Folate Conjugation to Polymeric Micelles via Boronic Acid Ester to

Mar 7, 2013 - The microscope is equipped with a mercury lamp, Bertrand lens, and side port (80/100%) and 12 V/100 W transmitted, light illumination, ...
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Folate Conjugation to Polymeric Micelles via Boronic Acid Ester to Deliver Platinum Drugs to Ovarian Cancer Cell Lines Wei Scarano,† Hien T. T. Duong,† Hongxu Lu,† Paul L. De Souza,‡ and Martina H. Stenzel*,† †

Centre for Advanced Macromolecular Design (CAMD), School of Chemical Engineering, 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: In this study, a novel technique was used for the reversible attachment of folic acid on the surface of polymeric micelles for a tumor-specific drug delivery system. The reversible conjugation is based on the interaction between phenylboronic acid (PBA) and dopamine to form a borate ester. The conjugation is fast and efficient and in vitro experiments via confocal fluorescent microscopy show that the linker is stable in for several hours. Reversible addition−fragmentation chain transfer (RAFT) polymerization was used to synthesize two various sized water-soluble block copolymer of oligoethylene glycol methylether methacylate and methyl acrylic acid (POEGMEMA35-b-PMAA200 and POEGMEMA26-b-PMAA90). The platinum drug, oxoplatin, was then subsequently attached to the polymer via ester formation leading to platinum loading of 12 wt % as determined by TGA. The platinum-induced amphiphilic block copolymers that consequently led to the formation of micelles of sizes 150 and 20 nm in an aqueous environment with the longer PMAA block forming larger micelles. The small micelles were in addition cross-linked using 1,8-diaminooctane to further stabilize their structure. The targeting ability of folate conjugated polymeric micelles was investigated against two types of tumor cell lines: A549 (-FR) and OVCAR-3 (+FR). The cell line growth inhibitory efficacy of material synthesized was evaluated by using SRB method. The results revealed that folate conjugated micelles showed higher activity in FR + OVCAR-3 cells but not in FR − A549 cells. Similar results were obtained for both small and large micelles without the conjugation of folate. Comparing large and small micelles it can be observed that larger micelles are more efficient, which has been attributed to the lower stability of the smaller micelles. Micelle stabilization via cross-linking could indeed increase the toxicity of the drug carrier.

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INTRODUCTION Platinum drugs have been the mainstay of chemotherapy regimens for many types of cancer including testicular, head, neck, and ovarian cancers over the past few decades.1−4 cisDiaminedichloroplatinum(II) (cisplatin, cis-PtCl2(NH3)2), the original platinum drug, and its second-generation analogues and derivatives, such as oxaliplatin and carboplatin, have wellestablished roles.5 Although they are relatively potent and clinically active, a number of side effects can limit their utility, including nephrotoxicity, cumulative neurotoxicity, ototoxicity (loss of hearing), and extreme emetogenic (vomiting) potential.6 Another issue for many chemotherapy drugs is drug-resistance.7 Due to these limitations, the search for improved analogues and platinum-based drug delivery has been underway for some time.8,9 Among a number of clinically screened platinum complexes, octahedral complexes with platinum(IV) centers including oxoplatin, satraplatin, tetraplatin, iproplatin, and ormaplatin appear to be potent anticancer agents particularly against tumors that are resistant to cisplatin.10−12 Platinum(IV) drugs are prodrugs because they © 2013 American Chemical Society

are believed to be reduced intra- or extracellularly by biological reductants such as glutathione, ascorbic acid, and cysteine to the more reactive platinum(II) complexes.12 The reduction rate, however was found to be dependent on the nature of axial and carrier ligands as reported by Choi and co-workers.13−15 The reduction rate and mechanism of platinum(IV) complexes has been reviewed and discussed in detail in the literature.16,17 Even though the class of octahedral platinum(IV) complexes has been shown to have anticancer activity, the issue of improved targeted drug delivery still remains. One of the more promising strategies is to temporarily attach drugs to a nanocarrier. Platinum(IV) drugs have recently been conjugated to single-wall carbon nanotubes.18,19 Polymeric micelles are self-assembled block copolymers with a core−shell structure and with diameters of approximately 10−100 nm. They have been proven to be promising vehicles for the targeted delivery Received: September 23, 2012 Revised: March 6, 2013 Published: March 7, 2013 962

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Scheme 1. Synthesis Approach to Platinum(IV) Complex Containing P(OEGMEMA)-b-P(MAA) Block Copolymers Using Dopamine Terminated Dithiobenzoate RAFT Agent, Followed by Attachment of cis,cis,trans-Diammine-dichloridodihydroxido-platinum(IV) Complex (Oxoplatin) and Self-Assembly into Micelles, Which Were Crosslinked Using 1,8Diaminooctane for Structural Stability

delivery of metal-based drugs such as platinum25−30 and gold drugs.31 Although polymeric micelles as drug delivery vehicles have passive tumor-targeting abilities, the therapeutic index of these platinum complexes can be further improved by using an active targeting approach. Active targeting can be achieved by functionalizing the surface of the micelles with targeting ligands such as small molecules, antibodies, and peptides. It is wellknown that folate-binding proteins (FBP) are selectively overexpressed on the surface of cancer cells. This could be targeted by designing an appropriate drug delivery system incorporating folate that could guide carriers directly to the cancer cells, where the drug carrier system is then internalized

of drugs, proteins, genes, and imaging agents. Because the molecular weight of polymeric micelles may reach more than 106 g mol−1, they surpass the typical renal threshold (ca. 40 kDa).20,21 In addition, their nanoscopic size is less than 200 nm, rendering them less recognizable by the phagocytic cells of the reticuloendothelial system (RES), hence, prolonged blood circulation.22 This “stealth” property of micelles, in combination with the passive accumulation in the solid tumor through the enhanced permeability and retention (EPR) mechanism23,24 make these core−shell particles the potential choice for drug delivery purposes. Although more commonly used for hydrophobic drugs, polymer micelles can be employed for the 963

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via receptor-mediated endocytosis.32 A major advantage of targeting folate receptors (FR) is that they are inaccessible to FR-directed therapeutics in the blood plasma prior to malignant transformation. Only after the transformation do they become generally accessible to intravenous drugs bearing folate moieties due to selective expression on the apical membrane surface.33 FR targeting moieties can be attached in different ways but it is advantageous to utilize folate-nanoparticle conjugates instead of drugs directly conjugated to folate as it has the potential to deliver large quantities of drug via the receptor mediated endocytosis pathway.34 Most folate targeting moieties are permanently attached to the drug delivery system.34−38 This requires the synthesis of a folate-containing initiator or chain transfer agent or the postmodification of the polymer. Possibly more versatile would be the postmodification of the final drug delivery system using boronic acid esters. Boronic acids bind to alcohols with high affinity via reversible boronate ester formation. It is possibly one of the strongest single-pair reversible functional group interactions in an aqueous environment39 and it has already been utilized for various biomedical applications,40−44 reviewed recently by Cambre and Sumerlin.39 The reaction consists of two steps. First, one of the two donor atoms of the bidentate ligand binds to the trigonal boronic acid to form a tetrahedral intermediate and the second step involves the chelating ring closure of the intermediate to form the 1:1 complex.45 The conjugation of folates to the drug carrier via boronic acid esters has the advantage of being a quick procedure that allows the attachment of various amounts of folates to a drug carrier within a short period of time. It should also be noted here that boronic acids esters are reversible. The equilibrium is shifted to the free acid at lower pH value, which means that the conjugation is potentially reversible. It is though currently unknown if this may be of advantage. The determination of pKa and binding constant of various boronic acids are carried out by using competitive assay containing the catechol dye Alizarin Red S (ARS).46,47 The fluorescence of ARS increases significantly upon binding with boronic acids; therefore, by monitoring the change in fluorescence intensity as the boronic acid concentration increases, the binding constant (Ka) can be calculated using Benesi−Hildebrand method.48 Most studies have revolved around phenyl boronic acid (PBA) and its related species. Introducing electron withdrawing groups onto the benzene ring lowers the pKa value of the phenyl boronic acid which subsequently favors the formation of the complex.48 On the other hand, electron donating groups increase the pKa value, however, the effect of electron donating groups is negligible compared with the electron withdrawing groups. In this paper, we describe a method to prepare micelles capable of actively targeting ovarian cancer cells by decorating the surface of self-assembled polymeric micelles with folate. The aim of this project is to compare the therapeutic efficacy of polymeric micelles for the delivery of platinum(IV) anticancer compounds in correlation to the size of the micelle and the amount of folate conjugated to the surface, which is attached via a reversible boronic acid linker. A polymeric micelle system comprised of block copolymers with POEGMEMA as micelle corona and PMAA as core is used to covalently incorporate cis,cis,trans-diammine-dichlorido-dihydroxido-platinum(IV) complex (oxoplatin; Scheme 1). POEGMEMA block was chosen because of its similar low-fouling properties to PEO.

