Enhanced Delivery of the RAPTA-C Macromolecular

Article pubs.acs.org/Biomac

Enhanced Delivery of the RAPTA‑C Macromolecular Chemotherapeutic by Conjugation to Degradable Polymeric Micelles Bianca M. Blunden,†,‡ Hongxu Lu,† and Martina H. Stenzel*,† †

Centre for Advanced Macromolecular Design, University of New South Wales, Sydney, New South Wales 2052, Australia Cooperative Research Centre (CRC) for Polymers, 8 Redwood Drive, Notting Hill, Victoria 3618, Australia

‡

S Supporting Information *

ABSTRACT: Macromolecular ruthenium complexes are a promising avenue to better and more selective chemotherapeutics. We have previously shown that RAPTA-C [RuCl2(p-cymene)(PTA)], with the water-soluble 1,3,5-phosphaadamantane (PTA) ligand, could be attached to a polymer moiety via nucleophilic substitution of an available iodide with an amide in the PTA ligand. To increase the cell uptake of this macromolecule, we designed an amphiphilic block copolymer capable of selfassembling into polymeric micelles. The block copolymer was prepared by ring-opening polymerization of D,L-lactide (3,6dimethyl-1,4-dioxane-2,5-dione) using a RAFT agent with an additional hydroxyl functionality, followed by the RAFT copolymerization of 2-hydroxyethyl acrylate (HEA) and 2chloroethyl methacrylate (CEMA). The Finkelstein reaction and reaction with PTA led to polymers that can readily react with the dimer of RuCl2(p-cymene) to create a macromolecular RAPTA-C drug. RAPTA-C conjugation, micellization, and subsequent cytotoxicity and cell uptake of these polymeric moieties was tested on ovarian cancer A2780, A2780cis, and Ovcar-3 cell lines. Confocal microscopy images confirmed cell uptake of the micelles into the lysosome of the cells, indicative of an endocytic pathway. On average, a 10-fold increase in toxicity was found for the macromolecular drugs when compared to the RAPTA-C molecule. Furthermore, the cell uptake of ruthenium was analyzed and a significant increase was found for the micelles compared to RAPTA-C. Notably, micelles prepared from the polymer containing fewer HEA units had the highest cytotoxicity, the best cell uptake of ruthenium and were highly effective in suppressing the colony-forming ability of cells.

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INTRODUCTION RAPTA-C is a ruthenium metallodrug that is weakly cytotoxic in vitro but very selective and efficient on metastases in vivo.1−5 However, it is limited by the high dose required for an effective treatment. It is inactive against primary tumors,6 but effective at reducing metastases combined with excellent clearance rates from vital organs.7,8 A predisposition toward protein binding may be responsible for the selective antimetastatic effect of RAPTA-C.4 It effectively inhibits cell growth by triggering G2/ M phase arrest and apoptosis in cancer cells, and also slowing cell division.6 The complex binds to oligonucleotides with loss of chloride and sometimes the arene also.7,9 RAPTA-C significantly inhibits the progression of cancer in animal models by reducing the number and weight of solid metastases with low general toxicity.6 To exploit the therapeutic benefits that can be harnessed from RAPTA-C, the drug should be protected from degradation before reaching the target, and passively delivered to cancer cells. Earlier work has shown that RAPTA-C can be successfully attached to polymers, creating macromolecular ruthenium drugs, without losing their activity.10 However, a linear polymer chain is often not readily taken up by cancer © 2013 American Chemical Society

cells, giving IC50 values that are less favorable than those of the free drug.11 The creation of a drug carrier in the nanosize range enables fast endocytosis. Micelles present a unique size that leads to increased circulation times,12 and the potential to exploit the EPR effect,13 which are important properties for therapeutic moieties. Covalent attachment of a therapeutic agent is a useful avenue to delay drug release until the micelle reaches a target site.14 Micelles, wherein a drug is covalently bound have been investigated, for example PEG-b-poly(ε-caprolactone) with chemically conjugated docetaxel,15 and a number of poly(ethylene oxide)-based polymers containing cisplatin.16−22 Water-soluble metalla-cages encapsulating ruthenium drugs have been investigated and shown to considerably improve the cytotoxicity with respect to RAPTA-C, thought to be due to the EPR effect.23 The advantages of nanosized drug carriers are evident and they have been successfully used with an array of drugs. The Received: June 23, 2013 Revised: November 7, 2013 Published: November 8, 2013 4177

dx.doi.org/10.1021/bm4013919 | Biomacromolecules 2013, 14, 4177−4188

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Scheme 1. Synthesis of Amphiphilic Polymers, End-Group Modification of Cl to I Using the Finkelstein Reaction, Two-Step Conjugation of RAPTA-C, and Subsequent Micellization and Degradation

