Anticancer Drugs to Isocyanates - American Chemical Society

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Biomacromolecules 2010, 11, 2290–2299

Core-Cross-Linked Micelles Synthesized by Clicking Bifunctional Pt(IV) Anticancer Drugs to Isocyanates Hien T. T. Duong,† Vien T. Huynh,† Paul de Souza,‡ and Martina H. Stenzel*,† Centre of Advanced Macromolecular Design (CAMD), The University of New South Wales, Sydney NSW 2052, Australia, and St. George Hospital Clinical School, The University of New South Wales, St. George Hospital, Sydney NSW 2217, Australia Received April 13, 2010; Revised Manuscript Received June 14, 2010

Most low molecular weight platinum-based anticancer drugs have a short circulation time in the bloodstream. One of the potential strategies to improve the targeted delivery of cisplatin and prolong its circulation is via the use of nanocarriers. An improved drug delivery system was developed via reversible addition-fragmentation chain transfer (RAFT) polymerization. In a one-pot reaction, the incorporation of anticancer drug and core crosslinking was simultaneously carried out by using the highly effective reaction of isocyanate groups in the core of the polymeric micelles poly(oligo(ethylene glycol) methyl ether methacrylate)-block-poly(styrene-co-3-isopropenylR,R-dimethylbenzyl isocyanate) (POEGMA-block-P(STY-co-TMI)) with amine groups in the prepared platinum(IV) drug. The micelles with platinum(IV) incorporated with a size of 36 nm were very stable in water. In a reductive environment, in this study simulated using ascorbate, the drug was released at a slow rate of 82% in 22 days and at the same time the cross-linked micelle broke down into free block copolymers as evidenced using inductively coupled plasma-mass spectrometer (ICP-MS), size exclusion chromatography (SEC), and dynamic light scattering (DLS). The in vitro study also revealed the promising antitumor activity of prepared platinum(IV) drugs encapsulated into the micelle structure.

Introduction Among the methods for cancer treatments, chemotherapy using platinum drugs as antineoplastic agents has been an important technique for controlling many types of cancer including testicular, head, neck, and ovarian cancers over the past few decades.1-3 The activity of cis-diamminedichloroplatinum(II) (cisplatin, cis-PtCl2(NH3)2; Scheme 1), the parent of all the platinum drugs, and its second-generation analogues and derivatives, such as oxaplatin and carboplatin, have been welldocumented in the literature over the years.4-8 The compound cis-PtCl2(NH3)2 (cisplatin) was first discovered by M. Peyrone in 1845 and known for a long time as Peyrone’s salt.9 Its high antitumor activity was first reported by Barnett Rosenberg,4,10 and cisplatin was approved for clinical use by the United States Food and Drug Administration (FDA) in 1978. Cisplatin represents the tetragonal (square) planar platinum(II) (Pt(II)) complex with the general structure cis-[Pt(am)2L2], where “am” is NH3 or primary amine and L is the leaving group. While two amine or amine ligands coordinate tightly to the platinum metal center in the cisoid arrangement, leaving groups typically chloride or carboxylate groups are weakly bound and easily replaced by other nucleophile such as water and alcohol. Cisplatin is administered to cancer patients intravenously and it remains intact due the relatively high concentration of chloride ions (∼100 mM) in the blood plasma. The neutral compound then enters the cell by either passive diffusion or active uptake by the cell. Inside the cell (cytoplasm), due to a much lower concentration of chloride ion (∼3-20 mM), the neutral cisplatin molecule undergoes hydrolysis in which a chlorine ligand is replaced by a molecule of water, generating a positively charged

species.11,12 The resulting reactive diaquated complexes then bind covalently with nucleophilic sites on guanine present in DNA forming the intrastrand and interstrand cross-linked adducts.13 This coordination complex not only inhibits replication and transcription of DNA, but also leads to programmed cell death (apoptosis).14 Although cisplatin and its analogues and derivatives are very potent and successful antineoplastics, they can cause a number of side effects that can limit their use in cancer treatment, including nephrotoxicity, cumulative neurotoxicity, ototoxicity (hearing loss), and severe emetogenesis (vomiting).15 In addition, frequently observed drug resistance16 led to the quest to develop improved Pt anticancer agents.17-19 Among the number of the prepared and biomedically screened platinum complexes, octahedral complexes with platinum(IV) (Pt(IV)) center, including satraplatin, tetraplatin, iproplatin, and ormaplatin, have been tested as potential anticancer agents particularly against cisplatin resistant tumor cell lines.20-23 Pt(IV) complexes with octahedral structure have advantages over the Pt(II) complexes because of their kinetic inertness and thus toxicity is less compared to Pt(II) counterparts.24,25 Pt(IV) drugs are considered prodrugs because they need to be reduced intra- or extra-cellularly by biological reductants such as glutathione, ascorbic acid, and cystein to Pt(II) complexes in order to become reactive.26,27 The reduction rate, however, was found dependent on the nature of axial and carrier ligands.28 A more detailed discussion of the reduction rate and mechanism of Pt(IV) complexes has been reviewed elsewhere.27 Scheme 1. Cisplatin

* To whom correspondence should be addressed. E-mail: m.stenzel@ unsw.edu.au. † CAMD (http:// www.camd.unsw.edu.au). ‡ St. George Hospital Clinical School. 10.1021/bm100396s  2010 American Chemical Society Published on Web 07/27/2010