Poly(methacrylic acid) (PMAA) is an attractive polymer because of its unique responsiveness to pH and ionic strength. Both blocks of the copolymers are hydrophilic; however, with the conjugation of oxoplatin to the polymer backbone, the block copolymer takes on an amphiphilic character. Subsequent to the formation of micelles, boronic acid linker is added to finally achieve the targeting drug delivery system.

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EXPERIMENTAL SECTION

Materials. Toluene (Aldrich; purum), N,N-dimethyl acetamide (DMAc; Aldrich, 99.9%), dimethyl sulfoxide (DMSO; Ajax, 98.9%), N,N-dimethylformamide (DMF; Aldrich), diethyl ether (Et2O; Ajax Fine Chem, 99%), potassium carbonate (Univar, anhydrous), petroleum spirit (BR 40−60 °C; Ajax Finechem, 90%), ethyl acetate (EtOAc; Ajax Finechem, 99.5%), methanol, tetrahydrofuran (THF), dichloromethane (DCM; Ajax Finechem, 99%), alizarin Red (ARS, Aldrich, 98%), cis-diammineplatinum(II) dichloride (CDDP; Sigma Aldrich, 99.9%), dopamine hydrochloride (Fluka, 95.8%), phenyl boronic acid (PBA; Aldrich, 95%), 3-amino phenyl boronic acid hydrochloride (3-APBA; Aldrich, 98%), 3-carboxy phenyl boronic acid (3-CPBA; Aldrich 99%), 9-amino acridine hydrochloride monohydrate (Aldrich, 98%), 1,8-diazabicycloundec-7-ene (DBU, Aldrich 98%), folic acid (FA; Aldrich, 97%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC; Aldrich, 98%), fluorescein Omethacrylate (Aldrich, 97%) were used without any further purification. Oligo(ethylene glycol) methyl ether methacrylate (OEGMEMA, Mn = 300 g mol−1; Aldrich) were destabilized by passing them over a column of basic alumina and stored at −7 °C. 2,2Azobisisobutyronitrile (AIBN; Fluka, 98%) was purified by recrystallization from methanol. Deionized (DI) water produced by Milli-Q water purification system and has a resistivity of 17.9 mΩ/cm. Fluorescence Binding Studies. The experimental setup for fluorescence binding studies has been described in the literature.49 For ARS-boronic acid fluorescent measurement, a stock solution of ARS (9.0 × 10−5 M) in 0.10 M sodium phosphate mono basic buffer made fresh (within the last 7 days) and stored in the refrigerator, was diluted 10-fold with 0.10 M sodium phosphate monobasic buffer to give final concentration of 9.0 × 10−6 M ARS in 0.10 M sodium phosphate buffer at pH of 7.4 (solution A). PBA was added to a portion of solution A to make a 9.0 × 10−6 M ARS, 2.0 × 10−3 M PBA solution in sodium phosphate buffer (solution B). The pH was checked again and corrected if necessary. Solution B was titrated into solution A in order to make mixtures with a constant concentration of ARS and a range of concentrations of PBA. A total of 12 different concentrations were made in order to cover as much of binding curve as possible which gives an increasing concentration of ARS-PBA complex at 0.25, 0.75, 1, 2.5, 5, 7.5, 10, 30, 50, 70, 100, and 222 equiv of PBA. Each mixture was allowed to stand for 5 min before any measurements were taken. The intensity of the excitation was recorded at 468 nm and emission at 572 nm. The experiments were carried out in triplicates. Competitive Binding Assay. A solution of 9.0 × 10−6 M ARS and 2.0 × 10−3 M of PBA in 0.10 M sodium phosphate buffer was prepared and pH adjusted (solution B). Diol of choice (dopamine/3,4dihydroxy benzoic acid) was dissolved in a portion of solution B, which gives a final concentration of 0.20 × 10−3 M diol, 9.0 × 10−6 M ARS, and 2.0 × 10−3 M of PBA in 0.10 M sodium phosphate buffer at pH of 7.4 (solution C). Various volume of solution C was added to solution B to make 13 mixtures of a constant concentration of ARS and PBA and a range of concentrations of diols at 0.25, 0.75, 1, 2.5, 5, 7.5, 10, 30, 50, 70, 100, 200, and 222 equiv of PBA. Synthesis. Synthesis of Boronate Folic Acid. Folic acid (1 g, 2.267 mmol) was dissolved in DMF (5 mL) and the solution was stirred for 20 min followed by the addition of NHS (260 mg, 2.260 mmol) and EDC (440 mg, 2.838 mmol) which was left to stir for further 1 h in ice bath. A solution of 3-APBA (411 mg, 2.375 mmol) deprotected by TEA (330 uL, 2.27 mmol) in DMF (2 mL) was then added to the solution of folic acid and was left to stir for 1 h in an ice bath and 24 h at rt. The solution was then added dropwise into deionized water (50 964

