Block Copolymer Micelles with Pendant ... - ACS Publications

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Block Copolymer Micelles with Pendant Bifunctional Chelator for Platinum Drugs: Effect of Spacer Length on the Viability of Tumor Cells Vien T. Huynh,†,‡ Jing Yang Quek,† Paul L. de Souza,‡ and Martina H. Stenzel*,† †

Centre for Advanced Macromolecular Design (CAMD), The University of New South Wales, Sydney NSW 2052, Australia Liverpool Hospital Clinical School, and Molecular Medicine Research Group, University of Western Sydney, Sydney NSW 2170, Australia

‡

S Supporting Information *

ABSTRACT: Three monomers with 1,3-dicarboxylate functional groups but varying spacer lengths were synthesized via carbon Michael addition using malonate esters and ethylene(MAETC), butylene- (MABTC), and hexylene (MAHTC) glycol dimethacrylate, respectively. Poly[oligo-(ethylene glycol) methylether methacrylate] (POEGMEMA) was prepared in the presence of a RAFT (reversible addition−fragmentation chain transfer) agent, followed by chain extension with the prepared monomers to generate three different block copolymers (BP-E80, BP-B82, and BP-H79) with similar numbers of repeating units, but various spacer lengths as distinguishing features. Conjugation with platinum drugs created macromolecular platinum drugs resembling carboplatin. The amphiphilic natures of these Pt-containing block copolymers led to the formation micelles in solution. The rate of drug release of all micelles was similar, but a noticeable difference was the increasing stability of the micelle against dissociation with increasing spacer length. The platinum conjugated polymer showed high activity against A549, OVCAR3, and SKOV3 cancer cell lines exceeding the activity of carboplatin, but only the micelle based on the longest spacer had IC50 values as low as cisplatin. Cellular uptake studies identified a better micelle uptake with increasing micelle stability as a possible reason for lower IC50 values. The clonogenic assay revealed that micelles loaded with platinum drugs, in contrast to low molecular weight carboplatin, have not only better activity within the frame of a 72 h cell viability study, but also display a longer lasting effect by preventing the colony formation A549 for more than 10 days.

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INTRODUCTION cis-Diaminedichloroplatinum(II) (cisplatin, CDDP) was first described in 1845 and has since then been known as Peyrone’s salt. However, it was not until 1965 that its high bioactivity against Escherichia coli was discovered by Barnett Rosenberg.1 In 1968, after successful intraperitoneal experiments in mice bearing a standard murine transplantable tumor,2 cisplatin entered clinical trial and was approved for clinical use by the United States Food and Drug Administration (FDA) in 1978.3 Cisplatin consists of two different types of ligands. One ligand, usually based on N, binds strongly to the Pt ion.4−6 All classic platinum complexes have at least one N containing ligand, with the activity of the platinum drug increasing according to NR3 (inactive, R = alkyl), NHR2 < NH2R < NH3.7 The other ligand is the leaving group, which is typically chloride or carboxylate. This leaving group should be moderately bound to platinum. Highly labile ligands such as NO3− would result in high toxicity, while strong ligands such as N3−, SCN−, or CN− cause the platinum complex to be inactive.6,8 © 2012 American Chemical Society

Cisplatin is administered to cancer patients intravenously and it remains intact due to the relatively high concentration of chloride ions (∼100 mM) in the blood plasma. This compound enters the cells via either passive diffusion or active uptake.3 Inside the cell (cytoplasm), due to a much lower concentration of chloride ion (∼3−20 mM), the neutral cisplatin undergoes hydrolysis in which a chloride ligand is replaced by a molecule of water, generating a positively charged species.9,10 The resulting active complexes then bind to DNA forming intrastrand and interstrand cross-linked adducts.11 This coordination complex not only inhibits replication and transcription of DNA, but also programmed cell death (apoptosis).12 Although cisplatin is successful in the treatment of a variety of solid tumors such as ovarian, bladder, testicular, head and neck, small-cell, and non-small-cell lung cancers,3,13,14 it is difficult to ignore the many side effects including nephrotoxicity, Received: December 4, 2011 Revised: February 6, 2012 Published: February 10, 2012 1010

dx.doi.org/10.1021/bm2017299 | Biomacromolecules 2012, 13, 1010−1023

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we aim at answering the question if micelles need to be very stable against disassembly to achieve the best possible outcome in regard to delivering the drug inside the tumor cell.

neurotoxicity, nausea, and vomiting, which results in restriction of its use.3,15 Moreover, the inherent drug resistance of tumors to chemotherapy has become one of the main driving forces to develop improved Pt anticancer agents.16−19 This includes the development of a number of new platinum complexes with modifications to the ligand structure,20−29 the development of Pt(IV) prodrugs,30−32 and formation of platinum macromolecular complexes.33−35 Increasing activity can be seen in the latter approach, where the drug is either attached via the permanent ligand, the leaving ligand, or the Pt(IV) prodrug36−42 to produce platinum polymeric complexes. This approach is of particular interest when the polymer is processed into nanosized carrier, which has the added advantage that the drug can be targeted passively via enhanced permeability and retention (EPR) effect to the tumor cells. In addition, nanosized carriers enter the cells via an endocytotic pathway and can therefore bypass the Pgp drug efflux resistance mechanism.43 It also should be mentioned that nanoparticles can protect cisplatin from undesirable binding events, mainly with thiols in peptides and proteins, which may inactivate the drug on its way to the tumor. Polymeric micelles, self-assembled block copolymers with a core−shell structure, have widely been used for the delivery of drugs, proteins, genes, and imaging agents.44,45 The high water content of the hydrophilic shell and the small scale (10−100 nm) of polymeric micelles permit a long circulation time in the bloodstream.46 Furthermore, micelles are small enough (50 kDa) to avoid renal excretion.46,47 While the behavior of micelles in water is reasonably well understood,48 there is only limited knowledge on how micelles behave in biological media and how they interact with cells. Understanding of the interaction of micelles with cells especially can be the key to a successful drug delivery system. While micelles based on pluronics can enhance the drug uptake even at low concentrations,49,50 other block copolymers have a very distinctive drop in cell uptake below the critical micelle concentration (CMC).51,52 It, therefore, seems paramount to aim at the use of micelles that are stable even at low concentrations. A potential way of increasing micelle stabilities is by cross-linking,53,54 but can also be achieved by simply altering the composition of the micelle.48 More voluminous hydrophobic blocks lead, in general, to more thermodynamically stable micelles, while targeting the glassy core may lead to increased kinetic stability.55 The aim of this study was to investigate the effect of the stability of the micelle on the performance as drug carrier by varying not the number of repeating units of the hydrophobic but the overall hydrophobicity of the block copolymer. This idea is nurtured by the fact that the length of hydrocarbon side chains has significant influences on the overall nature of the polymeric micelle interactions,56,57 yet only little attention has been paid to the relationship between micelle stability and drug loading and releasing, cellular uptake, and cancer cell proliferation ability. For this purpose, different spacer length monomers were prepared via Michael addition reaction, as displayed in Scheme 1. Subsequently, RAFT polymerization technique was employed to produce different block copolymers with similar hydrophilic and hydrophobic block length, but with a varied spacer length. Conjugation to CDDP results in the formation of platinum drug containing micelles. This study addressed the important question on the correlation between the stability of a micelle and its activity as a drug carrier. Although the focus here will be on platinum drugs,