Core-Cross-Linked Micelles

Even though octahedral Pt(IV) complexes are considered advanced platinum anticancer drugs, the targeted drug delivery to tumor sites is still challenging as the drugs can affect both healthy cells and cancer cells. One of the most promising strategies to improve targeting is by using polymeric nanocarriers. Polymeric micelles, self-assembled block copolymers with a core-shell structure and diameters of 10-100 nm, are wellknown as potential vehicles for the targeted delivery of drugs, proteins, genes, and imaging agents. Because the molecular weight of polymeric micelles may reach more than 106 g/mol, they surpass the renal threshold (ca. 40 kDa).29 In addition, their nanoscopic size is typically less than 200 nm and they are therefore less recognizable by the phagocytic cell of the reticuloendothelial system (RES). Thus, polymeric micelles usually lead to a prolonged blood circulation.30,31 The “stealth” property of micelles, which leads to a delayed detection by the reticuloendothelial system (RES), in combination with the passive accumulation in the solid tumor through the enhanced permeability and retention (EPR) mechanism32 make these core-shell nanoparticles potentially the preferred choice for drug delivery purposes. While polymeric micelles have a superior thermodynamic and kinetic stability compared to low molecular weight surfactants, the benefit of cross-linking of micelle has however repeatedly been highlighted. Shell- or corecross-linked micelles are found to be stable against disassociation at very low concentration and their aggregation is independent from external influences such as changes in ionic strength and pH values.33-36 It has recently been shown that cross-linking of micelles can potentially enhance cellular uptake.37 In this study, we explore a route to Pt(IV) containing crosslinked micelles. While gold nanoparticles and carbon nanotubes have been surface modified with Pt(IV) drugs,38,39 the encapsulation of Pt(IV) drugs into cross-linked micelles has yet to be explored. The advantage of encapsulation of platinum drugs into the center of a core-shell nanoparticle is their protection against deactivation by proteins and peptides. Therefore, a Pt(IV) containing difunctional amine anticancer drug was prepared from the parent compound cisplatin. The prepared Pt(IV) drug was conjugated to the polymer backbone while simultaneously crosslinking the core of the micellar structure of poly(oligo(ethylene glycol) methyl ether methacrylate)-block-poly(styrene-co-3isopropenyl-R,R-dimethylbenzyl isocyanate) (POEGMA-blockP(STY-co-TMI)) micelles37 by utilizing the versatility of reactive isocyanate functional group (Scheme 2). The reduction of the Pt(IV) complex in the micelle core in the cytoplasm by biological reductants will not only release cytotoxic cisplatin, but will also cleave the cross-linker of the micelle (Scheme 3).

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vacuum filtration and washed with ice cold water, ethanol, and diethyl ether. After filtration, the solvent was removed under reduced pressure to give the expected product as bright yellow powder (0.8 g, 78.5%). Synthesis of cis,cis,trans-Diaminedichlorodisuccinatoplatinum(IV) [2; Pt(NH3)2Cl2(OOCCH2CH2COOH]. The platinum complex (2) was prepared according to the literature procedures.42,43 Succinic anhydride (228 mg, 2.278 mmol) was added to a suspension of (1; 200 mg, 0.600 mmol) in DMF (4 mL), and the reaction mixture was stirred at 70 °C for 24 h. During this reaction, the solid material was dissolved to form a yellow-brown solution. DMF was then removed under reduced pressure. The residue was dissolved in acetone and filtered to give a clear, yellow solution. This solution was concentrated under reduced pressure, and subsequent purification by addition of diethyl ether led to precipitation of a pale-yellow solid. The product was dried in a vacuum oven for 2 days. Yield: 131.5 mg (85%). Synthesis of Activated Pentafluorophenol Ester (3). A mixture of platinum(IV) complex (2; 100 mg, 0.19 mmol), dicyclohexyl carbodiimide (77 mg, 0.38 mmol), and pentafluorophenol (69 mg, 0.38 mmol) in DMF was stirred at 30 °C overnight. After that, the mixture was cooled to 0 °C, and dicyclohexyl urea was separated and removed by filtration. DMF was then removed under reduced pressure to form yellow-brown oil. The crude product was purified by column chromatography (cyclohexane/ethyl acetate 3:1) to yield a brown solid. Yield: 82 mg (49.8%). 1H NMR (300 MHz, DMF-d7, 298 K) δ/ppm ) 2.97 (t, J ) 6.8 Hz, 2H, -CH2), 3.18 (t, J ) 6.8 Hz, 2H, -CH2), 7.14-7.04 (m, 6H, -NH3). 19F NMR (300 MHz, DMF-d7, 298 K) δ/ppm ) -165.0-164.7 (m, 2F), -160.3 (t, J ) 21 Hz, 1F), -153.5 (d, J ) 21 Hz, 2F). 13C NMR (300 MHz, DMF-d7, 298 K) δ/ppm ) 30.3 (C-2), 31.1 (C-3), 125.7 (C-7), 130.9 (C-8), 140.2 (C-5), 143.5 (C-6), 169.8 (C-4), 179.9 (C-1).

Synthesis of Platinum(IV) Containing Difunctional Amine (4). To a solution of Pt(IV) compound (3; 50 mg, 0.08 mmol) in DMF, ethylendiamine (48 mg, 0,8 mmol) was added. The mixture was stirred at room temperature overnight and then cooled. Ammonium salt formed was removed by filtration. The unreacted ethylene diamine was removed by azeotropic distillation using toluene/methanol mixture. The compound (4) was precipitated in diethyl ether, yielding a brown solid that is soluble in water. Yield: 37 mg (75%). 1H NMR (300 MHz, D2O, 298 K) δ/ppm ) 2.20-2.29 (m, 4H, -CH2CH2-), 2.89 (t, J ) 5.9 Hz, 2H, -CH2), 3.26 (t, J ) 5.9 Hz, 2H, -CH2). 13C NMR (300 MHz, D2O, 298 K) δ/ppm ) 31.9 (C-3), 32.6 (C-2), 36.8 (C-6), 39.2(C-5), 176.9 (C-4), 181.1 (C-1).

Materials and Methods Materials. All reagents were purchased from Sigma-Aldrich with the highest purity and were used as supplied, unless otherwise noted. Oligo(ethylene glycol) methyl ether methacrylate (OEGMA; Mn ) 300 g mol-1) and styrene were destabilized by passing them through a column of basic alumina. 2,2-Azobisisobutyronitrile (AIBN; Fluka, 98%) was purified by recrystallization from methanol. RAFT agent CDB was synthesized according to the previously reported procedure.40 Synthesis of cis,cis,trans-[Pt(NH3)2Cl2(OH)2] (Oxoplatin; 1). cis,cis,trans-[Pt(NH3)2Cl2(OH)2] was prepared according to a procedure described in the literature.41 A mixture of cisplatin (1.0 g, 3.05 mmol) and H2O2 30 w/v (3.5 mL, 30.5 mmol) in the aluminum covered roundbottom flask was heated at 70 °C for 5 h. The heat was then removed and the reaction mixture was stirred overnight. The product was recrystallized in situ at 4 °C overnight. The product was obtained by

Synthesis of POEGMA Macro-RAFT Agent. OEGMA (2.58 g, 8.60 × 10-3 mol), CDB RAFT agent (2.34 × 10-2 g, 8.60 × 10-5 mol), and AIBN (2.83 × 10-3 g, 1.72 × 10-5 mol) were dissolved in 20 mL of toluene in a round-bottom flask with a magnetic stirrer bar. The flask was then sealed with a rubber septum and purged with nitrogen for 30 min. The reaction mixtures were then immersed in a preheated oil bath at 70 °C. After 8 h, the polymerization was terminated by placing the samples in an ice bath for 5 min. The polymer was