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mL) to form a yellow precipitate. The precipitate was collected by filtration and then washed five times with acetone (20 mL). The yellow precipitate was left to dry in a vacuum oven at rt. Yield: 952 mg (68%). 1 H NMR (300 MHz, DMSO-d6) δ (ppm): 10.01 (s, 1H, H-1), 8.65 (s, 1H, H-3), 8.21 (s, 1H, H-7), 7.92 (d, 1H, H-13), 7.80 (d, 1H, H14), 7.71 (d, 1H, H-6), 7.61 (d, 1H, H-11), 7.50 (d, 1H, H-12), 7.22 (br, 1H, H-10), 6.85 (br, 1H, H-15), 6.63 (d, 1H, H-5), 4.49 (d, 2H, H-4), 2.37 (m, 2H, H-8), 2.16 (m, 2H, H-9). (Peak assignment can be found in the Supporting Information, Figure S1.) ESI-MS: Calcd, 560.19 g mol−1; measured, 560.20 g mol−1. Synthesis of Acridine Phenylboronic Acid. In solution 1,3carboxyphenylboronic acid (0.1 g, 0.6 mmol) and DBU (100 mg, 0.65 mmol) was dissolved in DMSO (2 mL). In another glass vial, 9amino acridine hydrochloride monohydrate (165 mg, 0.66 mmol) and TEA (0.1 mL) were dissolved in DMSO (1.5 mL) and left to stir for 1 h at room temperature before adding to solution. The reaction mixture was left to stir for 48 h at 40 °C before precipitated into DI water and centrifuged at 7000 rpm for 5 min. The supernatant was removed by decanting and the remaining solvent was removed by freeze-drying to obtain a yellow powder. The product was then purified via gel column chromatography using 7:1 dichloromethane/methanol as eluent. The third fraction was collected and the solvent removed under reduced pressure to obtain a yellow powder. Yield: 180 mg (53%). 1 H NMR (300 MHz, DMSO-d6) δ (ppm): 8.80 (dd, 2H, H-1), 8.44 (s, 1H, H-2), 8.23 (br, 2H, H-3), 8.04−8.01 (t, 4H, H-4), 7.59 (dd, 2H, H5), 7.48 (t, 1H, H6). Synthesis of Dopamine-Terminated RAFT Agent (CPADBD). The RAFT agent 4-cyanopentanoic acid dithiobenzoate (CPADB) was synthesized according to literature.50 Pentafluorophenol-activated RAFT agent was synthesized according to the literature.51 To a 100 mL round-bottom flask was added CPADB (0.2 g, 0.72 mmol) and DCC (0.18 g, 0.86 mmol). The solids were then dissolved in anhydrous DCM (30 mL). The reaction mixture was cooled to 0 °C in an ice bath and pentafluorophenol (0.145 g, 0.78 mmol) was added to the flask. In another vial, DMAP (20 mg, 0.164 mmol) was dissolved in anhydrous DCM (10 mL) and was then added dropwise into the round-bottom flask with fast stirring. The reaction was left to stir in an ice bath for 2 h and then left to stir under room temperature overnight. The solid was then filtered off and the solvent was removed under reduced pressure on a rotary evaporator. The crude product was purified via gel column chromatography using chloroform as eluent. The first fraction was collected and the solvent removed under reduced pressure to obtain a pink/red oil, which was placed in the freezer for crystallization. Yield: 243 mg (76%). 1 H NMR (300 MHz, CDCl3) δ (ppm): 7.80 (d, 2H, H-1), 7.50 (t, 1H, H-3), 7.30 (d, 2H, H-2), 2.31 (m, 2H, H-5), 2.70 (m, 2H, H-6), 1.85 (s, 3H, H-4). 19 F-NMR (300 MHz, CDCl3) δ (ppm): −152.5 (t, 2F, F-1), −157.3 (t, 2F, F-2), −161.8 (t, 2F, F-3). (Peak assignment can be found in the Supporting Information, Figure S2.) Dopamine (70 mg, 0.45 mmol) was dissolved in DMF (3 mL) and TEA (45 mg, 0.45 mmol) was slowly added to the reaction mixture which was left to stir for 10 min. Then CPADB-PFP (0.2 mg, 0.45 mmol) crystals were dissolved in DMF (2 mL) and was added to the dopamine solution and left to stir at room temperature for overnight. The reaction mixture was then dripped slowly into an aqueous phosphate buffer solution (20 mL, pH 4.0) to obtain a pink precipitate. The solution was then centrifuged and water is decanted followed by freeze-drying. The product was redissolved in acetone to precipitate out the phosphate buffer salt; alternatively, the product could be purified using gel chromatography using chloroform as eluent first then change to chloroform/ethyl acetate (1:1) to obtain the product in the second fraction. Yield: 162 mg (87%). 1 H NMR (300 MHz, CDCl3) δ (ppm): 7.85 (d, 2H, H-1), 7.50 (d, 2H, H-3), 7.35 (t, 1H, H-2), 6.79 (d, 1H, H-10), 6.65 (d, 1H, H-12) 6.51 (dd, 1H, H-11), 5.56 (br, 1H, H-7), 2.70 (m, 2H, H-6), 2.50 (m, 2H, H-9), 2.30 (m, 2H, H-5), 2.20 (m, 2H, H-8) 1.85 (s, 3H, H-4).

(Peak assignment can be found in the Supporting Information, Figure S3.) ESI-MS: Calcd, 414.11 g mol−1; measured, 414.10 g mol−1. RAFT Polymerization of OEGMEMA Using Dopamine-Terminated 4-Cyanopentanoic (CPADBD). Two separate reaction mixtures of OEGMEMA (2.00 g, 6.67 mmol), CPADBD (55.30 × 10−3 g, 1.33 × 10−4 mol), fluorescein o-methacrylate (2.00 × 10−3 g, 2 × 10−6 mol) and AIBN (4.37 × 10−3 g, 2.67 × 10−5 mol) were dissolved in 7.5 mL of toluene. The reaction mixtures were placed in an ice bath and purged with nitrogen for 30 min. The two reaction mixtures were then immersed in a preheated oil bath at 65 °C for 2 and 2.3 h, respectively. The polymerization was terminated by placing the samples in an ice bath for 5 min. 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 monomer during the course of polymerization was determined. The polymer was 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. The samples were stored in a freezer prior to any modifications. POEGMEMA with 26 repeating units (conversion = 52%, Mn(theo) = 7800 g mol−1, Mn(SEC) = 7284 g mol−1) and POEGMEMA with 35 repeating units (conversion = 65%, Mn(theo) = 9750 g mol−1, Mn(SEC) = 12571 g mol−1) were used as a so-called macroRAFT agent to control the chain extension with MAA. The number of repeating units of POEGMEMA was calculated from the monomer conversion obtained from 1H NMR. Synthesis of POEGMEMA-b-PMAA. Method 1. The POEGMEMA (35 repeating units) macroRAFT agent (1.00 × 10−1 g, 7.95× 10−6 mol), MAA (0.21g, 2.4 × 10−3 mol), and AIBN (1.31 × 10−4g, 7.9 × 10−7 mol) were dissolved in 2 mL of methanol. The reaction mixtures were placed in an ice bath and purged with nitrogen for 30 min. The polymerizations was carried out in an oil bath at 70 °C. The copolymers were purified by precipitating in cold diethyl ether, centrifuged, dried under reduced pressure, and kept at 2 °C prior to further experiments. The monomer conversion was calculated from the 1H NMR of the reaction mixture by comparing the intensity of vinyl proton peaks for MAA (at ca. 6.00 and 5.63 ppm) to that of the unchanged (−CH2CH2O−) POEGMEMA peak at 3.66 ppm. After 8 h, the conversion was determined to be 67%. The block copolymer with Mn(theo) = 29500 g mol−1 (Mn(SEC) = 29800g mol−1 ) POEGMEMA35-b-PMAA200 was used for reaction with oxoplatin. Method 2. The POEGMEMA (26 repeating units) macroRAFT agent (1.00 × 10−1 g, 1.30 × 10−5 mol), t-BMA (0.455 g, 3.2 × 10−3 mol), and AIBN (4.10 × 10−4 g, 2.56 × 10−6 mol) were dissolved in 1.25 mL of DMSO. The reaction mixtures were placed in an ice bath and purged with nitrogen for 30 min. The polymerizations were carried out in an oil bath at 70 °C. After 8 h, the copolymers were purified by precipitating in cold diethyl ether, centrifuged, dried under reduced pressure, and kept at 2 °C prior to further experiments. The conversion is calculated using 1H NMR using peaks stated in method 1 to be 35% and the block copolymer with Mn(theo) = 20600 g mol−1, Mn(SEC) = 17400 g mol−1. The copolymer was then dissolved in 2 mL of methanol and added 3 drops of HCl (32%); the reaction mixture was then heated to 70 °C overnight. The copolymer was then purified by precipitating in cold diethyl ether, centrifuged, dried under reduced pressure to give final block copolymer with Mn(theo) = 20700 g mol−1, Mn(SEC) = 17400g mol−1. (POEGMEMA26-b-PMAA90 was used for incorporation with oxoplatin. Chemical Modification of the Block Copolymers for SEC Analysis. For size exclusion chromatography, carboxylic acid groups of POEGMEMA-b-PMAA polymers were modified into methacrylate units the using trimethylsilyldiazomethane as a methylating reagent. A total of 10 mg of each sample was dissolved in a mixture of THF and water at room temperature (about 10 mL). The yellow solution of trimethylsilyldiazomethane in hexane was added dropwise at room temperature into the polymer solution. Upon addition, bubbles (nitrogen) appeared and the solution became instantaneously colorless. Addition of the methylation reagent was continued until the solution became yellow and no more gas formation was observed. Then, an excess of methylating reagent was added and the solution was stirred overnight at room temperature. The solvent was then 965