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EXPERIMENTAL SECTION

Materials. Unless otherwise specified, all chemicals were reagent grade and were used as received: di-tert-butyl malonate (Aldrich, 98%), ethylene glycol dimethacrylate (EGDMA, Aldrich, 98%), 1,4-butylene glycol dimethacrylate (BGDMA, Aldrich, 98%), 1,6-hexylene glycol dimethacrylate (HGDMA, Aldrich, 98%), potassium carbonate (Univar, anhydrous), 18-crown-6 (Sigma-Adrich, 99%), tetrahydrofuran (THF, anhydrous, 98%, Aldrich), diethyl ether (Et2O anhydrous, Ajax Finechem, 99%), petroleum ether (BR 40−60 °C; Ajax Finechem, 90%), ethyl acetate (ETOAc, AjaxFinechemicals, 99.5%), N,N-dimethylacetamide (DMAc; Aldrich, HPLC grade), magnesium sulfate (Ajax Finechem, 70%), toluene (Aldrich; purum), 1,4-dioxane (Sigma-Aldrich, 99%), dichloromethane (DCM; Ajax Finechem, 99%), trifluoroacetic acid (Sigma-Aldrich, 99%), chloroform-d (CDCl 3; Cambridge Isotape Laboratories), cis-dichlorodiaminoplatinum(II) (CDDP; Sigma-Aldrich; 99.9%), silver nitrate, pyrene, and ethanol. 2,2-Azobisisobutyronitrile (AIBN; Fluka, 98%) was purified by recrystallization from methanol. Oligo(ethylene glycol) methylether methacrylate (OEGMEMA; MW = 300 g mol−1; Aldrich) was deinhibited by passing through a column of basic aluminum oxide. The RAFT agent (4-cyanopentanoic acid)-4-dithiobenzoate (CPADB) was synthesized according to literature58,59 and recrystallized from toluene to yield a fine pink powder. Deionized (DI) water produced by a Milli-Q water purification system and has a resistivity of 17.9 mΩ/cm. Synthesis. Synthesis of Monomers with Pendant Carboxylic Functional Groups. 1,1-Di-tert-butyl 3-(2-(Methacryloyloxy)ethyl)butane-1,1,3-tricarboxylate MAETC (Scheme 2). To synthesize 1,1di-tert-butyl 3-(2-(methacryloyloxy)ethyl)butane-1,1,3-tricarboxylate (MAETC), ethylene glycol dimethacrylate (EDMA, 2.657 g, 13.4 mmol, 3 equiv), potassium carbonate (0.679 g, 4.9 mmol, 1.1 equiv), and 18-crown-6 (0.039 g, 0.15 mmol, 0.033 equiv) as a catalyst were added to 0.7 mL of anhydrous THF, and the mixture was cooled to 0 °C in an ice bath before degassing under nitrogen for 30 min. Di-tert-butyl malonate (0.966 g, 4.5 mmol, 1 equiv) was added dropwise with vigorous stirring. The slurry suspension was stirred for 30 min while maintaining the temperature at 0 °C, then stirred for a further 48 h at 40 °C. The color changed from colorless to yellowish. Deionized (DI) water (15 mL) was added into the slurry before it was washed 3 times with diethyl ether (3 × 30 mL) using a separation funnel. The diethyl ether phases were combined and evaporated using a rotary evaporator. The residual oily liquid was purified by silica gel column chromatography using petroleum spirit/ethyl acetate mixture as gradient eluent with the ratio changing from 20:1 to 8:1 v/v. The collected fractions were examined by TLC (stained by permanganate solution). The monomer (Rf = 0.3) was obtained as an oily yellowish liquid (56% in yield) and characterized by 1H and 13C NMR spectroscopy and ESI-MS. The liquid solidified into crystals when left in a freezer overnight. 1 H NMR (300.17 MHz, CDCl3, 25 °C): δ (ppm) = 6.12 (s, 1H, H1), 5.58 (s, 1H, H2), 4.34 (t, 4H, H4 and H5, J = 1.08 Hz), 3.2−3.25 (dd, 1H, H9, J = 6.45 Hz, J = 8.91 Hz), 2.45−2.57 (qq, 1H, H6, J = 6.99 Hz), 2.15 (dd, 1H, H8, J = 6.54 Hz), 1.9 (dd, 4H, H8 and H3, J = 6.12 Hz), 1.43 (s, 18H, H10), 1.18 (d, 3H, H7, J = 7.02 Hz). 13C NMR (75.48 MHz, CDCl3, 25 °C): δ (ppm) = 175.37 (Cg), 168.41 (Cn), 168.21 (Cp), 166.99 (Cd), 135.79 (Cc), 126.01 (Ca), 81.50 (Co), 62.33 (Ce), 61.99 (Cf), 51.64 (Cm), 37.08 (Ch), 31.96 (Cl), 27.79 (Cq), 18.18 (Cb), 17.30 (Ck). See Figure S1 in the Supporting Information for the full spectra. ESI-MS Calcd m/z for C21H34O8, 414.23; experimental, 437.1 (Na+). Melting point, 93 °C, measured by DSC (Supporting Information, Figure S2). The other two monomers, including 1,1-di-tert-butyl 3-(4-((2, 3-dimethylbut-2-enoyl)oxy)butyl)butane-1,1,3-tricarboxylate 1011


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Scheme 1. Monomer Synthesis via Carbon Michael Addition and Subsequent Synthesis of Block Copolymers via RAFT Polymerization Prior to Conjugation to Pt Drugs and Formation of Micelles

J = 6.99 Hz), 2.15 (dd, 1H, H10, J = 6.54 Hz), 1.9 (dd, 4H, H10 and H3, J = 6.12 Hz), 1.65 (m, 4H, H5 and H6), 1.43 (s, 18H, H12), 1.18 (d, 3H, H9, J = 7.02 Hz). 13C NMR (75.48 MHz, CDCl3, 25 °C): δ (ppm) = 176.5 (Ci), 169.4 (Co), 169.2 (Cp), 168 (Cd), 136.5 (Cc), 126.01 (Ca), 81.50 (Cq), 64.33 (Ce), 61.99 (Ch), 52.5 (Cf), 37.5 (Ck), 31.96 (Cm), 28.5 (Cr), 26 (Cf and Cg), 19 (Cb), 17.30 (Cl). See Figure S3 in the Supporting Information for the full spectra.

(MABTC; Scheme 3) and 1,1-di-tert-butyl 3-(6-((2,3-dimethylbut2-enoyl)oxy)hexyl)butane-1,1,3-tricarboxylate (MAHTC; Scheme 4) were synthesized using the same procedure above and they were characterized by 1H and 13C NMR spectroscopy and ESI-MS.MABTC 1 H NMR (300.17 MHz, CDCl3, 25 °C): δ (ppm) = 6.12 (s, 1H, H1), 5.58 (s, 1H, H2), 4.25 (t, 2H, H4), 4.15 (t, 2H, H5, J = 1.08 Hz), 3.2− 3.25 (dd, 1H, H11, J = 6.45 Hz, J = 8.91 Hz), 2.45−2.57 (qq, 1H, H8, 1012


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Scheme 2. MAETC Label for1H NMR (Left) and 13C NMR (Right)

Scheme 4. MAHTC Label for 1H NMR (Left) and 13C NMR (Right)

Scheme 3. MABTC Label for 1H NMR (Left) and 13C NMR (Right)