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Scheme 2. Preparation of Platinum(IV) Incorporated Cross-Linked POEGMA-block-P(STY-co-TMI) Micelles

purified three times by precipitation in petroleum spirits (boiling range of 40-60 °C). After centrifugation (7000 rpm for 15 min), the polymer was dried under reduced pressure at room temperature for 24 h. The samples were stored in a freezer prior to modification. By comparing the intensity of vinyl proton peaks (6.1 and 5.6 ppm) to that of aliphatic proton peaks (1.1-1.3 ppm), the conversion of the monomer during the course of polymerization was determined. After 8 h, a conversion of 58% was obtained. The molecular weight of the POEGMA macro RAFT agent was measured to be 17900 g mol-1 (PDI ) 1.18) by DMAc SEC, Mn(theo) ) 17600 g mol-1 by 1H NMR. Synthesis of POEGMA-b-P(STY-co-TMI). POEGMA with 59 repeating units (Mn(theo) ) 17600 g mol-1, Mn(SEC) ) 17900 g mol-1) was used as a so-called macroRAFT agent for chain extension with styrene and TMI. The number of repeating units of POEGMA was

calculated from the monomer conversion obtained from 1H NMR. The POEGMA macroRAFT agent (4.35 × 10-1 g, 2.47 × 10-5 mol) was dissolved in 6 mL (5.45 g, 5.2 × 10-2 mol) of STY and 2 mL (2.04 g, 1.01 × 10-2 mol) of TMI in a round-bottom flask. The reaction mixture was purged with nitrogen for 1 h in an ice bath to avoid the evaporation of styrene. The polymerization was carried out in an oil bath at 100 °C for 2.75 h. The polymerizations were terminated by placing the samples in an ice bath for 5 min. The copolymer was purified using membrane dialysis for 2 days against acetone, to remove unreacted styrene and TMI. The prepared polymer solutions (in acetone) were kept at 2 °C prior to further experiments. After 2.75 h, a conversion of 4.5% for STY corresponds to 95 STY repeating units and a conversion of 5% for TMI corresponds to 20 TMI repeating units. The molecular weight

Core-Cross-Linked Micelles Scheme 3. Reduction of Platinum(IV) Incorporated in the Micelles to Cisplatin


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rinsed five times with 1% acetic acid to remove unbound dye. Then the cultures were air-dried until no conspicuous moisture was visible. Bound dye was shaken for 10 min. The absorbance at 515 nm of each well was measured using a microtiter plate reader scanning spectrophotometer. Inhibition of proliferation was expressed as a percentage of control cells (medium only). All experiments were repeated three times.

Analysis

of the POEGMA59-block-P(STY95-co-TMI20) was measured to be 34800 g mol-1 (PDI ) 1.19) by DMAc SEC, Mn(theo) ) 31500 g mol-1 by 1H NMR. Self-Assembly of POEGMA59-block-P(STY95-co-TMI20). Distilled water was added dropwise to POEGMA59-block-P(STY95-co-TMI20) solution in acetone (20 mg mL-1) under moderate stirring at room temperature, followed by the evaporation of acetone under vacuum. The water was added at a very slow rate with the help of a pump at a set speed to obtain reproducible results. The targeted final micelle concentration was 4 mg mL-1. Synthesis of Core-Cross-Linked POEGMA59-block-P(STY95-coTMI20) Micelles. A POEGMA59-block-P(STY95-co-TMI20) micellar solution (4 mg mL-1; 10 mL) in distilled water was used for the corecross-linking reaction. The Pt(IV) complex (4; 0.015 g, 2.43 × 10-5 mol) was added and the mixture was stirred overnight. The core-crosslinked polymer was purified using membrane dialysis (molecular weight cut off of 3500 Da) for 2 days against distilled water to remove unreacted Pt(IV) drug (4). Reduction of Platinum(IV) Drug Incorporated POEGMA59-blockP(STY95-co-TMI20) Cross-Linked Micelles in the Presence of Sodium Ascorbate. The reduction of Pt(IV) complex incorporated polymeric micelles was carried out in distilled water but not buffer to avoid buffer coordination to platinum.44 The concentration of the stock sodium ascorbate solution is 5 mM. The concentration of micelles for this experiment was 1 mg mL-1, which corresponds to the concentration of platinum in the complex is 0.34 mM. The reduction of Pt(IV) complex incorporated micelles by sodium ascorbate was monitored over 22 days at 37 °C using an incubator. After each time intervals, inductively coupled plasma- mass spectrometer (ICP-MS) was used to determine the platinum concentration in the solution. Cytotoxicity Test by Using Sulforhodamine B Colorimetric Assay (SRB Assay). The cytotoxicity of block copolymer POEGMA59block-P(STY95-co-TMI20), (4) and cross-linked micelles with (4) incorporated was tested by a standard sulforhodamine B colorimetric assay (SRB assay).45 The SRB assay was established by the U.S. National Cancer Institute for rapid, sensitive, and inexpensive screening of antitumor drugs in microtiter plates. The lung cancer cell line A549 was kindly provided by Dr. Paul de Souza from St. George Hospital, Sydney, Australia. Briefly, A549 cancer cells were grown in RPMI1640 media containing 10% FBS and antibiotics. All cells were incubated at 37 °C under a 5% CO2 atmosphere. For the cytotoxicity assay, A549 cells were seeded onto 96-well microtiter plates containing 0.2 mL of growth medium per well at densities of 2000 cells per well. The microtiter plates were left for 24 h at 37 °C and then exposed to various doses of 4, block copymer POEGMA59-block-P(STY95-coTMI20) and Pt(IV) loaded POEGMA59-block-P(STY95-co-TMI20) for 72 h (0-30 µg mL-1 on platinum basis). Cells were then fixed with trichloroacetic acid 10% w/v (TCA) before washing, incubated at 4 °C for 1 h, and then washed five times with tap water to remove TCA, growth medium, and low molecular weight metabolites. Plates were air-dried and then stored until use. TCA-fixed cells were stained for 30 min with 0.4% (w/vol) SRB dissolved in 1% acetic acid. At the end of the staining period, SRB was removed and cultures were quickly

