplatin

16.1 ( 2.7 27.7 ( 0.6 18.5 ( 1.2

6.7 ( 0.8 10.3 ( 0.4 25.2( 0.3

4.3 (0.3 6.7 (0.9 4.6 (0.4

9.7 ( 1.3 4.3 ( 0.8 3.7 ( 0.5

calculation of Pt-DNA adducts/whole cell uptake,d % 4 h incubation

20 h incubation

3.6 1.0 0.36

5.6 1.6 2

a The IC 50 values were calculated from the MB assay. The experiment was performed in two or more separate plates with triplicate cultures for each drug concentration. SD values refer to results from the separate plates. b The uptake values were obtained after incubation with 200 µM of the platinum compound solution. SD values represent two independent experiments carried out in triplicate cultures. 4h incubation means 4 h continuous exposure to platinum compound. 20 h incubation means 4 h continuous exposure to platinum compound followed by 16 h of postincubation in free medium. c DNA-associated Pt was measured after 4 h continuous exposure to a 200 µM platinum compound solution followed by 16 h of postincubation in drug-free medium. SD values represent two independent experiments carried out in triplicate cultures. d Pt-DNA adducts (see footnote c) expressed as a fraction of whole cell drug uptake (see footnote b). The calculations based on our data, that 40 × 106 cells give approximately 1 mg of DNA.

DISCUSSION

Many attempts have been made to improve the therapeutic index of platinum anticancer agents by conjugating them to large polymeric carriers. Such modifications usually improve the pharmacokinetics and tissue distribution of the drug, but significantly reduce the cell permeability (due to the high molecular weight of the conjugate) (10). Although some macromolecular prodrugs of low molecular weight drugs such as mitomycin C can exert cytotoxicity by releasing the free drug in the extracellular medium (31), this may prove to be problematic in the case of platinum anticancer agents since they act as nonspecific electrophiles, and should the Pt moiety be released from the carrier prior to cellular internalization, the Pt will be rapidly inactivated by

extracellular nucleophiles. In this work we attempted to overcome this shortcoming by developing folate-targeted PEG carriers, for efficient, rapid, active transport of the platinum-carrier conjugate into cancer cells, utilizing folate receptor-mediated endocytosis, aimed at increasing cellular uptake and improving cytotoxicity. To test our working hypothesis, we prepared two bifunctional FA-PEG conjugates: FA-PEG-Pt and FA-PEG-FITC (see Schemes 1-4). FA-PEG-FITC enabled us to visualize (by confocal microscopy) and to quantitate (by FACS) the effect of conjugating folate to the PEG polymer on the cellular uptake of the PEG. FAPEG-Pt made possible to examine whether increased cellular uptake results in increased DNA platination and

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Figure 6. Pt uptake of Pt-containing drugs by M109 HiFR cells. Cells were incubated with different concentration of carboplatin (2), FA-PEG-Pt (9), or PEG-Pt (b) in folate-free RPMI solution for 4 h at 37 °C. Cell-associated platinum concentrations were measured using ICPMS technique. Each point represents the mean of at least three determinations; bars represent ( SD when larger than the symbol.

improved cytotoxicity. The synthetic strategy that was employed in this study is depicted in Schemes 1-4. Although somewhat tedious, the preparation of the FMOC-PEG-NH2 lends flexibility to the design of the syntheses of the conjugates, and once the di-tert-butyl malonate is coupled to it, essentially any targeting or reporting groups can be attached to its other end. On the basis of this approach, any moiety terminating with a free carboxylate can now be attached to the monoprotected polymer by a simple condensation reaction. We find this method more convenient and more efficient than the previously reported approach of direct alkylation of the di-tert butyl malonate by a functionalized polymer (15). Because of the high efficiency and mild reaction conditions of the amine-carboxyl condensation reaction, our carboxyl-functionalized malonate ligand may be easily attached to any amine-containing molecule. NHSactivated folate was prepared as an efficient synthon for the conjugation of the FA moiety to PEG-NH2. A major concern in the chemistry of PEG conjugates is the purification and characterization of the conjugates. All the Pt-PEG conjugates were analyzed by RP-HPLC, displaying a single peak in the chromatogram that attests to the purity of the conjugates. In addition, MADLI-TOFMS served both to confirm the purity of the conjugates as well as to characterize them. The mode of platination was established by preparing the 15N labeled analogues of the platinated conjugates and measuring the [1H,15N] inverse detection HSQC NMR spectra that showed a single peak corresponding to the diamminedicarboxylate coordination sphere. The identity of the coordination sphere of the platinum was also corroborated by 195Pt analysis and by 1H NMR. Thus, we also provide here, for the first time, a complete chemical characterization of PEG-Pt conjugates including 1H NMR, 195Pt NMR, [1H,15N] inverse detection HSQC and MALDI-TOF-MS. Confocal microscopy and FACS were used to study the cellular uptake of the fluorescent PEG conjugates. In both techniques the intensity of cell-associated fluorescence provided the indication for cell uptake of the fluoresceinlabeled PEG conjugates (FA-PEG-FITC and PEG-FITC). The data obtained by confocal microscopy (see Figure 5) and by FACS (see Figure 4) indicated that the folate targeted PEG (FA-PEG-FITC) is taken up by the folate receptor-enriched M109 cancer cells approximately three times more efficiently than the untargeted PEG (PEGFITC). To prove that the FA-PEG conjugates entered the cell via the FR pathway, the uptake experiments were repeated in the presence of an excess of free folate. The