regular time intervals and analyzed via 1H NMR to determine the conversion, followed by precipitation in petroleum spirit. The final macroRAFT agent was characterized by GPC and 1H NMR. 1H NMR (300.17 MHz, CDCl3, 25 °C) δ (ppm) = 4.4− 4.2 (2nH, CH3COOCH2CH2O), 4.0−3.8 (11nH, CH2 of OEGMEMA monomer), 3.6−3.4 (3nH, CH3 of chain end of OEGMEMA), 1.4−1.2 (3nH, CH3 of the main chain), 1.2−0.9 (2nH, CH2 of the main chain); n is the degree of polymerization (DPn) of POEGMEMA (Supporting Information, Figure S5) Chain Extension of Poly(OEGMEMA) as MacroRAFT Agent with MAETC. A batch of POEGMEMA (4 g) was synthesized using acetonitrile as a solvent to target a theoretical molecular weight of 12000 g mol−1 by polymerizing OEGMEMA for 3 h using CPADB under identical conditions as those described previously (XNMR = 50%,Mn,theo = 15279 g mol−1, Mn,SEC = 11100 g mol−1, PDI = 1.06). MAETC (0.1225 g, 3.03 × 10−4 mol), POEGMEMA (Mn,theo = 15279 g mol−1, Mn,SEC = 11100 g mol−1, PDI = 1.06, 0.046 g, 3.03 × 10−6 mol) as macroRAFT agent, and AIBN (10−4 g, 6.06 × 10−7 mol) were dissolved in 0.6 mL of dioxane to result in [MAETC]/ [POEGMEMA]/[AIBN] = 100:1:0.2 and [MAETC] = 0.5 mol L−1. The vial was capped with a rubber septum and copper wire. Six vials of the solution were thoroughly deoxygenated using nitrogen purging for 45 min and then placed in an oil bath at 70 °C. Samples were taken every 1 h up to 6 h and quenched in an ice bath. The final copolymer was characterized by GPC and 1H NMR. 1H NMR (300.17 MHz, CDCl3, 25 °C) δ (ppm) = 4.3−4.0 (4mH, OCH2CH2O), 4.0−3.8 (2nH, COOCH 2 CH 2 OCH 2 CH 2 O), 3.8−3.5 (11nH, COO CH2CH2OCH2CH2O), 3.5−3.3 (3nH, OCH2CH2OCH2CH2OCH3), 3.3−3.1 (mH, tert-Bu-OOCCHCH2COO-Bu-tert), 2.6−2.4 (mH, OOCCHCH3-CH2), 2.3−2.0 (mH, OOCCHCH3CH2CH(COOBu-tert)2), 2.0−1.6 (mH + 3mH + 3nH, OOCCHCH3CH2CH(COOBu-tert)2, CH3 attached to the backbone of MAETC, CH3 attached to the backbone of OEGMEMA, respectively), 1.5−1.3 (18mH, OOCCHCH3CH2CH(COO(CH3)3)2), 1.3−1.1 (3nH, CH3 of the main chain), 1.1−0.7 (2nH + 2mH, CH2 of the main chain of OEGMEMA and CH2 of the main chain of MAETC, respectively); n and m are the degrees of polymerization (DP n) of POEGMEMA and PMAETC, respectively. Deprotection of Block Copolymers. Deprotection of tert-butyl groups of POEGMEMA-b-PMAETC was carried out similar to the procedure described previously.60 Briefly, the diblock copolymer (0.2 g) was fully dissolved in dichloromethane (DCM, 0.5 mL). Trifluoroacetic

ESI-MS Calcd m/z for C23H38O8, 442.26; experimental m/z, 465.1 (Na+). MAHTC . 1H NMR (300.17 MHz, CDCl3, 25 °C): δ (ppm) = 6.12 (s, 1H, H1), 5.58 (s, 1H, H2), 4.25 (t, 2H, H4), 4.15 (t, 2H, H5, J = 1.08 Hz), 3.2−3.25 (dd, 1H, H11, J = 6.45 Hz, J = 8.91 Hz), 2.45−2.57 (qq, 1H, H8, J = 6.99 Hz), 2.15 (dd, 1H, H10, J = 6.54 Hz), 1.9 (dd, 4H, H10 and H3, J = 6.12 Hz), 1.65 (m, 4H, H5 and H6), 1.43 (s, 18H, H12), 1.18 (d, 3H, H9, J = 7.02 Hz). 13C NMR (75.48 MHz, CDCl3, 25 °C): δ (ppm) = 176.5 (Cl), 169.4 (Cr), 169.2 (Cq), 168 (Cd), 136.5 (Cc), 126.01 (Ca), 81.50 (Cs), 64.33 (Ce), 61.99 (Ck), 52.5 (Cp), 37.5 (Cm), 31.96 (Co), 28.5 (Ct), 26 (Cf, Cg, Ch, and Ci), 19 (Cb), 17.30 (Cn). See Figure S4 in the Supporting Information for the full spectra. ESI-MS Calcd m/z for C25H42O8, 470.3; experimental m/z, 493.2 (Na+). Polymer Synthesis. RAFT Polymerization of OEGMEMA Using CPADB RAFT Agent. OEGMEMA (7.0 g, 2.3 × 10−2 mol), CPADB (0.065 g, 2.3 × 10−4 mol), and AIBN (0.0077 g, 4.67 × 10−5 mol) were dissolved in toluene (46.62 mL) in a 100 mL round-bottom flask to give [OEGMEMA]/[CPADB]/[AIBN] = 100:5:0.2 and [OEGMEMA] = 0.5 mol L−1. The solution was divided into aliquots, sealed with a rubber septum, and thoroughly deoxygenated using nitrogen purging for 45 min before being placed in an oil bath at 70 °C. Samples were taken out at 1013


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acid (3 equivalent compared to tert-butyl group) was then added to the polymer mixture followed by stirring at room temperature for 72 h. The final reaction mixture was subsequently dialyzed against acetone− water (1:1) using a dialysis membrane with a molecular weight cut off (MWCO) of 3500 Da, followed by dialysis against pure water. Subsequently, the remaining solution inside the bag was freeze-dried to yield a waxy polymer. The deprotected copolymer was characterized by 1H NMR and HSQC-NMR. 1H NMR (300.17 MHz, acetone-d6, 25 °C): δ (ppm) = 4.3−4.0 (4mH, OCH2CH2O), 4.0−3.8 (2nH, COOCH2CH2OCH2CH2O), 3.8−3.5 (11nH, OCOCH2 CH2OCH2CH2OCH3), 3.5−3.3 (3nH, OCH2CH2OCH2CH2OCH3), 3.3−3.1 (mH, tert-Bu-OOCCHCH2COO-Bu-tert), 2.6−2.4 (mH, OOCCHCH3CH2), 2.3−2.0 (mH, OOCCHCH3CH2CH(COOBu-tert)2), 2.0−1.6 (mH + 3mH + 3nH, OOCCHCH3CH2CH(COOBu-tert)2,CH3 attached to the backbone of MAETC, CH3 attached to the backbone of OEGMEMA, respectively), 1.5−1.3 (18mH, OOCCHCH3CH2CH(COO(CH3)3)2), 1.3−1.1 (3nH, CH3 of the main chain), 1.1−0.7 (2nH + 2mH, CH2 of the main chain of POEGMEMA and CH2 of the main chain of PMAETC, respectively); n and m are the degrees of polymerization (DPn) of POEGMEMA and PMAETC, respectively. See Figure S6 in the Supporting Information for the full spectra of HSQC-NMR. Polymer−Platinum Conjugates. Conjugation of cisplatin to the polymer was described in previous works, but some modifications have been introduced here.61,62 In a typical experiment, CDDP (10 mg) was suspended in 10 mL distilled water and mixed with silver nitrate ([AgNO3]/[CDDP] = 1.955) to form the aqueous complex. The solution was stirred in the dark at room temperature for 4 h. White precipitate of silver chloride was observed indicative of the proceeding reaction. The mixture was then centrifuged at 9000 rpm for 20 min to remove the AgCl precipitate and the supernatant was purified by passing through a 0.22 μm filter. Polymers with carboxyl functional groups (25 mg, dissolved in 2 mL of NaOH (1 mg mL−1)) were added to the above-mentioned CDDP aqueous solution and left to react in a water bath at 37 °C for 12 h with gentle shaking to result in polymer− CDDP conjugates. The prepared conjugate was purified by ultrafiltration using Sartorius Vivaspin 6 centrifugal filter devices with a molecular weight cut off of 3000 Da, followed by freeze-drying, yielding a yellow powder. Self-Assembly of Platinum Drug Conjugates into Micellular Structure. The powder-formed conjugation of deprotected POEGMEMA-b-PMAETC and cis-dichlorodiaminoplatinum(II) (60 mg) was dissolved in DMF (2 mL), which is a good solvent for both hydrophobic and hydrophilic blocks. Distilled water (8 mL) was added dropwise using a syringe pump (3 mL h−1) to 2 mL of conjugates in DMF (30 mg mL−1) under moderate stirring at room temperature. The mixture was then dialyzed against water for 48 h using membrane (MWCO 3500 Da) to remove DMF. The targeted final polymer concentration was 5 mg mL−1. Micelle Stability. To determine the critical micelle concentration (CMC) values, pyrene fluorescence measurements were employed. Briefly, a stock solution of pyrene was prepared by adding a known amount of pyrene in 20 wt % ethanol in water. The mixture was sonicated to yield a clear solution. The pyrene containing ethanol was diluted with DI water to achieve a final pyrene concentration of 2 μM and 0.5% of ethanol. Micelles of different concentrations were added to the mixture to produce final micelle concentrations ranging from 0 to 50 mg L−1. Fluorescence emissions were measured in Cary Eclipse Fluoresce spectrophotometer using 10 mm path length quart cuvette. Excitation was done at 237.96 nm and emissions were recorded in the 350−450 nm wavelength range. The slit widths for both excitation and emission were fixed at 2.5 nm. The scan rate was 600 nm min−1. All measurements were carried out at 298 K. It was assumed that the ethanol content in the solution does not affect the self-aggregation behavior of the amphiphilic polymer. Release of Platinum Drugs from Platinum−Polymer Conjugates. Micelles (2 mL, 5 mg mL−1) containing platinum drugs were dialyzed against pH 7.4 buffer solution (250 mL) at 37 °C. NaCl (0.9%) was added into these buffer solutions to trigger the platinum drug release. Cellulose tubing membrane with a molecular weight cut off of 3500 Da