Nuclear Magnetic Resonance (NMR) Spectroscopy. All NMR spectra were recorded using a Bruker 300 MHz spectrometer. All chemical shifts are reported in ppm (δ) relative to tetramethylsilane, referenced to the chemical shifts of residual solvent resonances. Size Exclusion Chromatography (SEC). The molecular weight and polydispersity of prepared polymers were obtained via size exclusion chromatography (SEC). The eluent was N,Ndimethylacetamide [DMAc; 0.03% w/v LiBr, 0.05% w/v 2, 6-dibutyl-4-methylphenol (BHT)] at 50 °C (flow rate of 1 mL min-1) with a Shimadzu modular system comprising an SIL-10AD autoinjector, a Polymer Laboratories 5.0 µL bead-size guard column (50 × 7.8 mm) followed by four linear PL (Styragel) columns (105, 104, 103, and 500 Å) and an RID-10A differential refractive-index detector. The SEC calibration was performed with narrow-polydispersity polystyrene standards ranging from 168 to 106 g/mol. A total of 50 µL of polymer solution (2 mg mL-1 in DMAc) was injected for every analysis. Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR). ATR-FTIR measurements of the NCO band at 2250 cm-1 were performed using a Bruker IFS66\S Fourier transform spectrometer by averaging 64 scans with a resolution of 4 cm-1. Surface Tensiometry. Surface tensiometry was used for the determination of the critical micelle concentration (CMC) of block copolymer and its cross-linked micelles by measuring the surface tension (γ) as a function of concentration. The instrument used was a NIMA DST 9005 Dynamic Surface Tensionmeter with a platinum-iridium (Pt/Ir) alloy Du Nou¨y ring at 25 °C. Dynamic Light Scattering (DLS). The average diameters and size distributions of the prepared micelles were measured by using a Brookhaven Zetaplus particle size analyzer (laser, 35 mW, λ ) 632 nm; angle 90°) at a polymer concentration of 2 mg mL-1. Samples were filtered to remove dust using a microfilter 0.45 µm prior to the measurements. Transmission Electron Microscopy (TEM). TEM micrographs were obtained using a JEOL 1400 transmission electron microscope. It was operated at an acceleration voltage of 80 kV. The samples were prepared by casting the micellar solution (1 mg mL-1) onto a Formvar-coated copper grid. No staining was applied. Thermogravimetric Analysis (TGA). TGA studies were carried out in an air atmosphere and the heating rate was fixed at 20 °C min-1 on a thermal analyzer TGA 2950HR V5.4A. The temperature range employed was 50-1000 °C. The mass of the samples used in this study was 10 mg. Inductively Coupled Plasma-Mass Spectrometer (ICPMS). The Perkin-Elmer ELAN 6000 inductively coupled plasma-mass spectrometer (Perkin-Elmer, Norwalk, CT, U.S.A.) was used for quantitative determinations of platinum. All experiments were carried out at an incident radio frequency power of 1200 W. The plasma argon gas flow of 12 L min-1 with an auxiliary argon flow of 0.8 L min-1 was used in all cases. The nebulizer gas flow was adjusted to maximize ion

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intensity at 0.93 L min-1, as indicated by the mass flow controller. The element/mass detected was 195Pt and the internal standard used was 193Ir. Replicate time was set to 900 ms, and the dwell time was set to 300 ms. Peak hopping was the scanning mode employed and the number of sweeps/readings was set to 3. A total of 10 replicates were measured at a normal resolution.

Results and Discussion Synthesis of Pt(IV) Prodrug Containing Difunctional Amine. Several Pt(IV) complexes have been prepared and entered clinical trial due to their advantages over their platinum(II) counterparts.27,46,47 Pt(IV) complexes are relatively kinetically inert to the ligand substitution due to their octahedral structure, which lowers their reactivity and the prospect of side reaction.27 We therefore aimed at synthesizing a difunctional Pt(IV) containing cross-linking agent, as depicted in Schemes 2 and 4, with an axial ligand flexible and reactive enough to act as a linker between two polymer chains. The amine end groups chosen are highly reactive in the presence of isocyanates, which will facilitate the covalent conjugation and simultaneous cross-linking of the core. The chosen approach will therefore cross-link the micelle permanently encapsulating the Pt(IV) complex until the drug is released as Pt(II) in a reductive environment. The reduction of Pt(IV) leads to the breakage of the core-cross-linked micelles into block copolymer and therefore facilitates excretion from the body. The cross-linker is based on the commercially available drug cisplatin. Cisplatin is readily oxidized by hydrogen peroxide to produce cis,cis,trans-diaminedichlorodihydroxyplatinum(IV) (1).42 Pt(IV) complex can be easily modified in the subsequent step to trans-dicarboxylatoplatinum(IV) (2) complex by the reaction with succinic anhydride. The preparation of transdicarboxylatoplatinum(IV) has been previously reported in DMSO and DMF solvents.42,43 In this study, the carboxylation reaction was carried out in DMF at 70 °C to avoid the formation of an undesired adducted compound, which can occur in DMSO.48 Two different coupling methodologies were attempted to synthesis the ester linked complexes with cis,cis,transdiaminedichloro disuccinatoplatinum(IV). The initial approach employed methyl ester as an intermediate compound. The esterlinked Pt(IV) complexes was obtained by the reaction of cis,cis,trans-diaminedichloro disuccinatoplatinum(IV) with methanol catalyzed by Amberlite IR-120 resin. While this product was obtained in high conversion, the subsequent reaction with an excess of ethylenediamine was observed to be unsuccessful. The alternative pathway, the preparation of the Pt(IV) crosslinker was achieved via amidation reaction of activated esters as depicted in Scheme 4.49 Recently, pentafluorophenyl esters has been extensively reported as an active intermediate for further functionalization.50-56 The ester can be synthesized in high yields from the reaction of carboxylic acid with pentafluorophenol in DMF at 30 °C using dicyclohexylcarbodiimide (DCC) as coupling reagent.56-58 Ultimately, the designed Pt(IV) cross-linker was obtained through the amidation of the activated ester with excess of ethylenediamine in DMF at room temperature to yield the desired product (4; Scheme 4). Synthesis of Block Copolymer POEGMA-block-P(STY-coTMI). Block copolymer POEGMA-block-P(STY-co-TMI) was synthesized using previously established conditions.37 POEGMA macroRAFT agent was prepared in toluene at 70 °C in the presence of cumyl dithiobenzoate (CDB) as a RAFT agent. The conversion of the monomer was determined using 1H NMR