Aronov et al.

reduction of cellular uptake of the FA-PEG conjugates in the presence of the free folate (see Figures 4, 5) clearly indicates that free folate successfully competes with the conjugates for the folic acid receptor sites thereby reducing the cellular uptake of the conjugates. Additional evidence to support this claim comes from the observation that the cellular accumulation of FA-PEG-Pt does not linearly depend on the platinum concentration in the medium, but at higher platinum concentrations the uptake is less efficient probably due to saturation of the folate receptors (Figure 6). While the FACS provides the quantitative data, confocal microscopy demonstrated that the conjugates actually entered into cytoplasm and did not merely adhere to the exterior of the cell membrane. Cytotoxicity studies were carried out on cancer cells enriched with folate receptors in order to evaluate the potency of the conjugates and to see whether higher cellular uptake indeed results in higher potency. Surprisingly, the cytotoxicity data showed that FA-PEG-Pt is less potent than PEG-Pt (see Table 1). The IC50 value of FA-PEG-Pt is higher by factor 1.7, than that of PEG-Pt (Table 1). The cell-associated Pt levels measured by ICP-MS (see Figure 6) were greater for the folatetargeted conjugate, pointing to a lack of correlation between the cellular Pt levels and cytotoxicity. These contrasting findings prompted us to measure the levels of Pt binding to cellular DNA. Surprisingly, it was found that PEG-Pt forms twice as many adducts with the cellular DNA than does FA-PEG-Pt despite the higher cellular uptake of the latter. One possible explanation for this unexpected phenomenon pertains to the nature of FRME process. In this process, the folatetargeted conjugates are not freely released into the cytosol but are directed to acidic cytoplasmic vesicles, such as endosomes and lysosomes. The release of FAPEG-Pt from the vesicle to the cytoplasm may be slow and inefficient, reducing the cytotoxic activity of the delivered complex. Observations made by Rui et al. (32) and Qualls and Thompson (33) with folate-targeted pHsensitive liposomes point to a major increase in cytosol delivery and biological activity of the encapsulated compounds when compared to stable liposomes. This supports the hypothesis that FRME leads to an acidic vesicular compartment with reduced access to other cell compartments unless the delivery system is unstable at low pH. Although our study deals with a polymer-drug complex, much smaller than a liposome, it is possible that these complexes remain to a large extent trapped in acidic vesicles following FRME, thus preventing their cytotoxic activity During the course of the study we noticed that despite its low cellular uptake, PEG-Pt is at least as cytotoxic as carboplatin. This was surprising since in the initial cell uptake studies (see Figure 6) carboplatin was internalized much more efficiently than PEG-Pt. To further explore this point, the cells were incubated with the Pt compounds for 4 h, the medium was replaced, and the cells were allowed to grow for an additional 16 h prior to measuring the cell-associated Pt levels. As can be seen from Table 1, the carboplatin levels after 4 h of incubation and 16 h of postincubation are dramatically lower than those measured without the postincubation, suggesting that carboplatin is being excreted from the cells while the PEG-Pt conjugate is retained in the cells once it is internalized. Since the IC50 measurements were performed with continuous exposure of the cells to the drugs, and the cell uptake and DNA binding were performed with a 4 h exposure and a 16 h postincubation, we have also looked at cell survival using the latter conditions.

Folate-Targeted PEG

The results indicate that the PEG-Pt is one and a half times more effective in inhibition of cell growth than FAPEG-Pt and four times more effective than carboplatin (data not shown). Both these facts indicate that the untargeted PEG-Pt conjugate is a potentially promising anticancer agent and deserves independent investigation. The findings of this study suggest that folate-targeted conjugates such as FA-PEG-Pt, may be problematic as a prodrug for the carboplatin family, because the conjugates or the active moieties are neutralized or blocked during the FRME process. Thus, the attempt to increase the levels of Pt-DNA adducts by active targeting with FA resulted in a different kind of detoxification mechanism: entrapment of the prodrugs in certain cellular compartments. ACKNOWLEDGMENT

This research was funded in part by the Israel Cancer Association through the Chaya and Naftali Bloch Memorial Fund. ABBREVIATIONS

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