was used to allow equilibrium of free platinum drug from inside and outside the dialysis membrane. Aliquots of 1 mL were taken in regular time intervals from the dialysate over 168 h. The amount of released Pt was determined using inductively coupled plasma mass spectroscopy (ICP-MS). To a 10 mL centrifuge tube, 1 mL of the dialysate was diluted five times by aqua regia 2% (HCl/HNO3 = 3:1). The solution was then digested at 60 °C for 3 h and then cooled to room temperature. The concentration of Pt released from the conjugate was expressed as a ratio of the amount platinum in the releasing solution (the solution outside the dialysis membrane) and that in the initial sample. The percentage of Pt released was calculated using the equation

V (t ) × C + Y %release = total Z where Vtotal(t) is the remaining volume in the releasing container at time t in mL; C is the concentration of platinum determined from ICP-MS in μg mL−1; Y is the amount of platinum that has already been collected in μg; and Z is the total amount of platinum at t = 0 present in the dialysis bag in μg. Cell Culture. Human prostate cancer cell lines DU145, PC3 and human ovarian cancer cell lines OVCAR3 and A2780 were grown in RPMI-1640 [2 × 10−3 M L-glutamine, 1.5 g L−1 sodium bicarbonate, 0.010 M 2-hydroxyethylpiperazinesulfonic acid (HEPES), 4.5 g L−1 glucose, 10−3 M sodium pyruvate] medium supplemented with 10% fetal bovine serum (FBS). The cells were grown in 5% CO2 at 37 °C. Cytotoxicity Assay. The sulforhodamine B (SRB) assay established by the U.S. National Cancer Institute for rapid, sensitive, and inexpensive screening of antitumor drugs in microplates was employed to screen the cytotoxicity and antitumor activities of polymers and polymeric platinum drugs, respectively.63 Human nonsmall lung cancer cells (NSCLC, A549) diluted in 100 μL of RPMI-1640 medium (2 mM L-glutamine, 1.5 g L−1 sodium bicarbonate, 10 mM HEPES, 4.5 g L−1 glucose, 1 mM sodium pyruvate) were seeded into the wells with 2000 cells/well. The microtiter plates were left for 24 h at 37 °C and then exposed to various doses of polymers and micelles for 72 h. Cell cultures were fixed with TCA (10%, w/v) and incubated at 4 °C for 1 h. The wells were 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/v) SRB dissolved in 1% (v/v) acetic acid. At the end of the staining period, SRB was removed and cultures were quickly rinsed five times with 1% (v/v) acetic acid to remove unbound dye. Subsequently, the cultured plates were air-dried until no conspicuous moisture was visible before bound dye was shaken in 100 μL of 10 mM Tris base for 5 min. The absorbance at 570 nm of each well was measured using microtiter plate reader scanning spectrophotometer (BioTek’s PowerWave HT Microplate Reader and KC4 Software). Each sample was replicated three times. Cellular Uptake. Cellular uptake experiments were performed according to a previously described method with some modifications.64 A549 cells were seeded into a 12-well plate at 16 × 103 cells per well and incubated for 24 h. The cells were treated with polymer-Pt micelles, including BP-27/Pt, BP-54/Pt, and BP-80/Pt and free CDDP at equal CDDP concentrations. After 2 and 24 h incubation at 37 °C, the medium was removed and rinsed with cold PBS (1 mL × 3). The cells were trypsinized and incubated with HNO3 (68%, v/v) at 65 °C for 20 h. Platinum content uptake was determined using inductively coupled plasma mass spectrometer (ICP-MS). A four-point standard curve was plotted between intensity versus a serial dilution of a certified reference standard ranging from 1 to 1000 ppb. The reported result of the sample is the average of three replicates. The amount of polymer taken up by the cells was qualitatively and quantitatively determined using fluorescence microscopy and fluorescence reader, respectively. To label polymers, fluorescein-o-methacrylate (2% mol of POEGMEMA) was copolymerized with OEGMEMA before chain extending with the synthesized monomers, including MAETC, MABTC, and MAHTC, to yield similar structures of BP-E80, BP-B82, and BP-H79. These block copolymers were conjugated to CDDP and formed micelles followed the procedure 1014


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mentioned above. Three different cell lines, including A549, OVCAR3, and SKOV3, were seeded into a 12-well plate at 16000 cells per well and incubated for 24 h. Those cells were subsequently treated with the fluorescein-label micelles for 2 and 15 h at 37 °C. All cells were washed three times with PBS before observing under microscopy. Cell uptake pictures were acquired by using fluorescence microscope with mercury lamp of λex 535 nm and λem 590 nm to track the fluorescence micelles. Fluorescence intensity of treated cell solution was measured by fluorescence reader at λex = 535 nm and λem = 590 nm. Colony Formation Assay. Unlike the cell proliferation assay, the colony formation assay measures the productive integrity of the cells following withdrawal of drug treatment. The assays were performed as described by Liebmannet et al.65 with some modifications. Briefly, A549 cells were exposed to carboplatin (8.5 and 241 μM), loaded (1.1 and 65 μM Pt), and unloaded (100 μg polymer mL−1) micelles for 72 h and then washed with phosphate buffer solution (PBS). Single survived cells were then plated in six-well plates with fresh PRMI 1640 medium, and the medium was changed every 3 days. Following 10 days of incubation, the cells were washed twice with cold PBS and incubated with methanol for 30 min at room temperature to fix the cells. Methanol was evaporated and cells were stained with 0.1% crystal violet for 3 min. The excess crystal violet was washed five times with tap water and air-dried overnight. The data were calculated based on eqs 1 and 2.

plating efficiency(PE) =

no. of colonies formed × 100% no. of cells seeded

surviving fraction(SF) =

no. of colonies formed after treatment no. of cells seeded × PE

quantum efficiency, and an ALV/LSE-5003 multiple tau digital correlator electronics system. The samples were filtered to remove dust using a microfilter (0.45 μm) prior to measurement. Transmission Electron Microscopy (TEM). Analyses were performed using a JEOL 1400 TEM with a beam voltage of 100 kV and a Gatan CCD for acquisition of digital images. Samples were prepared by placing a droplet of a 1 mg mL−1 polymer solution on a formamide and graphite-coated copper grid and draining the excess using filter paper after 60 s. To negatively stain the samples a droplet of 2% (w/v) phosphotungstic acid solution was placed on the copper grid for 30 s before being drained with filter paper. Thermo Gravimetric Analysis (TGA). Thermal decomposition properties of polymers were recorded using a Perkin-Elmer Thermogravimetric Analyzer (Pyris 1 TGA). Analyses were conducted over a temperature range of 30−700 °C with a programmed temperature increment of 20 K per min. Inductively Coupled Plasma−Mass Spectrometer (ICP-MS). 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 ratio 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 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 193 Ir. Replicate time was set to 900 ms and the dwell time 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. The samples were treated with aqua regia solution at 90 °C for 2 h to digest platinum.