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spectroscopy by comparing the intensity of vinyl proton peaks (6.1 and 5.6 ppm) to the aliphatic proton peaks (1.1-1.3 ppm; see Supporting Information). The POEGMA macroRAFT agent used for the block copolymerization with TMI and STY monomers was obtained after a monomer conversion of 58% (Mn(theo) ) 17600 g mol-1, Mn(SEC) ) 17900 g mol-1, PDI ) 1.18). The low-range conversion was chosen to avoid the formation of significant amount of dead polymer, which is frequently observed at very high monomer conversion of more than 90%.59 The chain extension of POEGMA was carried out with a mixture of TMI and styrene (Scheme 2). By adjusting the ratio of the two monomers, the number of isocyanate functional groups and thus the cross-linking density in the core can be carefully tailored. The cross-linking density will furthermore influence on the loading capacity of the micelle with higher TMI content resulting in more anchor points for the Pt(IV) cross-linker. This is contrary to the physical entrapment of the drug in the hydrophobic core, high cross-linking densities usually limit the amount of drug taken up by a micelle. POEGMA macroRAFT was used to control the polymerization of TMI and STY monomer by using the ratio between STY monomer and thiocarbonylthio concentration was therefore set to 2000 for STY and 400 for TMI. 1H NMR spectroscopy was employed to monitor the consumption of TMI and styrene individually through their nonoverlapping vinylic proton signals (see Supporting Information).60 The analysis of the 1H NMR spectra using the vinylic proton signals of TMI (5.7 ppm) and styrene (6 ppm) results in the individual monomer conversion of both monomers as a function of the reaction time. The block copolymer with Mn(theo) ) 31500 g mol-1, Mn(SEC) ) 34800 g mol-11, and PDI ) 1.19 (POEGMA59-block-P(STY95-co-TMI20), which was obtained after 2.75 h of copolymerization, was used for further experiments. Self-Assembly of POEGMA-block-P(STY-co-TMI) in Aqueous Solution. Eisenberg and co-workers have done extensive research on factors that may control the morphology of self-assembled aggregates such as absolute and relative block length, the water content of the solvent mixture, the nature and the presence of additives (ions, homopolymers, and surfactants), and the polydispersity (PDI) of the block copolymer.61 It was also found that micellization is, to a significant extent, controlled by the length of the hydrophobic block.37,62 The formation of aggregates was achieved by dissolving the block copolymer in acetone followed by the slow addition of water. Acetone was efficiently removed under vacuum, but also dialysis against water would be possible. The self-assembly into micelles of block copolymers POEGMA59-block-P(STY95-co-TMI20) in water was confirmed via DLS and TEM (Figure 1). The isocyanate group was found to be stable under these conditions for an extended period of time and premature cross-linking with water was negligible. Core-Cross-Linked POEGMA-block-P(STY-co-TMI) with Platinum(IV) Complex (4). Self-assembled structures in Figure 1 are dynamic structures with a tendency to dissociate at very low concentrations. The trend to dissociate is usually in correlation with the length of the hydrophobic block, with a short core-forming block resulting in less stable aggregates, while long block length can result in thermodynamically and kinetically very stable structures. The pendant isocyanate group represents a reactive anchor for functional groups, especially amino or hydroxyl functionalities, as well as water, leading to urea or urethane formation, respectively. The reaction is usually fast and efficient in the presence of amine, while the reaction with hydroxyl groups usually requires catalysts. However,


Figure 1. TEM analysis of the micelles (concentration of micelles 1 mg mL-1 in water) of POEGMA59-block-P(STY95-co-TMI20); scale bar ) 200 nm.

Figure 2. FT-IR spectra of POEGMA59-block-P(STY95-co-TMI20) before (top) and after (bottom) cross-linking reaction.

because the cross-linking process is carried out in an aqueous environment, a preliminary experiment was necessary to determine the amount of cross-linking occurring in a competing process with water. FTIR analysis was employed to evaluate the amount of isocyanate reacting with water. Within the time frame investigated, the polymers did not show any significant signs of cross-linking in the aqueous environment. The hydrophobic core, in which the isocyanate is embedded, is therefore efficiently repelling any water penetration. It also needs to be considered that the reaction between isocyanates and water is usually slow in the absence of catalysts, especially at room temperature.63,64 The cross-linking reaction was carried out using Pt(IV) complex with difuntional amine groups, which is watersoluble but also hydrophobic enough to be able to penetrate into the core of the micelle. The cross-linker (4) was added to the micellar solution of POEGMA59-block-P(STY95-co-TMI20) in water. The reaction between isocyanate groups and the crosslinker was monitored by ATR-FTIR spectroscopy and SEC. The declining intensity of the isocyanate group at 2250 cm-1 using FTIR can directly be related to the proceeding reaction. Figure 2 displays the FTIR spectra of purified block copolymer POEGMA59-block-P(STY95-co-TMI20) before cross-linking and after 12 h cross-linking time. Theoretically, with an excess of


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Figure 3. Comparison of SEC chromatograms of POGEMA homopolymer, un-cross-linked block copolymer and cross-linked micelles of POEGMA59-block-P(STY95-co-TMI20).

4, the signal corresponding to the isocyanate group should completely disappear. However, about 30% of isocyanate groups are remaining according to signal integration. Extended reaction time does not lead to any further changes. This can be assigned to bulkiness of the Pt(IV) cross-linker, which has limited accessibility to the core of the micelles in the aqueous media. The reaction with the Pt(IV) cross-linker (4) is evident by the urea formation and the presence of ester and amide groups between 1400 and 1700 cm-1, which correspond to 4 and the urea group (Figure 2). If there are concerns regarding excess isocyanates in the polymer, they can be destroyed using low molecular weight amines such as hexylamines, which have recently been shown to be able to penetrate into the core of such micelles yielding in the full conversion of all isocyanate groups.37 The cross-linked polymer was moreover characterized by SEC by dissolving the copolymer after the cross-linking reaction in DMAc, which is a good solvent for both the hydrophobic and hydrophilic sections of the copolymer. The SEC chromatograms of the prepared cross-linked polymers and the underlying block copolymers are displayed in Figure 3. Substantial shift in molecular weight between the block copolymer and the crosslinked micelle, 34800 g mol-1 compared to 680000 g mol-1, provides further evidence for the successful cross-linking of the micelles. The cross-linked sample (PDI ) 1.21) shows only one peak with a narrow molecular weight distribution indicating not only the complete cross-linking with the absence of free block copolymer, but also the formation of stable core-shell particles with a narrow size distribution. It should be noted that the SEC system was calibrated with linear polystyrene standards and the number average molecular weight obtained underestimates the actual molecular weight of the micelles. The typical surface active behavior of an amphiphilic block copolymer was analyzed using surface tensiometry. Determination of the critical micelle concentration (CMC) is a means by which the stability of a micelle can be evaluated. POEGMA59block-P(STY95-co-TMI20) was thoroughly purified by dialysis against acetone for 2 days and subsequently transferred into an aqueous environment. The CMC of un-cross-linked micelle solution was determined to be at 1.4 × 10-3 g L-1 (1.4 mg L-1). The typical behavior observed using amphiphilic molecules disappears when using the cross-linked micelle. The surface tension gradually decreases with increasing concentration indicative of a light enrichment of the cross-linked micelle at

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Figure 4. Surface tension vs concentration of un-cross-linked and cross-linked copolymer POEGMA59-block-P(STY95-co-TMI20).