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(0.5 mL) and added to Milli-Q water (9.5 mL) to give a concentration of 0.2 mg/mL. The solution was diluted further in a 10-fold dilution to give a final concentration of 20 μg/mL. The solution was then added to the micelle solution accordingly to result in a 10, 20, or 30% (mole ratio between folate and polymer concentration) folate content. The solution was stirred overnight before being used in biological tests. Attachment of Acridine−Boronic Acid Linker to Micelles. Acridine−boronic acid linker (2.5 mg) was dissolved in DMSO (1 mL) and added to Milli-Q water (4 mL) to give a concentration of 0.5 mg/mL. The solution was added to the micelle solution according to 20% (mole ratio between acidine and polymer concentration) acridine content. The solution was stirred overnight before further use. Sample Preparation for Laser Scanning Confocal Microscopy. Human ovarian carcinoma Ovcar-3 cells were seeded in 35 mm Fluorodish (World Precision Instruments) at a density of 60000 per dish and cultured for 3 days with RPMI 1640 medium supplemented with 10% fetal bovine serum. Micelles solution was loaded to Ovcar-3 cells at a working concentration of 50 μg/mL and incubated at 37 °C for designed periods (1, 6, and 24 h). After incubation, the cells were washed thrice with phosphate buffered saline (PBS, pH 7.4). Then the cells were stained with 100 nM LysoTracker Red DND-99 (Invitrogen) for 1 min. The dye solution was quickly removed and the cells were gently washed with PBS. Finally, the cells were mounted in PBS and observed under a laser scanning confocal microscope system (Zeiss LSM 780). The system equipped with a Diode 405−30 laser, an argon laser, and a DPSS 561−10 laser (excitation and absorbance wavelengths: 405, 488, and 561 nm, respectively) connected to a Zeiss Axio Observer.Z1 inverted microscope (oil immersion ×100/1.4 NA objective). The ZEN2011 imaging software (Zeiss) was used for image acquisition and processing. Cell Culture. The NIH:OVCAR-3 human ovarian carcinoma (FR +) and A549 (FR-) human lung carcinoma cell lines were kindly provided by Dr. Paul de Souza from the St. George Hospital, Sydney, Australia. The A549 and OVCAR-3 cell lines were grown in a ventilated tissue culture flask T-75 using Roswell Park Memorial Institute (RPMI-1640) media supplemented with 10% Fetal Bovine Serum (FBS), penicillin/streptomycin, and L-glutamine. The cells were incubated at 37 °C in a 5% CO2 humidified atmosphere and passaged every 2−3 days when monolayers at around 80% confluence were formed. Cell density was determined by counting the number of viable cells using a trypan blue dye (Sigma-Aldrich) exclusion test. For passaging and plating, cells were detached using 0.05% trypsin−EDTA (Invitrogen), stained using trypan blue dye, and loaded on the hemocytometer. All the experiments were done in triplicate. Cell Viability Assay. The cytotoxicity of POEGMEMA-b-PMAA, cisplatin, oxoplatin, and oxoplatin incorporated micelles was measured by a standard sulforhodamine B colorimetric proliferation assay (SRB assay). 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 cells were seeded at density of 5000 cells per well in 96-well microtiter plates containing 200 μL of growth medium per well and incubated at 37 °C in a 5% CO2 for 24 h. The medium was then replaced with fresh medium (200 μL) containing various concentrations of POEGMEMA-b-PMAA, cisplatin, oxoplatin, and oxoplatin incorporated micelles over an equivalent cisplatin concentration range of 0−350 μM. For folate-decorated, drug-loaded micelles 100 μL of A549 and OVCAR-3 cells were seeded in 96-well microtiter plates at a density of 5000 cells per well and to allow adhesion overnight. The growth medium was discarded and washed with warm PBS solution 3 times and the micelle solution containing folate targeting agent was diluted with PBS to give a volume of 100 μL and topped with folate-deficient RPMI 1640 medium to give a total volume of 200 μL. After 48 h incubation, cell were 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/v) SRB dissolved in 1% acetic acid. At the end of the staining period, SRB was removed and cultures were quickly rinsed five times

removed under reduced pressure and the methylated polymer was used for size exclusion chromatography. Synthesis of cis,cis,trans-[Pt(NH3)2Cl2(OH)2] (Oxoplatin). Oxoplatin was synthesized according to literature.52 A mixture of cisplatin (1.0 g, 3.05 mmol) and H2O2 30 w/v (3.5 mL, 30.5 mmol) in an aluminum foil covered round-bottom 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 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 a bright yellow powder. Investigation of the Solubility of PMAA Conjugated with Oxoplatin in Water. PMAA was reacted with oxoplatin using the same procedure as during the conjugation of oxoplatin to POEGMEMA-b-PMAA block copolymer. The solubility of the resulting polymer was tested in various solvents revealing that oxoplatin conjugated to PMAA is insoluble in water. Thus, the oxoplatin incorporated POEGMEMA-b-PMAA behaves the same manner as amphiphilic block copolymer, which can be self-assembled into to micelles with core−shell structure. Conjugation of Oxoplatin to POEGMEMA-b-PMAA Block Copolymer and Formation of Micelles. A total 30 mg POEGMEMA-b-PMAA polymer was dissolved in DMF (5 mL). Oxoplatin (25 mg, 0.07 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC; 25 mg, 1.30 × 10−4 mol), and 4-dimethylaminopyridine (DMAP; 6 mg, 4.9 × 10−5 mol) was added to the solution and it was left to stir in an oil bath at 40 °C for 24 h. The sample was then purified by centrifugation to remove the undissolved oxoplatin and then dialyzed against water for 48 h using membrane (MWCO3500) dialysis to remove free oxoplatin. The water is replaced every 6 h. During dialysis, micelle formation was induced because the hydrophilic PMAA takes on hydrophobic featured after oxoplatin conjugation. Release of Cisplatin from Micelles by Reduction of Oxoplatin in the Presence of Ascorbic Acid. The reduction of Pt(IV) complex incorporated polymeric micelles was carried out in distilled water at pH 5 (acidic nature in endosomes) by adding small amounts of HCl. The use of buffer had to be avoided because of coordination to platinum.53 The concentration of the stock ascorbic acid solution is 7.5 mM. The concentration of platinum in the complex is 0.75 mM. The reduction of oxoplatin incorporated micelles by ascorbic acid was monitored for 7 days at 37 °C in the incubator. After each time interval, the concentration of reduced cisplatin was determined by UV−vis analysis. Determination of Cisplatin (CDDP) Concentration Released after Reduction of Oxoplatin Using Colorimetry with o-Phenylenediamine (OPDA). Quantitative determination of released CDDP from oxoplatin incorporated POEGMEMA-b-PMAA by biological reductant was performed using a spectrophotometric method previously described.54 In this method, platinum(II) (cisplatin) reacts with ophenylenediamine at pH 6.5 to form a light blue complex in 15 min at 100 °C. This complex has a maximum at 703 nm, which is far beyond the wavelength of cisplatin, o-phenylenediamine, and ascorbic acid. The light blue solution was placed in the 1 cm path length cuvette and the absorption was measured at 703 nm using Varian Cary 300 UV− vis spectrophotometer. The amount of cisplatin in the conjugate was calculated in reference to standard solutions of free cisplatin in the concentration range of 0.1−5.5 ppm. Synthesis of Cross-Linked Micelles. A total of 15 mg POEGMEMA-b-PMAA, which has been conjugated to oxoplatin, was dissolved in DMF (5 mL), and 2 mL of distilled water was added dropwise to the solution. The sample was then dialyzed against distilled water for 48 h using membrane (MWCO-3500) dialysis. The water was replaced every 6 h. After that, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC; 3 mg, 1.9 × 10−3 mmol) and 3 mg of 1,8diaminooactane (3 mg, 2.0 × 10−3 mmol) was added to the solution and was left to stir overnight at room temperature to achieve core cross-linked micelles. Attachment of Folate−Boronic Acid Linker to Micelles. Folate− boronic acid linker (2 mg, 3 × 10−3 mmol) was dissolved in DMSO 966

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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 570 nm of each well was measured using a microtiter plate reader scanning spectrophotometer BioTek’s PowerWave HT microplate reader and KC4 software. All experiments were repeated three times. Dose−response curves were plotted (values expressed as percentage of control, medium only) and IC50 inhibitory concentrations were estimated by regression analysis. After a 48 h incubation, the culture medium was discarded and the live cells were fixed with 200 μL of trichloroacetic acid 10% w/v (TCA) for 1 h at 4 °C before washing five times with tap water. After removal of water, the TCA-fixed cells were stained for 30 min with 0.4% (w/vol) SRB dye dissolved in 1% acetic acid. The unbounded dye was then removed by washing five times with 1% acetic acid. The plates were left to air-dry overnight followed by the addition of 100 μL of 10 mM unbuffered Tris base to each well to dissolve bound dye.The absorbance at 570 nm of each well was measured using a microtiter plate reader scanning spectrophotometer BioTek’s PowerWave HT microplate reader and KC4 software. All experiments were repeated three times. Dose−response curves were plotted (values expressed as percentage of control, medium only) and IC50 inhibitory concentrations were estimated by regression analysis.

dissolved in solvent specified in the Experimental Section. The sample molecules were ionized with either Na+ or H+ ion. Fluorescence Microscope. LEICA DM IRB live cell microscope was used to obtain fluorescence photographic pictures for live cells. The microscope is equipped with a mercury lamp, Bertrand lens, and side port (80/100%) and 12 V/100 W transmitted, light illumination, centrable field and aperture diaphragms, four rotatable filter blocks (Zero Pixel Shift), flexible condenser system for Brightfield, and coarse and Fine focus with an accuracy of 0.05. Fluorescence. Fluorescence measurements performed on Agilent Cary Eclipse fluorescence spectroscopy with xenon flash lamp using a 1 cm path length, four-sided quartz cuvette. All measurements were obtained at room temperature and fluorescence spectra were recorded between 400 and 800 nm at λex = 468 nm, λem = 572 nm with entrance and exit slit width of 10 mm.