(1)

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RESULTS AND DISCUSSION Synthesis of Block Copolymers and Conjugation to Platinum Drugs. The main objective of this project was to investigate the effects of spacer length (distance between platinum moieties and polymer backbone) on the stability of the micelle and therefore on the performance as a drug delivery carrier. Malonate derivatives were chosen as a ligand for platinum conjugation due to their resemblance to carboplatin. The Michael addition reaction between ethylene glycol dimethacrylate crosslinker and di-tert-butyl malonate yielding a monomer with bidentate carboxylato groups (Scheme 1) was described in detail in an earlier study.60,66 Here, this approach was extended to various dimethacrylates (i.e., 1,4-butylene glycol dimethacrylate and 1,6-hexylene glycol dimethacrylate cross-linkers) to generate monomers with the same functional group, but different spacer lengths. Briefly, di-tert-butyl malonate was deprotonated in the presence of strong base, potassium carbonate in THF mixed with 18-crown-6 to enhance the solubility of potassium carbonate in organic solvents. The yield of the monomer synthesis was independent from the type of dimethacrylate reaching around 60% reproducibly. The monomer structures were confirmed by ESI-MS, 1H NMR, 13C NMR, DEPT-90, and DEPT-135. Displayed in Figure 1, the changes in the 1H NMR spectra consist of a double doublet at 3.25 ppm, which accounts for the methine group between two carboxylates. The coupling pattern is caused by differences in the chemical shift of both protons of the adjacent methylene groups (1.9 and 2.15 ppm, respectively). Further confirmation of the structures was obtained using 13C NMR, DEPT-90, and DEPT-135, which are shown in Supporting Information, Figures S1, S3, and S4. Mass spectrometry analysis via ESI-MS of the monomers (MAETC, MABTC, and MAHTC) gave a single peak at 437.1 (Na+), 465.1 (Na+), and 493.2 (Na+), respectively, which are in agreement with the theoretical molecular weight of 414.23 (437.1 Na+), 442.26 (465.1 Na+), and 470.3 (493.2 Na+), respectively.

Analyses. Size Exclusion Chromatography (SEC). SEC was implemented using a Shimadzu modular system comprised of a DGU-12A degasser, LC-10AT pump, SIL-10AD automatic injector, CTO-10A column oven, RID-10A refractive index detector, and SPD-10A Shimadzu UV/vis detector. A 50 × 7.8 mm guard column and four 300 × 7.8 mm linear columns (500, 103, 104, and 105 Å pore size, 5 μm particle size) were used for the analyses. N,N′-dimethylacetamide (DMAc; HPLC grade, 0.05% w/v of 2,6-dibutyl-4-methylphenol (BHT), 0.03% w/v of LiBr) with a flow rate of 1 mL min−1 and a constant temperature of 50 °C was used as the mobile phase with an injection volume of 50 μL. The samples were filtered through 0.45 μm filters. The unit was calibrated using commercially available linear polystyrene standards (0.5−1000 kDa, Polymer Laboratories). Chromatograms were processed using Cirrus 2.0 software (Polymer Laboratories). Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H and 13C NMR spectra were recorded using a Bruker ACF300 (300 MHz) spectrometer, using (CD3)2SO, CD3OD, or CDCl3 as solvents. All chemical shifts are stated in ppm (δ) relative to tetramethylsilane (δ = 0 ppm), referenced to the chemical shifts of residual solvent resonances (1H and 13 C). The number of scans was 16 as default for all polymer samples. For 195Pt NMR measurement, 195Pt resonances were externally referenced to Na2PtCl6 at 0 ppm. Spectra were obtained using a broadband observe 5 mm probe with z-axis gradient capability. The Bruker pulse program zgmultiscan was modified to execute a very short delay time (set at d1 = 2 ms) followed by a hard 90° pulse. The experiment was run in increments of 20000 scans (ns = 20000) over a 130 kHz sweep width (9 ms acquisition time). A loop counter parameter 13 = 100 was incorporated such that the initial iteration of 20000 scans was repeated 90 times to give an accumulated number of 2000000 scans (FIDs from each iteration were automatically combined and Fourier transformed to produce the frequency domain spectrum). Dynamic Light Scattering (DLS). The average hydrodynamic diameters Dh and size distributions of the prepared micelle solution in an aqueous solution (1 mg mL−1) were measured using a Malvern ZetasizerNano ZS instrument equipped with a 4 mV He−Ne laser operating at λ = 632 nm, an avalanche photodiode detector with high 1015


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Figure 1. 1H NMR of monomers synthesized including 1,1-di-tert-butyl 3-(2-(methacryloyloxy)ethyl)butane-1,1,3-tricarboxylate (MAETC), 1,1-ditert-butyl 3-(4-((2,3-dimethylbut-2-enoyl)oxy)butyl)butane-1,1,3-tricarboxylate (MABTC), 1,1-di-tert-butyl 3-(6-((2,3-dimethylbut-2-enoyl)oxy)hexyl)butane-1,1,3-tricarboxylate (MAHTC).

Table 1. Summary of Block Copolymers Used for Platinum Drug Conjugationa code

copolymers

Mntheo (g mol−1)

MnGPC (g mol−1)

PDI

No. of RU, NOEGMEMA

No. of RU, NMAETC

BP-E80 BP-B82 BP-H79

POEGMEMA50-b-PMAETC80 POEGMEMA50-b-PMABTC82 POEGMEMA50-b-PMAHTC79

48399 51523 52409

29800 28900 31200

1.12 1.12 1.13

50 50 50

80 82 79

a

MnGPC determined by DMAc SEC using PSt calibration; MnNMR calculated by the following equation using the NMR signal intensities I: MnNMR = [(ICH,3.0−3.5 ppm) − (ICH,3.0−3.5 ppm)t=0/((ICH,3.0−3.5 ppm) − (ICH,3.0−3.5 ppm)t=0) + (ICHCH2,5.5−6.5 ppm/2)] × [MAETC]/[MacroRAFT] × MWMAETC + MWMacroRAFT, MWMAETC correspond to molar mass of MAETC, MWMacroRAFT correspond to molar mass of homopolymer of POEGMEMA.

Synthesis of Block Copolymers and Conjugation to Platinum Drugs. The monomer OEGMEMA was chosen as the building block of the hydrophilic micelle shell. The oligoethylene oxide side chain can provide high biocompatibility without causing protein crystallization, which has been reported for long-chain poly(ethylene oxide) micelle coronas.67,68 POEGMEMA macroRAFT agent was prepared in acetonitrile at 70 °C in the presence of 4-cyanopentanoic acid dithiobenzoate (CPADB) as RAFT agent to target the molecular weight of 15000 g mol−1. The conversion of the monomer was determined using 1H NMR 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; Supporting Information, Figure S5). POEGMEMA macroRAFT agent used for the block copolymerization with the novel synthesized monomers was obtained after a monomer conversion of 50% (Mn,theo = 15279 g mol−1, Mn,sec = 11100 g mol−1, PDI of 1.06). Subsequent chain extension of POEGMEMA macroRAFT agent with the selection of monomers was carried out in 1,4dioxane after prior testing of the solvent for peroxides, which can potentially harm the RAFT end group (Scheme 1). The consumption of all monomers during the block copolymer synthesis follows pseudo-first-order kinetics (Supporting Information, Figure S7). The resulting block copolymers

displayed all narrow molecular weight distributions (8 lead to sufficient deprotonation to 1018


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Table 5. Release Constant τ (Hours) Obtained by Fitting % CDDP Release = A exp(−(t/τ)) + y0 Release Data Displayed in Figure 5 and IC50 Values of the Polymer Only and the PtConjugated Polymer against A549 Expressed in Platinum Concentration and Polymer Concentration

samples BP-E80 at pH 7.4 BP-B82 at pH 7.4 BP-H79 at pH 7.4 CDDP carboplatin

τ (hours)

R2

IC50 (polymer; μg mL−1)

44.13 49.42 51.07

0.989 0.998 0.993

a a a

IC50 (Ptpolymer) c[Pt] (μM)

IC50 (Ptpolymer) c[polymer] (μg mL−1)