Figure 5. Hydrodynamic diameter analysis via DLS (concentration of micelles solution of 1 mg mL-1) of copolymer POEGMA59-blockP(STY95-co-TMI20).

the water/air interface. The distinctive drop of the surface tension with the onset of the micelle formation is, however, absent (Figure 4). The formation of cross-linked micelles was evident from the DLS analysis. The particle sizes of un-cross-linked micelles in water is slightly smaller than that of cross-linked micelles (ca. 26.9 nm for the un-cross-linked micelle compared to ca. 36.2 nm for the cross-linked micelle; Figure 5A,C). DMAc, which is a good solvent for both blocks of the polymer, led to the disassociation of the micelle into unimers (free block copolymers), indicating the structural instability (Figure 5B). In contrast, unimers are fully absent when cross-linked copolymer was dissolved in DMAc revealing the stabilization of micelles by core cross-linking (Figure 5D). While DLS, SEC, FT-IR, and surface tensiometry evidently confirms the successful cross-linking via isocyanate groups and the absence of any free block copolymers, the amount of loaded drug Pt(IV) was quantified using TGA. Prior to analysis, the cross-linked micelle was thoroughly purified via dialysis to remove physically entrapped Pt(IV) drugs.

Duong et al.

Figure 6. TGA to determine the amount of platinum drug incorporated into the cross-linked micelle based on block copolymer POEGMA59block-P(STY95-co-TMI20) (black line) and TGA of cisplatin (gray line).

Thermogravimetric analysis (TGA) permits the quantification of thermally stable compounds, here Pt, while the surrounding polymer typically decomposes at temperature below 400 °C.66 In this study, the heating rate was fixed at 20 °C min-1, and the thermal decomposition of Pt(IV) incorporated block copolymer was monitored in the range 50-1000 °C. A control experiment using cisplatin results in a total weight loss of 35% at 372 °C (Figure 6), validating elemental platinum as the remainder in the thermally investigated range of 50-1000 °C. The results of thermal analysis of the cross-linked revealed total mass loss of conjugated polymer occurred at around 500 °C, and the amount of elemental platinum present in the coordinate complex is about 6.6% by mass, which corresponds to about 20.8% (w/ w) of Pt(IV) drug content in the micelles (Figure 6). Combining the results from 1H NMR, which quantifies the exact number of TMI repeating units per macromolecule (NTMI ≈ 20) and FT-IR, which quantifies the amount of reacted isocyanate units (NTMI, reacted≈ 14) and TGA results, the amount of 4 connected to only one TMI (Pt(IV) pendant group) or two TMI (Pt(IV) cross-linker) repeating units can be calculated. As depicted in Scheme 4, around two TMI repeating units act as a cross-linking point, while 12 TMI units operate as an anchor point for the drug without functioning as a cross-linker. The rather small percentage of cross-linker is, however, sufficient to form a fully cross-linked network in the core. A commonly accepted rule of thumb is that the presence of two cross-linking units per chain is required to obtain a network polymer. Reduction of Platinum(IV) to Platinum(II) by Sodium Ascorbate. The Pt(IV) drug cross-linked micelles show high stability in distilled water with no dissociation and precipitation for a prolonged period of time. In the reductive intracellular environment though, the Pt(IV) complexes is converted to the equivalent Pt(II) complexes, leading to the formation of the active drug, which will bind to DNA in a destructive manner. Concurrently with the release of the drug as cisplatin, the network in the core is cleaved, leading to the formation of free block copolymer (Scheme 5). To mimic the reductive intracellular environment, sodium ascobate (5 mM) was used as a reductant at 37 °C in distilled water,27 though it does not represent the main reductant in the cell. ICP-MS was used to monitor the amount of cisplatin released. ICP-MS is among the most common instrumental methods used for determination of trace levels of elements including platinum with high throughput. Around 80% of cisplatin was released from the cross-linked



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Scheme 4. Synthetic Pathway for the Preparation of Difunctional Pt(IV) Prodrug as Cross-Linker

Scheme 5. Schematic Drawing of the Structure in the Core before and after Drug Release

micelles in a sustained manner within 3 weeks. In addition, no initial burst of drug was observed (Figure 7). With the release, the cross-linker was cleaved at an equivalent rate, resulting in the gradual decline of the hydrodynamic diameter over 2 weeks (Figure 7). While the micelle structure was maintained in water due to the amphiphilic nature of the resulting block copolymer, the formation of free block copolymers, unimers, were clearly visible when dissolving a sample into DMAc, a good solvent for the block. The breakdown of the cross-linked micelle and the formation of free block copolymer was moreover observed using SEC. Samples of cross-linked micelles taken after 7, 10, and 14 days in a reductive environment demonstrate the destruction of the cross-linked micelle. A low molecular weight product is slowly emerging, associated with the free block copolymer, which coincides with the decline in intensity of the block copolymer (Figure 8).

Initial In Vitro Cytotoxicity Tests. To examine the cytotoxicity of the un-cross-linked block copolymer, Pt(IV) crosslinker (4), and Pt(IV) cross-linked micelles, A549 lung cancer cells were exposed to five various doses (0-40 µg/mL Pt) for 72 h. The cytotoxicity was evaluated using the sulforhodamine B (SRB) assay and the results are presented in Figure 9. It is noted that the concentration of block copolymer was equivalent to the amount of polymer present in the Pt(IV) loaded micelles as shown in Table 1. No toxicity was observed for the block copolymer itself. Coincidentally, the toxicity of Pt(IV) incorporated POEGMA59block-P(STY95-co-TMI20) micelles is comparable with the Pt(IV) cross-linker itself. However, the toxicity of the prodrug cannot be compared to the toxicity of the Pt(IV)-loaded micelle because a different mechanism determines their activity. Next to the

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Figure 7. Release of platinum(II) as evaluated by ICP-MS from the Pt(IV)-POEGMA59-block-P(STY95-co-TMI20) cross-linked micelles using ascorbate as reductant (open square) and the time-dependent change in the hydrodynamic diameter of the micelles in (square) water and (circle) DMAc (micelles concentration 1 mg mL-1).

Duong et al.

Figure 9. Cytotoxicity profile of un-cross-linked POEGMA59-blockP(STY95-co-TMI20) block copolymer, Pt(IV) crosslinker (4), and Pt(IV) cross-linked micelles on lung cancer cell A549 after 72 h. Table 1. Concentration of Pt and the Equivalent Concentration of Polymer in the Pt(IV) Micelle System Used for Cytotoxicity Tests µg Pt mL-1

µg polymer mL-1

5 10 20 41

20 39 79 157

studies need to be employed to quantify cellular uptake and the escape of these micelles from endosomes.