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RESULTS AND DISCUSSION A range of pathways have been investigated to conjugate folates to the surface of nanoparticles.55−58 The conjugation

cell viability(%) = (OD570,sample − OD570,blank ) /(OD570,control − OD570,blank ) × 100 Figure 1. Structural difference between α- and γ-NHS-folate isomers.67

Analysis. 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 SPD10A Shimadzu UV/vis detector. A Phenomenex 50 × 7.8 mm guard column and four Phenogel 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 LiBr, 0.05% w/v 2,6-dibutyl-4-methylphenol (BHT)) 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 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. Dynamic Light Scattering (DLS). The average hydrodynamic diameters and size distribution of prepared micelle solution in an aqueous solution were obtained using Malvern Nano-ZS as particle size analyzer (laser, 4 mW, λ = 632 nm; measurement angle 12.8 and 175°). Samples were filtrated to remove dust using a microfilter 0.45 μm prior to the measurements and run for at least three times at 25 °C. Transmission Electron Microscopy (TEM). The TEM micrographs were obtained using a JEOL 1400 transmission electron microscope. The instrument operates at an accelerating voltage of 100 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 5 °C min−1 on a thermal analyzer TGA 2950HR V5.4A. The temperature profile for analysis ranged between 50−1000 °C with a 5 min isothermal at 100 °C. The mass of the samples used in this study was 10 mg. ElectroSpray Ionization Mass Spectrometry (ESI-MS). ElectroSpray ionization mass spectrometry (ESI-MS) was performed on a Thermo Finnigan LCQ Decaquadrupole ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an atmospheric pressure ionization source operating in the nebulizer-assisted electrospray mode. The samples were prepared by using 0.2−0.25 mg

Table 1. Summary of Homopolymer and Block Copolymers Used for Platinum Drug Conjugation copolymer

Mntheo (g mol−1)

Mnsec (g mol−1)

PDI

POEGMEMA35 POEGMEMA35-b-PMAA200 POEGMEMA26 POEGMEMA26-b-PMAA90

9750 29500 7800 20700

12500 29800 7300 17400

1.15 1.16 1.14 1.37

Figure 2. Color change before and after conjugation of oxoplatin to POEGMEMA-b-PMAA.

Table 2. Summary of Average Hydrodynamic Diameter Dh and Polydispersity Index (PDI) of Micelles code 1 2 3

copolymer POEGMEMA35-b-PMAA200-Pt POEGMEMA26-b-PMAA90-Pt POEGMEMA26-b-PMAA90-Pt crosslinked

number mean (nm)

PDIDLSa

149.8 19.3 18.5

0.21 0.46 0.57

a

DLS number distribution can be found in Supporting Information, Figures S9−S11.

chemistries employed required often a reaction time of several hours, making it difficult to achieve a high throughput when attempting to generate micelles with a variety of folate density on the surface. Conjugation based on boronic acid ester is in contrast almost instantaneous. Micelles, which have folates 967

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Figure 3. TEM image of micelles (concentration of micelles 1 mg mL−1 in water): POEGMEMA35-b-PMAA200-Pt (left, scale bar = 500 nm) and POEGMEMA26-b-PMAA90-Pt (right, scale bar = 200 nm).

Figure 4. Release of platinum(II) complex from the oxoplatin incorporated POEGMEMA27-b-PMAA250 micelles by ascorbic acid (7.5 mM; open squares indicated the measured hydrodynamic diameter measure via DLS; closed squares refer to the amount of platinum drugs released).

Figure 6. Human lung carcinoma A549 viability after being exposed to a solution containing POEGMEMA35-b-PMAA200 and POEGMEMA26-b-PMAA90 after 48 h.

bound to their surface via boronic acid esters need to be based on block copolymer with an end functionality that is known to react with boronic acid in an efficient manner such as 1,2dihydroxy benzene (catechol). The preparation of catecholterminated RAFT agent was achieved via amidation reaction of

activated esters, as depicted in Scheme 1. Recently, pentafluorophenyl esters have been extensively reported as active intermediates for further functionalization.51,59−64 The

Figure 5. Competitive binding of a phenylboronic acid with Alizarin Red S. and a catechol. 968

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react with the dithioester core resulting in aminolysis of the RAFT agent. Hence, converting CPADB into a more active ester is necessary for successful attachment of dopamine to the RAFT agent. Neutralization of dopamine·HCl with an equivalent amount of TEA was also necessary in this reaction as the low pH would greatly reduce the coupling efficiency to form amide coupling. However, basic conditions (>pH 9) caused by the addition of extra TEA will also cause the destruction of the RAFT agent, thus, a neutral environment would be preferred. Synthesis of Folate Boronic Acid. For folic acid to bind to polymers via the formation of borate ester, the folate itself must be attached to a boronic acid species. The synthesis of boronic acid functionalized folic acid is achieved via EDC/NHS coupling method. Folic acid suffers from low solubility in most organic solvents; therefore, it is wise to allow complete dissolution in DMF prior to the addition of EDC and NHS. The solution was allowed to stir for 30 min for the reaction of EDC with the carboxyl group on folic acid to form an amine reactive O-acylisourea ester. This intermediate is unstable and susceptible to hydrolysis; therefore, it is short-lived in aqueous solution. However, in the presence of NHS, it stabilizes the amine reactive intermediate, which then allows the addition of the amine functional group on 3-ABPA to form a stable amide bond with folic acid. Thus, this approach increases the coupling efficiency between amine and carboxyl functional groups. In addition, neutralization of 3-ABPA·HCl with an equivalent amount of TEA was performed preceding the reaction with NHS-folic acid intermediate. It is important to note that 65% of the FA-NHS conjugate is γ-isomer (Figure 1), while the αisomer is considered biologically inactive.67 Unfortunately,

Figure 7. Human ovarian carcinoma OVCAR-3 viability after being exposed to a solution containing POEGMEMA35-b-PMAA200 and POEGMEMA26-b-PMAA90 after 48 h.

ester can be synthesized in high yields from the reaction of carboxylic acid with pentafluorophenol using dicyclohexylcarbodiimide (DCC) as coupling reagent.64−66 The product was purified via gel column chromatography. Ultimately, the designed catechol-terminated RAFT agent was obtained through the amidation of the activated ester with excess of dopamine in the presence of TEA in DMF at room temperature to yield the desired product. The pentafluorophenol ester is a facile leaving group when attacked by amines to achieve the amide functionality. Without the pentafluorophenol ester, the amine group of the dopamine would first

Table 3. Summary of IC50 Values of Various Micelle Sizes with 0, 10, 20, and 30 mol % of End Functionalities Conjugated to Folatea cell

folate functionalization (% mole)

OVCAR-3

drug only μM

IC50 (small micelle) μM

IC50 large micelle μM

IC50 (cross-linked micelle) μM

93 50 31 27

19 17 16 15

39 22 18 15

60 51 62 58

78 84 90 90

cisplatin 4 oxoplatin 30

(folate-free media) 0 10 20 30 A549

cisplatin 5 oxoplatin 87

(folate-free media) 0 10 20 30 OVCAR-3

cisplatin 3.8 oxoplatin 40

(normal media) 0 10 20 30 a

36 41 24 26

The cell viability vs the concentration, including the errors for each experiment can be found in the Supporting Information. 969

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Figure 8. Fluorescence microscopy images of PEGMEMA26-b-MAA90-Pt with A549 and OVCAR-3 cells: (A) A549 for 1 h, (B) A549 for 24 h, (C) OVCAR-3 for 1 h, (D) OVCAR-3 for 24 h. Scale bar = 50 μm.