58.8 44.1 10.3

54.8 39.9 10.0

Figure 7 depicts the dose-dependent inhibition of cell proliferation of all cancer cell lines studied. The 50% growthinhibitory effect (IC50) of the different micelles, cisplatin, and carboplatin were determined from Figure 7 and are summarized in Table 6. The IC50 values (in μM) range from 10−52 μM (A549), 13−28 μM (OVCAR3), and 28−87 μM (SKOV3) revealing different chemosensitivities of different tumor cell lines. The IC50 value of BP-H79/Pt micelles appeared to exhibit the best cytotoxic activity (the lowest IC50 values), followed by BP-B82/Pt and BP-E80/Pt. The performance of BP-H79/Pt is in fact truly outstanding with IC50 values well below that of the parent drug carboplatin, reaching values close to cisplatin. This is even more astonishing considering that after 72 h only 50− 60% of platinum complex has been released, although the drug release experiments may not be fully comparable to the reactions that actually take place in the cell. These results raise the question why BP-H79/Pt has such a unique position compared to BP-B82/Pt and BP-E80/Pt. This can be partly attributed to a slightly higher platinum drug release within the first 72 h, but it also needs to be considered that the most distinguishing feature of these three types of micelles are their stability, which is expressed by their CMC values. More insight can be gained when discussing the cellular uptake of these micelles in terms of micelle stability. Cellular Uptake. To investigate the interaction between the cells and the micelles, the micelles were labeled with green fluorescence marker. After 6 h incubation, the cells were washed with cold PBS. The washing process was duplicated three times to completely remove residual fluorescence micelles. The green fluorescence of the cells is therefore equivalent to the amount of micelles taken up by the cells. The micelles have been taken up by all studied cancer cells (A549, OVCAR3, and SKOV3), probably only via endocytosis taking their size into account, and could then be found enriched in the cytosol (Figure 8), where cisplatin can be released from the carrier and penetrate the nucleus to bind to the cell DNA. The micrographs presented in Figure 8 only confirm that the carrier has been taken up, but they are no proof for the simultaneous entry of the drug, which could have been released in the cell growth media. ICP-MS and fluorescence spectroscopy were employed to quantitatively determine the amount of platinum drug and drug carrier in the cell. Table 7 lists the amount of Pt, measured using ICP-MS, which has been found in a precise number of cells. Because the Pt content of each type of micelle is known in Table 3, the theoretical mass of polymer that enters together with the drug can be calculated. The actual mass of polymer can meanwhile

7.2 32.4

a

Cannot be determined from Figure 5, but IC50 values are estimated from 600−800 μg mL−1

generate a fully water-soluble polymer. The difference though is the release experiments have been carried out in the presence of NaCl (0.155 M). The presence of NaCl has a destabilizing effect on these micelles, thus, they start disassembling after Pt has been released. Cytotoxicity of Polymers. The biocompatibility of copolymers including BP-E80, BP-B82, and BP-H79 was examined prior to platinum conjugation regarding the viability of different cancer cell lines such as lung cancer cell line (A549), ovarian cancers (OVCAR 3 and SKOV3). All samples were filtered through 0.22 μm filter, followed by placing them under UV light for 20 min to sterilize before incubating with cancer cell lines. The sulforhodamine B (SRB) assay was employed to determine the cytotoxicity of the polymers after an incubation time of 72 h. Although very high polymer concentrations appeared to be toxic, these concentrations were well above the amounts typically used for drug delivery systems (Figure 6). In Vitro Activity Carboplatin vs Polymeric Platinum Micelles. To understand the possible relationship between various polymeric micelles containing platinum drugs and their cytotoxic activity, in vitro cytotoxicities of micelles BP-E80/Pt, BP-B82/Pt, and BP-H79/Pt against A549, OVCAR3, and SKOV3 were determined using the sulforhodamine-B (SRB) microculture colorimetric assay. These cytotoxicities were compared with their parent drug, carboplatin, which has a similar ligand structure observed from the same assay, and also with cisplatin, which is formed while cleaving the platinum complex from the polymer by ligand exchange.

Figure 6. Cell viability (in %) after exposing a solution containing the block copolymers BP-E80, BP-B82, and BP-H79 to (A) A549, (B) SKOV3, and (C) OVCAR3 for 72 h. 1019


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Table 6. IC50 Values of Platinum Polymeric Micelles and Carboplatin against Various Cancer Cell Linesa IC50/μM compounds

A549

OVCAR3

SKOV3

BP-E80/Pt BP-B82/Pt BP-H79/Pt carboplatin cisplatin

58.8 44.1 10.3 32.4 7.2

25.3 19 12.7 16.7

85.7 65.7 24.3 42.9

a

The concentration relates to the concentration of platinum drugs in each system.

Figure 8. Fluorescence microscopy images of BP-E80/Pt incubated with A549, OVCAR3, and SKOV3 for 2 h at 37 °C. The polymer concentration was 100 μg mL−1: (A) A549, (C) OVCAR3, and (E) SKOV3 (light microscopy, halogen lamp); (B) A549, (D) OVCAR3, and (F) SKOV3 with fluorescence light.

Figure 7. Cell viability (%) after being exposed to CDDP, carboplatin, and platinum polymeric micelles including BP-E80/Pt, BP-B82/Pt, BP-H79/Pt; (A) A549, (B) OVCAR3, and (C) SKOV3.

the cell membrane.51 The enhancement of stability using a more hydrophobic core-forming block not only promotes endocytosis, but increased cellular uptake can lead directly to a more efficient drug delivery carrier in terms of cytotoxicity resulting in lower IC50 values. Colony Formation. Clonogenic assay, an in vitro cell survival assay, has been employed as described by Franken et al.76 to investigate the cell proliferation capacity of the cancer cells after drug exposure. The difference between this assay and the SRB assay is that the SRB assay only estimates cell growth inhibition relative to control, whereas the clonogenic assay is perhaps more representative of the clinical situation, where

be obtained from the fluorescence intensity of the polymer. Both values seem to be in good agreement within errors, indicating that the drug enters accompanied by the polymer. The most noticeable result is the significant increase in cell uptake of BP-H79/Pt compared to the other micelles. This reinforces the earlier suggestion described in the introduction of this article that the stability of the micelle can play a major role in cellular uptake.51,52 Block copolymer micelles that are potentially unstable tend to dissociate and are not efficiently taken up, often being observed to disassemble and partly integrate with 1020


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Table 7. Cellular Uptake with A549 Lung Cancer Cell Lines, Pt Content was Determined Using ICP-MS and Polymer Content was Analyzed Using Fluorescence Spectrometer Pt content ngPt/106 cellsa

[polymer]theob ng polymer/106 cells

[polymer]c ng polymer/106 cells

polymer−drug conjugates

2h

15 h

2h

15 h

2h

15 h

BP-E80/Pt BP-B82/Pt BP-H79/Pt

21 ± 2.5 27 ± 3.0 65 ± 5.5

42 ± 3.0 44 ± 4.0 145 ± 4.0

99 126 323

202 205 723

97 ± 5.20 127 ± 6.94 300 ± 4.97

197 ± 7.12 200 ± 6.38 650 ± 7.22

a

Pt content determined by extracting 106 cells, followed by Pt content analysis via ICP-MS. bTheoretical polymer concentration were calculated based on the Pt content determined by ICP-MS and the loading efficiency in Table 3. cActual polymer concentrations were measured by fluorescence spectrometry of the labeled polymer.