Conclusion

Figure 8. Time-dependent change in molecular weight of the POEGMA59-block-P(STY95-co-TMI20) cross-linked micelles after 0 (black straight line), 7 (dotted line), 10 (dashed line), and 14 (gray straight line) days.

redox potential, the cellular uptake of the drug is one of the major factors that determines the toxicity. Lipophilicity was named as one of the main factors that determines the uptake of drugs. A better accumulation into cells and usually higher toxicity was found with hydrophobic Pt(IV) drugs into the cell.67,68 The drug employed here is, however, highly hydrophilic and shows good solubility in water, which could potentially be the cause for the rather low toxicity of the drug. Micelles, decorated with PEG, moieties are, in contrast, readily taken up by cells.37,69 But now the drugs are embedded in a polymer matrix in the core and are only slowly released. According to the experiments depicted in Figure 7, only about 5% of Pt drug has been released as cisplatin after 72 h. In future work, we aim to vary the amount of TMI and therefore the amount of Pt(IV) in the system and test the effect of micelles loaded with higher amounts of Pt(IV) on a range of cancer cell lines. This work will be complemented by cell uptake studies. Some preliminary experiments showed that it is indeed possible for the nanoparticles to enter the cells,37 but further

New type of polymeric micelles with covalently incorporated Pt(IV) drugs were developed. Platinum(IV) drug was incorporated into the core of the micelles, which led not only to the loading of the micelle, but also to cross-linking. The drug-loaded micelles were stable for prolonged period of time, but in the presence of reductant, the drug was slowly released as cisplatin. Simultaneously, the cross-linked micelles were cleaved into block copolymers, allowing the easy excretion from the body. In vitro studies using A549 human lung cancer cells demonstrated that the drug-loaded micelles are toxic to the cell line. However, further cell testing is required to understand the detailed mechanism. Acknowledgment. M.H.S. acknowledges funding from the ARC (Australian Research Council) and an ARC Future Fellowship. H.T.T.D. thanks the Australian government for Endeavour Postgraduate Award. H.T.T.D. is also thankful to Lei Tao and Cyrille Boyer for valuable discussion and Wei Scarano for helping with column chromatography. The authors would like to thank the Centre for Advanced Macromolecular Design (CAMD) and UNSW Analytical Centre for support. Supporting Information Available. NMR spectrum used to determine the conversions of polymerizations. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) (2) (3) (4)

Kelland, L. Nat. ReV. Cancer 2007, 7 (8), 573–584. Boulikas, T.; Vougiouka, M. Oncol. Rep. 2004, 11 (3), 559–595. Roth, B. J. Semin. Oncol. 1996, 23 (5), 633–644. Rosenberg, B. Interdiscip. Sci. ReV. 1978, 3 (2), 134–147.

Core-Cross-Linked Micelles (5) Lippert, B. Cisplatin: Chemistry and Biochemistry of a Leading Anticancer Drug; Verlag Helvetica Chimica Acta and Wiley-VCH: Zurich, Switzerland, and Weinheim, Germany, 1999. (6) Jakupec, M. A.; Galanski, M.; Keppler, B. K., Tumour-inhibiting platinum complexes-state of the art and future perspectives. ReViews of Physiology, Biochemistry and Pharmacology; Springer-Verlag Berlin: Berlin, 2003; Vol 146, pp 1-53. (7) Galanski, M.; Jakupec, M. A.; Keppler, B. K. Curr. Med. Chem. 2005, 12 (18), 2075–2094. (8) Hambley, T. W. Coord. Chem. ReV. 1997, 166, 181–223. (9) Peyrone, B. Ann Chem. Pharm. 1845, 51, 129. (10) Rosengerg, B.; VanCamp, L.; Trosko, J. E.; Mansour, V. H. Nature 1969, 222, 385. (11) Neuse, E. W. S. Afr. J. Sci. 1999, 95 (11-12), 509–516. (12) Zwelling, L. A.; Kohn, K. W. Cancer Treat. Rep. 1979, 63 (9-10), 1439–1444. (13) Zaludova´, R.; Za´kovska´, A.; Kasparkova´, J.; Balcarova´, Z.; Kleinwa¨chter, V.; Vra´na, O.; Farrell, N.; Brabec, V. Eur. J. Biochem. 1997, 246 (3), 508–517. (14) Lippard, B. Coord. Chem. ReV. 1999, 182, 263. (15) Pinzani, V.; Bressolle, F.; Haug, I. J.; Galtier, M.; Blayac, J. P.; Balme`s, P. Cancer Chemother. Pharmacol. 1994, 35 (1), 1–9. (16) Godwin, A. K.; Meister, A.; O’Dwyer, P. J.; Huang, C. S.; Hamilton, T. C.; Anderson, M. E. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 3070– 3074. (17) Cleare, M. J.; Hydes, P. C.; Malerbi, B. W.; Watkins, D. M. Biochimie 1978, 60, 835–850. (18) Dabrowiak, J. C.; Bradner, W. T. Prog. Med. Chem. 1987, 24, 129– 158. (19) Kelland, L. R. Crit. ReV. Oncol./Hematol. 1992, 15, 191–219. (20) Schilder, R. J.; LaCreta, F. P.; Perez, R. P.; Johnson, S. W.; Brennan, J. M.; Rogatko, A.; Nash, S.; McAleer, C.; Hamilton, T. C.; Roby, D.; Young, R. C.; Ozols, R. F.; O’Dwyer, P. J. Cancer Res. 1994, 54, 709–717. (21) Gurney, H.; Crowther, D.; Anderson, H.; Murphy, D.; Prendiville, J.; Ranson, M.; Mayor, P.; Swindell, R.; Buckley, C. H.; Tindall, V. R. Ann. Oncol. 1990, 1, 427–433. (22) Trask, C.; Silverstone, A.; Ash, C. M.; Earl, H.; Irwin, C.; Bakker, A.; Tobias, J. S.; Souhami, R. L. J. Clin. Oncol. 1991, 9, 1131–1137. (23) Kelland, L. R.; Abel, G.; McKeage, M. J.; Jones, M.; Goddard, P. M.; Valenti, M.; Murrer, B. A.; Harrap, K. R. Cancer Res. 1993, 53, 2581– 2586. (24) Dolman, R. C.; Deacon, G. B.; Hambley, T. W. J. Inorg. Biochem. 2002, 88 (3-4), 260–267. (25) Hall, M. D.; Alderden, R. A.; Zhang, M.; Beale, P. J.; Cai, Z. H.; Lai, B.; Stampfl, A. P. J.; Hambley, T. W. J. Struct. Biol. 2006, 155 (1), 38–44. (26) Shi, T. S.; Berglund, J.; Elding, L. I. J. Chem. Soc., Dalton Trans. 1997, (12), 2073–2077. (27) Hall, M. D.; Hambley, T. W. Coord. Chem. ReV. 2002, 232, 49. (28) Choi, S.; Filotto, C.; Bisanzo, M.; Delaney, S.; Lagasee, D.; Whitworth, J. L.; Jusko, A.; Li, C.; Wood, N. A.; Willingham, J.; Schwenker, A.; Spaulding, K. Inorg. Chem. 1998, 37 (10), 2500–2504. (29) Bontha, S.; Kabanov, A. V.; Bronich, T. K. J. Controlled Release 2006, 114 (2), 163–174. (30) Liu, H.; Farrell, S.; Uhrich, K. J. Controlled Release 2000, 68 (2), 167–174. (31) Kwon, G.; Suwa, S.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. J. Controlled Release 1994, 29 (1-2), 17–23. (32) Maeda, H. AdV Enzyme Regul. 2001, 41 (1), 189–207. (33) O’Reilly, R. K.; Hawker, C. J.; Wooley, K. L. Chem. Soc. ReV. 2006, 35 (11), 1068–1083. (34) Wooley, K. L.; Hawker, C. J. Nanoscale objects: Perspectives regarding methodologies for their assembly, covalent stabilization, and utilization. Functional Molecular Nanostructures; Springer: New York, 2005; Vol. 245, pp 287-305. (35) Read, E. S.; Armes, S. P. Chem. Commun. 2007, (29), 3021–3035.