Figure 9. Cytotoxicity profile of cross-linked small micelles (20 nm) against OVCAR-3 ovarian cancer cells for 48 h in folate free RPMI 1640 media.

Figure 10. Cytotoxicity profile of cross-linked small micelles (20 nm) against OVCAR-3 ovarian cancer cells for 48 h in normal RPMI 1640 media.

separation of α- and γ-isomers is deemed difficult as reported in literature. Synthesis of Block Copolymers. Two different micelle sizes were targeted to compare the effect of the nanoparticle size on the performance of the drug carrier. The molecular weight of the underlying block copolymer plays a crucial role in determining the size of the micelle. In particular, the hydrophobic, core-forming block is responsible for size changes and shape transitions.68,69 Longer hydrophobic blocks can lead to substantial micelle size increases due to their space requirement, which ultimately increases the aggregation number. Two different block copolymers were therefore

envisaged: Both with a POEGMEMA block of similar size, but a PMAA block of different length. The RAFT agent was subsequently employed in the polymerization of OEGMEMA. The monomer poly(oligo(ethylene glycol) methyl ether methacrylate (OEGMEMA) was chosen as the hydrophilic shell of the micelle is due to its water solubility as well as excellent biocompatibility. Methacrylic acid (MAA) was selected as the core of the micelle due to its unique responsiveness to pH and ionic strength, but mostly as a functional polymer capable of reacting with oxoplatin. The molecular weights of the two block copolymers are listed in Table 1. The block copolymers were prepared using two 970

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Scheme 2. Micelles Conjugated to 9-Amino Acridine via Boronic Acid Ester and the Relationship between Phenylboronic Acid and Its Diol Ester49

Figure 11. Confocal microphotographs of OVCAR-3 cells after incubation with micelles at 37 °C for 1, 6, and 24 h. PEGMEMA26-b-tBMA90 micelles (green) were labeled with fluorescein. Acridine (yellow) was added to the micelles. Lysosomes (red) were stained with LysoTracker Red DND-99. Blue triangles indicate the intracellular parts with only acridine but without fluorescein-labeled polymers. Scale bar = 5 μm.

pathways: Either t-butyl methacrylate was polymerized, followed by deprotection (Scheme 1) or MAA was polymerized directly. However, postmodification was required to protect the carboxylic group via esterification to obtain better SEC data since PMAA had a high tendency to aggregate and interact with the column material. The measured values of the block copolymers prepared either way are close to the theoretical value. In addition, the molecular weight distribution is narrow (Supporting Information, Figure S4). This is an important point considering that catechol is prone to oxidation,70 which may lead to higher molecular weights. In addition, significant chain transfer events that may be promoted by catechol seem to absent as evidenced by the SEC distribution (Supporting Information, Figure S4). Both block copolymers were subsequently conjugated to oxoplatin in DMF as solvent using EDC as coupling agent

(Figure 2). Thermogravimetric analysis (TGA) was employed to determine the Pt content. A control experiment monitoring the thermal decomposition of only cisplatin was carried out revealing an experimental total weight loss of 35 wt % at 372 °C confirming that only elemental platinum remained (see Supporting Information, Figure S5). Oxoplatin led to a total weight loss of 49 wt % at 372 °C, again confirming that only elemental platinum remains (see Supporting Information, Figure S6). The results of thermal analysis revealed total mass loss of polymer occurred at around 420 °C and the amounts of elemental platinum present in the coordinate complex is about 12 wt % for both polymer variations: PEGMEMA35-bPMAA200-Pt and PEGMEMA26-b-PMAA90-Pt (TGA trace analysis can be found in Supporting Information, Figures S7 and S8). These numbers are equivalent to a conjugation of 971

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acid, which was conjugated to folic acid, and dopamine, which has been reacted with the RAFT agent. To quantify the affinity between both, Alizarin Red S (ARS) as fluorescence indicator was employed.46−49 In the presence of boronic acid or boronate functional groups the catechol portion of the ARS forms a boronate ester that emits fluorescence upon excitation. The formation of this boronate ester can be observed as fluorescence intensity increases with the addition of boronic acid to an ARS solution until equilibrium is reached (Supporting Information, Figure S12). It can also be observed that there is a change in λmax corresponding to the visible color change from a deep red solution containing ARS in 0.1 M phosphate buffer solution to a more orange yellow color with the addition of boronic acid. As expected, the fluorescence intensity decreases within minutes as dopamine was added to the PBS solution of PBA and ARS, presumably through the removal of fluorescence quenching mechanism. In this competitive assay, the fluorescence intensity was measured directly for the equilibrium between PBA and ARS in a PBS solution. In the presence of dopamine, it sets up a second equilibrium between PBA and the catechol itself, leaving more free ARS in the solution, therefore, causing the decrease in fluorescence (Figure 5). The competitive binding assay, which was carried out at pH 7, shows that, with equal ratio between dopamine and PBA and 200 more equivalence of ARS, the fluorescence is decreased by 30% and the resulting binding constant (Ka), which can be calculated using the Benesi−Hildebrand method, was calculated to be 448 M−1. According to the measured equilibrium constant, the affinity between both building blocks is high, indicating the very efficient formation of boronic ester. Cell Growth Inhibition Study. To confirm the cytotoxic effect of these folate-decorated, Pt-loaded micelles in vitro, growth-inhibition tests were carried out using two cell lines: (FR−) A549 and (FR+) OVCAR-3 cells. A549 FR negative cell line was employed as a negative control for all experiments. The stability of the micelles in cell growth media was briefly studied using the scattering intensity. Over a period of several hours, no changes were observed, while visual inspection after 24 h does not reveal any formation of precipitate. The toxicity of the polymer at various concentrations was investigated using both A549 and OVCAR-3 cell lines prior to the platinum conjugation (Figures 6 and 7). It can be deduced that the RAFT-terminated polymers have little or no effect on the growth of A549 in the concentration ranged used. Compared to the A549 cell lines, OVCAR-3 cells are more sensitive toward these carboxyl group bearing polymers, indicating that these cells are more susceptible to pH changes in the environment. The cytotoxicity of oxoplatin incorporated micelles has been compared with oxoplatin and cisplatin itself. Comparison to the toxicity free drug allows conclusion about the efficiency of the drug carrier and the release of the drug via reduction. As expected, cisplatin proves to be more cytotoxic toward both cell lines compared to oxoplatin as oxoplatin is a platinum(IV) compound which requires an extra reduction step to form the active diaquo-diamino platinate(II), which is the active species to bind to DNA to cause cell apoptosis. A549 is a cell line generally less susceptible than OVCAR-3 cells therefore the IC50 (Table 3) value for cisplatin and oxoplatin against A549 cells is much higher compared to OVCAR-3 cells (Supporting Information, Figures S13 and 14).