Figure 9. Colony formation of A549 cells (A) without drug treatment, after 3 days of incubation with (B) BP-H79 micelles at 100 μg mL−1, (C) carboplatin at IC10 (8.5 μM), (D) carboplatin at IC90 (241 μM), (E) BP-H79/Pt micelles at IC10 (1.1 μM), and (F) BP-H79/Pt micelles at IC90 (65 μM). After treatment cells were regrown for 10 days in PRMI 1640 mediun and changed medium every 3 days.

regrowth of cancer is common after treatment with chemotherapy. Lung cancer cells (A549) were incubated 3 days with platinum-polymeric micelles, polymeric micelles, or carboplatin. Single cell suspensions were regrown in PRMI medium including 10% FBS for 10 days with medium changes every 3 days. Two concentrations, IC10 and IC90, of polymer−platinum conjugates or carboplatin, were employed in these experiments. IC10 and IC90 are the inhibitory concentrations required to inhibit the growth of 10 and 90% of cancer cells. These concentrations were chosen based on the cytotoxicity results and it is expected to result in the same amount of cells deaths for all samples after exposure of the polymer-platinum conjugates or carboplatin at these concentrations (IC10 or IC90). Figure 9A depicts the proliferative capacity of a nontreated A549 single cell after a 10 day incubation, which resulted in the formation of a large colony of A549 cells. While the unloaded polymeric micelles did not affect cell proliferation, significant loss of cell proliferative capacity was observed when A549 were exposed to platinum-polymeric micelles or carboplatin. Interestingly, platinum-polymeric micelles showed a better ability to restrict the cell regrowth compared to carboplatin (Table 8). This also needs to be seen under the light that more carboplatin is initially required to achieve the same percentage of cell death. The success of drug loaded micelles in contrast to admin-

Table 8. Plating Efficiency and Surviving Fraction of A549 after 3 Days Exposure to Polymer, Polymer−Pt Conjugates, or Carboplatin, Followed by a 10 Day Incubation in PRMI 1640 Medium Pt plating efficiency concentration/μM (PE)/%

samples BP-H79/Pt at IC90 BP-H79/Pt at IC10 carboplatin at IC90 carboplatin at IC10 BP-H79 micelles control

65 1.1 241 8.5

surviving fraction (SF) 0 0.066 0.112 0.225 0.993

61.4

istration of the free drug lies therefore not only in the possibility of being able to lower the amount of drug to achieve the same outcome, but also in its ability to prevent the long-term formation of colonies. This post-treatment effect could be explained by the efficient uptake of the micelle by the cell. The micelle remains lodged within the cell for an extended period of time, where the slow release of the drug from the micelles continues beyond the 3 days of drug incubation leading eventually to cell death. This post-treatment effect appears to be absent when carboplatin was used. 1021


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(4) Knox, R. J.; Friedlos, F.; Lydall, D. A.; Roberts, J. J. Cancer Res. 1986, 46 (4Part 2), 1972−1979. (5) Macquet, J. P.; Butour, J. L. J. Natl. Cancer I 1983, 70 (5), 899− 905. (6) Cleare, M. J.; Hoeschele, J. D. Bioinorg. Chem. 1973, 2 (3), 187− 210. (7) Deubel, D. V.; Lau, J. K. C. Chem.Eur. J. 2005, 11 (9), 2849− 2855. (8) Ho, Y. P.; Au-Yeung, S. C. F.; To, K. K. W. Med. Res. Rev. 2003, 23 (5), 633−655. (9) Neuse, E. W. S. Afr. J. Sci. 1999, 95 (11−12), 509−516. (10) Zwelling, L. A.; Kohn, K. W. Cancer Treat. Rep. 1979, 63 (9−10), 1439−44. (11) Zaludova, R.; Zakovska, A.; Kasparkova, J.; Balcarova, Z.; Kleinwachter, V.; Vrana, O.; Farrell, N.; Brabec, V. Eur. J. Biochem. 1997, 246 (2), 508−17. (12) Lippert, B. Coord. Chem. Rev. 1999, 182 (1), 263−295. (13) Reedijk, J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (7), 3611− 3616. (14) Boulikas, T.; Vougiouka, M. Oncol. Rep. 2003, 10 (6), 1663− 1682. (15) Reedijk, J. Eur. J. Inorg. Chem. 2009, 10, 1303−1312. (16) Godwin, A. K.; Alton, M.; O’Dwyer, P. J.; Huang, C. S.; Hamilton, T. C.; Anderson, M. E. Proc. Natl. Acad. Sci. U.S.A. 1992, 89 (7), 3070−3074. (17) Cleare, M. J.; Hydes, P. C.; Malerbi, B. W.; Watkins, D. M. Biochimie 1978, 60 (9), 835−850. (18) Dabrowiak, J. C.; Bradner, W. T. Prog. Med. Chem. 1987, 24, 129−58. (19) Kelland, L. R. Crit. Rev. Oncol./Hematol. 1993, 15 (3), 191−219. (20) Calvert, A. H.; Harland, S. J.; Newell, D. R.; Siddik, Z. H.; Jones, A. C.; McElwain, T. J.; Raju, S.; Wiltshaw, E.; Smith, I. E.; Baker, J. M.; Peckham, M. J.; Harrap, K. R. Cancer Chemother. Pharmacol. 1982, 9 (3), 140−147. (21) Bitha, P.; Carvajal, S. G.; Citarella, R. V.; Child, R. G.; Delossantos, E. F.; Dunne, T. S.; Durr, F. E.; Hlavka, J. J.; Lang, S. A.; Lindsay, H. L.; Morton, G. O.; Thomas, J. P.; Wallace, R. E.; Lin, Y. I.; Haltiwanger, R. C.; Pierpont, C. G. J. Med. Chem. 1989, 32 (8), 2015− 2020. (22) Weiss, R. B.; Christian, M. C. Drugs 1993, 46 (3), 360−377. (23) Evans, B. D.; Raju, K. S.; Calvert, A. H.; Harland, S. J.; Wiltshaw, E. Cancer Treat. Rep. 1983, 67 (11), 997−1000. (24) Connors, T. A.; Jones, M.; Ross, W. C. J.; Braddock, P. D.; Khokhar, A. R.; Tobe, M. L. Chem.-Biol. Interact. 1972, 5 (6), 415− 424. (25) Rose, W. C.; Crosswell, A. R.; Schurig, J. E.; Casazza, A. M. Cancer Chemother. Pharmacol. 1993, 32 (3), 197−203. (26) 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 (11), 2581−2586. (27) Farrell, N.; Appleton, T. G.; Qu, Y.; Roberts, J. D.; Fontes, A. P. S; Skov, K. A.; Wu, P.; Zou, Y. Biochemistry 1995, 34 (47), 15480−15486. (28) Farrell, N. Cancer Invest. 1993, 11 (5), 578−589. (29) Farrell, N.; Qu, Y.; Hacker, M. P. J. Med. Chem. 1990, 33 (8), 2179−2184. (30) Cubo, L.; Hambley, T. W.; Miguel, P. J. S.; Carnero, A.; Navarro-Ranninger, C.; Quiroga, A. G. Dalton Trans. 2011, 40 (2), 344−347. (31) Daly, H. L.; Hall, M. D.; Failes, T. W.; Zhang, M.; Foran, G. J.; Hambley, T. W. Aust. J. Chem. 2011, 64 (3), 273−278. (32) Hall, M. D.; Mellor, H. R.; Callaghan, R.; Hambley, T. W. J. Med. Chem. 2007, 50 (15), 3403−3411. (33) Huynh, V. T.; Chen, G.; Souza, P. d.; Stenzel, M. H. Biomacromolecules 2011, 12 (5), 1738−1751. (34) Sood, P.; Thurmond, K. B.; Jacob, J. E.; Waller, L. K.; Silva, G. O.; Stewart, D. R.; Nowotnik, D. P. Bioconjugate Chem. 2006, 17 (5), 1270−1279.

CONCLUSIONS Three different block copolymers with malonate ligands were prepared as macromolecular ligands for platinum drugs leading to structures with strong resemblance to carboplatin, a FDA approved drug. The targeted monomers with the same neighboring carboxylate functional groups but containing varying spacer lengths with two, four, and six methylene groups between ligand and vinyl functionality were obtained via carbon Michael addition. Upon Pt conjugation, the block copolymers took on amphiphilic properties resulting in the formation of micelles. The micelles differentiate from each other by the spacer size between the ligating malonate group and the polymer backbone. The increasing spacer distance led to lower hydrophilicity and consequently to an increase in micelle stability evidenced by the decreasing CMC. The micelle stability was found to be the key to a successful drug delivery system. The uptake of the drug carrier by the tumor cell lines was significantly enhanced with increasing stability, which then, in turn, leads to a substantial increase in activity. The activity of the platinum drug loaded into the most stable drug carrier was improved compared to the parent drug carboplatin and almost as efficient as cisplatin, the pure active species. The drug carrier results in higher cell death after a set period of time, and also a prolonged effect even after the tissue culture medium was removed, suggesting that the trapped micelles act slowly within the tumor cells for many more days, preventing clonogenic growth of the tumor cells.