2299

(36) Stenzel, M. H. Chem. Commun. 2008, 30, 3486–3503. (37) Duong, H. T. T.; Nguyen, T. L. U.; Stenzel, M. Polym. Chem. 2010, 1, 171. (38) Dhar, S.; Daniel, W. L.; Giljohann, D. A.; Mirkin, C. A.; Lippard, S. J. J. Am. Chem. Soc. 2009, 131 (41), 14652–14653. (39) Dhar, S.; Liu, Z.; Thomale, J. r.; Dai, H.; Lippard, S. J. J. Am. Chem. Soc. 2008, 130 (34), 11467–11476. (40) Le, T. P.; Moad, G.; Rizzardo, E.; Thang, S. H. PCT Int Appl WO 9801478 A1 980115, 1998. (41) Ellis, L. T.; Er, H. M.; Hambley, T. W. Aust. J. Chem. 1995, 48 (4), 793–806. (42) Rieter, W. J.; Pott, K. M.; Taylor, K. M. L.; Lin, W. B. J. Am. Chem. Soc. 2008, 130 (35), 11584. (43) Reithofer, M.; Galanski, M.; Roller, A.; Keppler, B. K. Eur. J. Inorg. Chem. 2006, 2006 (13), 2612–2617. (44) Lempers, E. L. M.; Bloemink, M. J.; Reedijk, J. Inorg. Chem. 1991, 30 (2), 201–206. (45) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82 (13), 1107–1112. (46) Fojo, T.; Farrell, N.; Ortuzar, W.; Tanimura, H.; Weinstein, J.; Myers, T. G. Crit. ReV. Oncol. Hematol. 2005, 53 (1), 25–34. (47) Reithofer, M.; Galanski, M.; Roller, A.; Keppler, B. K. Eur. J. Inorg. Chem. 2006, (13), 2612–2617. (48) Fischer, S. J.; Benson, L. M.; Fauq, A.; Naylor, S.; Windebank, A. J. Neurotoxicology 2008, 29 (3), 444–452. (49) Bailey, P. D.; Collier, I. D.; Morgan, K. M. Amides; Pergamon: Cambridge, 1995; Vol. 5. (50) Boyer, C.; Davis, T. P. Chem. Commun. 2009, (40), 6029–6031. (51) Roth, P. J.; Wiss, K. T.; Zentel, R.; Theato, P. Macromolecules 2008, 41 (22), 8513–8519. (52) Nilles, K.; Theato, P. J. Polym. Sci., Part A: Polym. Chem. 2009, 47 (6), 1696–1705. (53) Roth, P. J.; Theato, P. Chem. Mater. 2008, 20 (4), 1614–1621. (54) Vogel, N.; Theato, P. Macromol. Symp. 2007, 249, 383–391. (55) Roleira, F. M. F.; Borges, F.; Andrade, L. C. R.; Paixao, J. A.; Almeida, M. J. M.; Carvalho, R. A.; da Silva, E. J. T. J. Fluorine Chem. 2009, 130 (2), 169–174. (56) Boyd, E.; Chavda, S.; Eames, J.; Yohannes, Y. Tetrahedron: Asymmetry 2007, 18 (4), 476–482. (57) Stevens, T. E.; Graham, W. H. J. Am. Chem. Soc. 1967, 89 (1), 182. (58) Green, M.; Berman, J. Tetrahedron Lett. 1990, 31 (41), 5851–5852. (59) Stenzel, M. H. Macromol. Rapid Commun. 2009, 30 (19), 1603–1624. (60) Barner, L.; Barner-Kowollik, C.; Davis, T. P. J. Polym. Sci., Part A: Polym. Chem. 2002, 40 (8), 1064–1074. (61) Soo, P. L.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 2004, 42 (6), 923–938. (62) Hamley, I., Block Copolymers in Solution: Fundamentals and Applications; John Wiley and Sons, Ltd: New York, 2005. (63) Ni, H.; Nash, H. A.; Worden, J. G.; Soucek, M. D. J. Polym. Sci., Part A: Polym. Chem. 2002, 40 (11), 1677–1688. (64) Han, J. L.; Yu, C. H.; Lin, Y. H.; Hsieh, K. H. J. Appl. Polym. Sci. 2008, 107 (6), 3891–3902. (65) Withey, A. B. J.; Chen, G. J.; Nguyen, T. L. U.; Stenzel, M. H. Biomacromolecules 2009, 10 (12), 3215–3226. (66) Howell, B. A.; Fan, D.; Rakesh, L. J. Therm. Anal. Calorim. 2006, 85 (1), 17–20. (67) Hall, M. D.; Amjadi, S.; Zhang, M.; Beale, P. J.; Hambley, T. W. J. Inorg. Biochem. 2004, 98 (10), 1614–1624. (68) Ang, W. H.; Khalaila, I.; Allardyce, C. S.; Juillerat-Jeanneret, L.; Dyson, P. J. J. Am. Chem. Soc. 2005, 127 (5), 1382–1383. (69) Zhang, L.; Nguyen, T. L. U.; Bernard, J.; Davis, T. P.; BarnerKowollik, C.; Stenzel, M. H. Biomacromolecules 2007, 8 (9), 2890–2901.

BM100396S

Anticancer Drugs to Isocyanates - American Chemical Society

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