oxoplatin at approximately every eleventh MAA unit for POEGMEMA35-b-PMAA200 and every seventh MAA unit for POEGMEMA26-b-PMAA90. The maximum loading can be understood when taken into account that the polar and bulky complex will cause chain stretching of the backbone to a point where it is entropically unfavorable, thus, no more Pt drug can be conjugated to the polymer. This could also explain the higher loading of the smaller block copolymer. Meanwhile, a control experiment was carried out using PMAA only in the reaction with oxoplatin to evaluate the solubility in water. Although PMAA is water-soluble, the presence of pendant oxoplatin renders the polymer insoluble in water making it hydrophobic. Upon dialysis of the product against water, micellization occurs as the oxoplatin incorporated block copolymer behaves the same manner as an amphiphilic polymer, in which drug incorporated PMAA is hydrophobic and POEGMEMA is hydrophilic. The oxoplatin-incorporated micelles are stable in distilled water. There is no observation of disassociation or precipitation for a long period of time. It should be mentioned here that there is a possibility that oxoplatin could possibly act as cross-linker. However, dissolving the block copolymer after oxoplatin conjugation in a good solvent such as DMF resulted in hydrodynamic diameters of well below 10 nm, indicative of single chains. Also, SEC analysis resulted in just a monomodal peak albeit a rather broad peak of the Pt-conjugated polymer. Cross-linking cannot be fully excluded, but it seems that the majority of oxoplatin drugs only react via one axial ligand. The size distribution of the oxoplatin-incorporated micelles is shown in Table 2, with an average hydrodynamic size of about 150 nm, a relative narrow distribution (PDI = 0.21) for PEGMEMA35-b-PMAA200, and an average size of 20 nm for PEGMEMA26-b-PMAA90 with a PDI of 0.46. From TEM it can be observed that the nanoparticles have spherical morphology, with low polydispersity (Figure 3). The particle sizes measured by TEM and DLS are in good agreement with the sizes obtained from DLS being slightly larger than the sizes from TEM due to the hydration of the corona. In a reductive environment, the oxoplatin will be reduced to platinum(II) complex resulting in the release of cisplatin (Figure 4). In this study, the release was carried out at pH 5, which reflects the acidic nature of the cell interior. The reductive cell environment was simulated using 7.5 mM ascorbic acid and the amount of release cisplatin was determined using UV−vis spectroscopy. The redox potential at pH = 6 of a similar derivative of oxoplatin with only one hydroxyl axial ligand converted to ester was reported to be −0.49 V.71 Cisplatin was released gradually, and no initial burst release was observed. The release seems to slow down after 20 h. This can be understood considering the open and oxygen-saturated system, which may inhibit further reduction. The current setup can therefore only be used as guidance. Important though is the change in hydrodynamic diameter during the release. Upon release of cisplatin, the PMAA block is converted back into a water-soluble block, resulting in the disappearance of the micelles. Water-soluble block copolymers can then be cleared from the body with ease while high molecular weight micelle may evade clearance for a prolonged time. Binding of Micelles to Folate. The binding between the folate modified phenyl boronic acid and the dopamine functionality, which is located on the surface of these micelles, was tested using the underlying building blocks: phenyl boronic 972

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the polymers seem to accumulate into the nucleus probably because of the disassembly into single polymer chains that are small enough to reach the nucleus. Cross-Linked Micelles. Because the low stability of the micelles based on POEGMEMA26-b-PMAA90-Pt was held responsible for the rather low cytotoxicity, stabilization of the micelle should lead to higher cell toxicity. Cross-linking of micelles proves to be an excellent method for enhancing the stability and structural integrity of small micelles, which ultimately helps the prodrugs to be uptaken into the cell via receptor mediated endocytosis before the platinum compound is released. The micelles were cross-linked in the core using established procedures (Scheme 1). After self-assembly of the oxaliplatin conjugated block copolymer POEGMEMA26-bPMAA90-Pt in the aqueous solution a diamine cross-linker was used to react with the free carboxyl functional groups on the polymer backbone via EDC/NHS coupling reaction thus improve their structural stability. The subsequent cell viability studies using OVCAR-3 cells were carried out after various amounts of folate were conjugated to the surface. Again, a dependency of the cell viability and the resulting IC50 on the folate concentration was observed (Figure 9 and Table 3). However, this time the IC50 values were noticeably smaller. It can be confirmed that the structural integrity of small micelles are very important to achieve a high cellular uptake via RME and therefore attain high toxicity toward tumor cells. In normal RPMI1640 media, which contains free folate, a competitive process takes place. Both folate in the cell growth media and on the surface of the micelle compete for the folate receptors. The cellular uptake of the micelles should therefore be reduced. As a result, fewer drugs are delivered and the dose required to inhibit cell growth should increase. Indeed, as depicted in Figure 10 and summarized in Table 3, the dependency of the IC50 on the folate concentration decreases, although it has not been fully eliminated. Within error, the micelle with the highest folate concentration still conveys the highest toxicity. Since this is a competitive process, it seems that even more free folate would be required to fully occupy the folate receptor. However, the higher IC50 values confirm that folate receptors indeed play a determining role in this process. Stability of Boronic Acid Ester. The conjugation of boronic acid with diols has the advantage that it is quick and efficient. However, the conjugation is potentially reversible leading to the loss of the bioactive group. This may or may not be an advantage. To test the stability under in vivo conditions a control experiment has been developed, which employs the yellow fluorescent boronic acid derivatized 9-amino acridine instead of folic acid (Scheme 2). The micelles employed were based on POEGMEMA as the hydrophilic block and t-butyl acrylate (PtMA) as the hydrophobic block, labeled with fluorescein. The replacement of the Pt-containing block was necessary to enable investigations over several hours without cell death. The stability of the boronic acid ester was then monitored using confocal fluorescent microscopy. The aim was to investigate if the yellow fluorescent acridine is colocalized with the green fluorescent micelles. The micelle’s penetration and distribution is shown in Figure 11. The attachment of fluorescein to the polymer backbone was achieved using fluorescein methacrylate (3% mole) in the PEGMEMA block (green). Acridine phenyl boronic acid can be localized because of their yellow fluorescence while the lysosomes were stained red with LysoTracker DND-99. After

The solution containing platinum incorporated micelles were divided into four separate samples each were added appropriate amount of PBA-FA linker to give 0, 10, 20, and 30 mol % of folic acid on the surface of the micelles. Conjugation occurs within minutes of addition. Those samples were then diluted with PBS to give a concentration variance between 300 and 600 μM of platinum content for final cell growth inhibition testing. Using SRB assay, we compared the cytotoxicity (IC50 values) of both large and small micelles synthesized from POEGMEMA35b-PMAA200 and POEGMEMA26-b-PMAA90, respectively, in FRpositive OVCAR-3 cells and the FR-negative A549 cells over an incubation period of 48 h. The cell viability of both cell lines was recorded against the amount of oxaliplatin present in the micelle solution in dependence of the folate conjugation (Supporting Information, Figures S15−19). The error analysis is listed in Tables S1−S6 in the Supporting Information. The IC50 value was determined from these graphs and the values are listed in Table 3. As can be noted, for both platinum containing large and small micelles prepared from POEGMEMA35-b-PMAA200 (150 nm) and POEGMEMA26-b-PMAA90 (20 nm), the IC50 values are dependent on the amount of FA on the surface for the folate receptor positive OVCAR-3 cells. In contrast, the IC50 of these folate-decorated micelles values were not affected by the amount of FA on the FR negative A549 cells. Convincingly, the results indicate that the higher the amount of FA the lower the IC50 value for the test against FR positive cells which intern supports the fact that the prodrugs are up taken via receptormediated endocytosis on the OVCAR-3 cells. However, by comparing the results on OVCAR-3 cell lines between large and small micelles; it clearly shows a large variation of IC50 values between 0 and 30% of FA for small micelles compared to the large ones. The large micelles show only a very small dependency on the amount of folate on the micelle surface (19 vs 15 μM), while the small micelles show a clear improvement in cell toxicity with increasing amount of folate (from 93 μM (no FA) to 27 μM (30% FA)). This may be explained that large micelles of more than 100 nm enter the cell via other endocytosic pathways, while receptor-mediated endocytosis is more efficient with smaller nanoparticles sizes. Recent theoretical work, which has been supported experimentally, suggests that the optimum particle diameter for receptor-mediated endocytosis is indeed around 50 nm,72 although this depends on the ligand density.73 Particles above 100 nm are treated unfavorably during receptor-mediated endocytosis.74 Although the folate concentration dependency of the IC50 value suggests receptor-mediated uptake, the overall values are noticeably smaller to the bigger micelles. It needs to be considered that the small micelles of size approximately 20 nm with the rather short hydrophobic block are more susceptible to disassembly. Recent work has indeed shown that micelles of low stability and below the CMC do not enter the cells and therefore having a less impact on cell growth inhibition.75,76 The lower stability of the smaller micelle, which is based on (POEGMEMA26-b-PMAA90-Pt) can be observed by fluorescence microscopy. The cells were incubated with small micelles for 1 and 24 h with A549 (FR−) and OVCAR-3 (FR+) cells (Figure 8). It can be observed that after 1 h of incubation the fluorescent labeled polymers are located on the surface of the cell membranes suggesting some interaction of the membrane with the bilayer structure, a possibility which has been suggested in earlier publications.77 After 24 h of incubation, 973