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ASSOCIATED CONTENT

* Supporting Information S 13

C, DEPT-135, and DEPT-90 NMR spectrum of all purified monomers, DSC curve of MAETC, 1H NMR spectrum of POEGMEMA before purification, HMQC of diblock copolymer before and after deprotection, molecular weight distribution obtained from SEC of all block copolymers, first-order kinetic plot of homo- and block copolymerization, 195Pt NMR spectra, polymer concentrations used for cell work. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.S. thanks the ARC (Australian Research Council) for funding in form of a Future Fellowship (FT0991273) and a Discovery Project (DP1092694). V.T.H. thanks the Australian government for Endeavour Postgraduate Award. The authors would like to thank the Centre for Advanced Macromolecular Design (CAMD) and UNSW Analytical Centre for support. The authors like to thank especially Drs. Jim Hook and Don Thomas from the NMR department for their help.

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REFERENCES

(1) Rosenberg, B.; Van Camp, L.; Krigas, T. Nature 1965, 205 (4972), 698−699. (2) Rosenber., B; Vancamp, L.; Trosko, J. E.; Mansour, V. H. Nature 1969, 222 (5191), 385. (3) Kelland, L. Nat. Rev. Cancer 2007, 7 (8), 573−584. 1022


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(35) Rice, J. R.; Gerberich, J. L.; Nowotnik, D. P.; Howell, S. B. Clin. Cancer Res. 2006, 12 (7), 2248−2254. (36) Haxton, K. J.; Burt, H. M. J. Pharm. Sci. 2009, 98 (7), 2299− 2316. (37) Komane, L.; Mukaya, E.; Neuse, E.; van Rensburg, C. J. Inorg. Organomet. Polym. Mater. 2008, 18 (1), 111−123. (38) Nishiyama, N.; Kataoka, K. J. Controlled Release 2001, 74 (1−3), 83−94. (39) Mizumura, Y.; Matsumura, Y.; Hamaguchi, T.; Nishiyama, N.; Kataoka, K.; Kawaguchi, T.; Hrushesky, W. J. M.; Moriyasu, F.; Kakizoe, T. Jpn. J. Cancer Res. 2001, 92 (3), 328−336. (40) Nishiyama, N.; Okazaki, S.; Cabral, H.; Miyamoto, M.; Kato, Y.; Sugiyama, Y.; Nishio, K.; Matsumura, Y.; Kataoka, K. Cancer Res. 2003, 63 (24), 8977−8983. (41) Bontha, S.; Kabanov, A. V.; Bronich, T. K. J. Controlled Release 2006, 114 (2), 163−174. (42) Duong, H. T. T.; Huynh, V. T.; de Souza, P.; Stenzel, M. H. Biomacromolecules 2010, 11 (9), 2290−2299. (43) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. Adv. Drug Delivery Rev. 2002, 54 (5), 759−779. (44) Stenzel, M. H. Chem. Commun. (Cambridge, U.K.) 2008, 30, 3486−3503. (45) Nishiyama, N.; Kataoka, K. Pharmacol. Ther. 2006, 112 (3), 630−648. (46) Kwon, G. S. Crit. Rev. Ther. Drug Carrier Syst. 2003, 20 (5), 357−403. (47) Leroux, J. C.; Gaucher, G.; Dufresne, M. H.; Sant, V. P.; Kang, N.; Maysinger, D. J. Controlled Release 2005, 109 (1−3), 169−188. (48) Hamley, I. W. Block Copolymers in Solution: Fundamentals and Applications; John Wiley & Sons, Ltd: Chichester, England, 2005; p 1− 300. (49) Rapoport, N.; Marin, A.; Luo, Y.; Prestwich, G. D.; Muniruzzaman, M. J. Pharm. Sci. 2002, 91 (1), 157−170. (50) Muniruzzaman, M.; Marin, A.; Luo, Y.; Prestwich, G. D.; Pitt, W. G.; Husseini, G.; Rapoport, N. Y. Colloids Surf., B 2002, 25 (3), 233−241. (51) Zastre, J.; Jackson, J.; Bajwa, M.; Liggins, R.; Iqbal, F.; Burt, H. Eur. J. Pharm. Biopharm. 2002, 54 (3), 299−309. (52) Barz, M.; Luxenhofer, R.; Zentel, R.; Kabanov, A. V. Biomaterials 2009, 30 (29), 5682−5690. (53) van Nostrum, C. F. Soft Matter 2011, 7 (7), 3246−3259. (54) O’Reilly, R. K.; Hawker, C. J.; Wooley, K. L. Chem. Soc. Rev. 2006, 35 (11), 1068−1083. (55) Lavasanifar, A.; Samuel, J.; Kwon, G. S. Adv. Drug Delivery Rev. 2002, 54 (2), 169−190. (56) Shi, W.; Peterson, D. S.; Palmer, C. P. J. Chromatogr., A 2001, 924 (1−2), 123−135. (57) Akbay, C.; Shamsi, S. A. Electrophoresis 2004, 25 (4−5), 635− 644. (58) Mitsukami, Y.; Donovan, M. S.; Lowe, A. B.; McCormick, C. L. Macromolecules 2001, 34 (7), 2248−2256. (59) Oae, S.; Okabe, T.; Yagihara, T. Tetrahedron 1972, 28 (12), 3203. (60) Huynh, V. T.; de Souza, P.; Stenzel, M. H. Macromolecules 2011, 44 (20), 7888−7900. (61) Ye, H.; Jin, L.; Hu, R.; Yi, Z.; Li, J.; Wu, Y.; Xi, X.; Wu, Z. Biomaterials 2006, 27 (35), 5958−5965. (62) Haxton, K. J.; Burt, H. M. Dalton Trans. 2008, 43, 5872−5875. (63) 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. (64) Xing, R.; Wang, X.; Zhang, C.; Zhang, Y.; Wang, Q.; Yang, Z.; Guo, Z. J. Inorg. Biochem. 2009, 103 (7), 1039−1044. (65) Liebmann, J. E.; Cook, J. A.; Lipschultz, C.; Teague, D.; Fisher, J.; Mitchell, J. B. Br. J. Cancer 1993, 68 (6), 1104−1109. (66) Gu, W.; Gaborieau, M.; Huynh, V. T.; deSouza, P. L.; Stenzel, M. H. Polymer 2011, 52, 5993−6002. (67) Boyer, C.; Stenzel, M. H.; Davis, T. P. J. Polym. Sci., Part A: Polym. Chem. 2011, 49 (3), 551−595.

(68) Gregory, A.; Stenzel, M. H. Prog. Polym. Sci. 2012, 37 (1), 38− 105. (69) Cameron, N. S.; Corbierre, M. K.; Eisenberg, A. Can. J. Chem. 1999, 77 (8), 1311−1326. (70) Berners-Price, S. J.; Ronconi, L.; Sadler, P. J. Prog. Nucl. Magn. Reson. Spectrosc. 2006, 49 (1), 65−98. (71) Acharya, K. R.; Bhattacharyya, S. C.; Moulik, S. P. J. Photochem. Photobiol., A 1999, 122 (1), 47−52. (72) Blume, A.; Tuchtenhagen, J.; Paula, S. Application of titration calorimetry to study binding of ions, detergents, and polypeptides to lipid bilayers. In Trends in Colloid and Interface Science VII; Laggner, P., Glatter, O., Eds.; Springer: Berlin/Heidelberg, 1993; Vol. 93, pp 118− 122. (73) Prasad, M.; Donald, A. M.; Palepu, R.; Moulik, S. P. J. Surf. Sci. Technol. 2003, 19 (3−4), 125−138. (74) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99 (7), 2039−2044. (75) Cai, S.; Vijayan, K.; Cheng, D.; Lima, E.; Discher, D. Pharm. Res. 2007, 24 (11), 2099−2109. (76) Franken, N. A. P.; Rodermond, H. M.; Stap, J.; Haveman, J.; van Bree, C. Nat. Protoc. 2006, 1 (5), 2315−2319.

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NOTE ADDED AFTER ASAP PUBLICATION This article posted ASAP on February 29, 2012. Scheme 2 and Figure 2 have been revised. The correct version posted on March 5, 2012.

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