dx.doi.org/10.1021/bm400121q | Biomacromolecules 2013, 14, 962−975

Biomacromolecules

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(6) Shen, J.; Huang, C.; Jiang, L.; Gao, F.; Wang, Z.; Zhang, Y.; Bai, J.; Zhou, H.; Chen, Q. Biochem. Pharmacol. 2007, 73, 1901. (7) Min, Y.; Mao, C.-Q.; Chen, S.; Ma, G.; Wang, J.; Liu, Y. Angew. Chem., Int. Ed. 2012, 51, 6742. (8) 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, 38. (9) Wong, E.; Giandomenico, C. M. Chem. Rev. 1999, 99, 2451. (10) Chin, C. F.; Wong, D. Y. Q.; Jothibasu, R.; Ang, W. H. Curr. Topics Med. Chem. 2011, 11, 2602. (11) Hall, M. D.; Martin, C.; Ferguson, D. J. P.; Phillips, R. M.; Hambley, T. W.; Callaghan, R. Biochem. Pharmacol. 2004, 67, 17. (12) Hall, M. D.; Davies, M. S.; Dillon, C.; Berners-Price, S.; Hambley, T. W. J. Inorg. Biochem. 2001, 86, 245. (13) Choi, S.; Cooley, R. B.; Voutchkova, A.; Leung, C. H.; Vastag, L.; Knowles, D. E. J. Am. Chem. Soc. 2005, 127, 1773. (14) Choi, S.; Vastag, L.; Larrabee, Y. C.; Personick, M. L.; Schaberg, K. B.; Fowler, B. J.; Sandwick, R. K.; Rawji, G. Inorg. Chem. 2008, 47, 3920. (15) Choi, S. H.; Delaney, S.; Jewett, K. J.; Orbai, L. J. Inorg. Biochem. 1999, 74, 98. (16) Hall, M. D.; Hambley, T. W. Coord. Chem. Rev. 2002, 232, 49. (17) Zhang, J. Z.; Wexselblatt, E.; Hambley, T. W.; Gibson, D. Chem. Commun. 2012, 48, 847. (18) Feazell, R. P.; Nakayama-Ratchford, N.; Dai, H.; Lippard, S. J. J. Am. Chem. Soc. 2007, 129, 8438. (19) Li, J.; Yap, S. Q.; Chin, C. F.; Tian, Q.; Yoong, S. L.; Pastorin, G.; Ang, W. H. Chem. Sci. 2012, 3, 2083. (20) Miyata, K.; Christie, R. J.; Kataoka, K. React. Funct. Polym. 2011, 71, 227. (21) Seymour, L. W.; Miyamoto, Y.; Maeda, H.; Brereton, M.; Strohalm, J.; Ulbrich, K.; Duncan, R. Eur. J. Cancer 1995, 31, 766. (22) Stolnik, S.; Illum, L.; Davis, S. S. Adv. Drug Delivery Rev. 1995, 16, 195. (23) Fang, J.; Nakamura, H.; Maeda, H. Adv. Drug Delivery Rev. 2011, 63, 136. (24) Maeda, H. P. Proc. Jpn. Acad., Ser. B 2012, 88, 53. (25) Cabral, H.; Nishiyama, N.; Okazaki, S.; Koyama, H.; Kataoka, K. J. Controlled Release 2005, 101, 223. (26) Plummer, R.; Wilson, R. H.; Calvert, H.; Boddy, A. V.; Griffin, M.; Sludden, J.; Tilby, M. J.; Eatock, M.; Pearson, D. G.; Ottley, C. J.; Matsumura, Y.; Kataoka, K.; Nishiya, T. Br. J. Cancer 2011, 104, 593. (27) Uchino, H.; Matsumura, Y.; Negishi, T.; Koizumi, F.; Hayashi, T.; Honda, T.; Nishiyama, N.; Kataoka, K.; Naito, S.; Kakizoe, T. Br. J. Cancer 2005, 93, 678. (28) Huynh, V. T.; Chen, G. J.; de Souza, P.; Stenzel, M. H. Biomacromolecules 2011, 12, 1738. (29) Huynh, V. T.; de Souza, P.; Stenzel, M. H. Macromolecules 2011, 44, 7888. (30) Huynh, V. T.; Quek, J. Y.; de Souza, P. L.; Stenzel, M. H. Biomacromolecules 2012, 13, 1010. (31) Pearson, S.; Scarano, W.; Stenzel, M. H. Chem. Commun. 2012, 48, 4695. (32) Hayama, A.; Yamamoto, T.; Yokoyama, M.; Kawano, K.; Hattori, Y.; Maitani, Y. J. Nanosci. Nanotechnol. 2007, 8, 1. (33) Lu, Y.; Low, P. S. Adv. Drug Delivery Rev. 2002, 54, 675. (34) Garcia-Bennett, A.; Nees, M.; Fadeel, B. Biochem. Pharmacol. 2011, 81, 976. (35) Leamon, C. P.; Cooper, S. R.; Hardee, G. E. Bioconjugate Chem. 2003, 14, 738. (36) Leamon, C. P.; Low, P. S. Drug Discovery Today 2001, 6, 44. (37) Xue, Y.; Tang, X.; Huang, J.; Zhang, X.; Yu, J.; Zhang, Y.; Gui, S. Colloids Surf., B 2011, 85, 280. (38) Yoo, H. S.; Park, T. G. J. Controlled Release 2004, 96, 273. (39) Cambre, J. N.; Sumerlin, B. S. Polymer 2011, 52, 4631. (40) Stolowitz, M. L.; Ahlem, C.; Hughes, K. A.; Kaiser, R. J.; Kesicki, E. A.; Li, G. S.; Lund, K. P.; Torkelson, S. M.; Wiley, J. P. Bioconjugate Chem. 2001, 12, 229. (41) Moffatt, S.; Wiehle, S.; Cristiano, R. J. Gene Ther. 2006, 13, 1512.

incubation with the OVCAR-3 cells for 1 h, the micelles were uptaken into the cells and can be located in the lysosomes (red). The detachment of acridine phenyl boronic acid and polymeric micelles cannot be observed at 1 and 6 h. After 24 h, small amounts of of acridine has been detached from the polymers (blue triangle) indicating that the boronic acid is stable for a prolonged period of time but finally may succumb to some cleavage.

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CONCLUSION In this study, complete water-soluble block copolymer was used for the incorporated of Pt (IV) drugs, which allows the fast dissociation of micelles once uptaken into the cells. The cytotoxicity of the polymer used in this study was tested and shown to be nontoxic to cells at the range of drug testing. The folate decorated micelles were tested against FR− A549 and FR + OVCAR-3 cells; result showed these micelles are selectively taken into cancer cells by folate-receptor-mediated endocytosis. However, the IC50 values for small micelles (20 nm) were generally higher than the IC50 value for large micelles (150 nm), regardless of the amount of folate attached on the surface. By cross-linking the small micelles, it shows a greater structural stability therefore allowed to enter the cell as a whole and consequently resulting in lower IC50 values compared to the un-cross-linked version.

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ASSOCIATED CONTENT

S Supporting Information *

Assigned 1H NMR spectra for borate folic acid, pentafluoroactivated RAFT agent, and dopamine-terminated RAFT agent. TGA analysis of cisplatin-, oxoplatin-, and platinum-attached polymers. Fluorescence profile of ARS in presence of ARS. Size measurements of large and small cross-linked and un-crosslinked micelles using DLS. Cytotoxicity study of cisplatin, oxoplatin, large micelles, small crosslinked, and un-cross-linked micelles against A549 and OVCAR-3 cells. Tabulated results containing error % for all cell growth inhibition studies against A549 and OVCAR-3 cells. 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 The authors would like to thank the Australian Research Council (ARC) for funding. M.H.S., W.S., H.L., and H.T.T.D. acknowledge the Centre for Advanced Macromolecular Design (CAMD), St George Research Education Centre (CPT), UNSW Analytical Centre for support and UNSW Electron Microscope Unit for support. The authors would also like to thank Quentin Li for his help during the experiments.

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REFERENCES

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