While polymer−drug conjugates and the use of micelles are well established techniques, we present here for the first time a functioning drug carrier for RAPTA-C. In contrast to more established drugs, the design of a drug carrier for ruthenium drugs needs to fulfill a range of prerequisites to achieve high bioactivity: This includes the suitable conjugation technique to ensure that the drug is still active even when chemically altered. In addition, the drug carrier in itself needs to be degradable as discussed above. We therefore utilized the existing knowledge on micelle design for drug delivery to design a carrier specifically for this drug. Therefore, 2-chloroethyl methacrylate (CEMA) as the reactive building block for RAPTA-C conjugation was copolymerized with 2-hydroxyethyl acrylate (HEA) due to its hydrophilic nature. To ensure degradation of

drug is often physically encapsulated or, when conjugated to the polymer chain, can be released again upon a trigger or by simple hydrolysis. A range of polymer−drug conjugates are available that rely on the concept of having a cleavable linker between polymer and drug.24 However, the polymer−RAPTAC conjugate reported earlier by our group is unlikely to degrade into its components and is therefore termed a macromolecular drug. Macromolecular drugs need to be sufficiently agile to reach their intracellular target, but this prerequisite cannot be fulfilled by a nanosized carrier, that is, for example, unlikely to enter the nucleus. It is therefore essential to design a carrier that can take on a nanosized micellar structure, but is also able to degrade into smaller polymer chains over time to increase the activity of the macromolecular drug. 4178


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conducted using a Bruker DMX 600 MHz spectrometer (1H, 600.13 MHz) with a QNP probe and an Avance III 500 MHz spectrometer (1H, 500.13 MHz) with a TBI probe. The 600 and 500 MHz spectrometer parameters are 8390 Hz sweep width, 1.95 s acquisition time, and 2 s recycle delay and 6009 Hz sweep width, 5.45 s acquisition time, and 5 s recycle delay, respectively. The pulse program WATERGATE was used for water suppression.33 Samples were analyzed in the solvents DMSO-d6 and D2O. 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 13C). 31P spectra were calibrated to H3PO4 (85%; δ = 0 ppm). X-ray Crystallography. Crystals were grown from a DCM/heptane layered solution in the glovebox. The single crystal of the compound for analysis was mounted on a Bruker APEXII CCD single crystal diffractometer equipped with graphite monochromated MoK radiation. Dynamic Light Scattering (DLS). Particle sizes were determined using a Brookhaven Zetaplus particle size analyzer (laser: 35 mW, λ: 632 nm, angle: 90°) and a solution of 1 mg mL−1 polymer in distilled water at 25 °C. Five measurements, with three runs consisting of two scans of 2 min in each measurement, were taken of each sample. Samples were purified from dust using a micro filter (0.45 μm) before analyzing. The mean diameter was obtained from the arithmetic mean using the relative intensity of each particle size. Transmission Electron Microscopy (TEM). The TEM micrographs were obtained using a JEOL1400 transmission electron microscope, consisting of a dispersive X-ray analyzer interfaced to the column and a Gatan CCD facilitating the acquisition of digital images. It was operated at an accelerating voltage of 80 kV. Samples were prepared by casting a 1 mg mL−1 polymeric micelle solution onto a copper grid. The grids were air-dried and then negatively stained with phosphotungstic acid for 2 min and air-dried again. Thermogravimetric Analysis (TGA). Thermal decomposition properties of products were recorded using a TG5000 Thermogravimetric Analyzer. The sample (99%, Aldrich), triethylamine (NEt3; >99%, Aldrich), fluorescein Omethacrylate (F; 97%, Aldrich), toluene (>99.4%, Ajax), N,Ndimethylacetamide (DMAc; 99.9%, Aldrich), N,N-dimethylformamide (DMF; 99%, Aldrich), dimethylsulfoxide (DMSO; >98.9%, Ajax Finechem), silica gel (DAVISIL), ethyl acetate (EtOAc; 95%, Ajax Finechem), n-hexane (95%, Ajax Finechem), dichloromethane (DCM; >99.8%, Aldrich), sodium iodide (NaI; >99.5%, Aldrich), dichlororuthenium(II) (p-cymene) dimer (RuCl2(p-cymene) dimer; 97%, Aldrich), 1,3,5-triaza-7-phosphaadamantane (PTA; 97%, Aldrich), diethyl ether (99%, Ajax Finechem), tin(II) 2-ethyl-hexanoate (SnOct2; 95%, Aldrich), and 3,6-dimethyl-1,4-dioxane-2,5-dione (lactide; Aldrich) were used without any further purification. 2Hydroxyethyl acrylate (HEA; 96%, Aldrich) was destabilized by passing over basic aluminum oxide. Acetone (HPLC grade, Aldrich) was dried for 24 h over 3 Å molecular sieves, activated under vacuum at 100 °C for 12 h. Deuterated dimethylsulfoxide-d6 (DMSO-d6; Cambridge Isotope Laboratories) was degassed by bubbling with Argon for 30 min. 2,2-Azobis(isobutyronitrile) (AIBN; 98%, Fluka) was purified by recrystallization from methanol. The monomer 2chloroethyl methacrylate (CEMA)10 was synthesized according to a literature procedure. The RAFT agent benzyl (2-hydroxyethyl) carbonotrithioate31 was prepared according to literature procedures. Milli-Q water was obtained from an UltraPure water purification system. Analyses. Size Exclusion Chromatography (SEC) with RI and UV/ Vis Detectors. SEC was implemented using a Shimadzu modular system comprising a DGU-12A degasser, LC-10AT pump, SIL-10AD automatic injector, CTO-10A column oven, RID-10A refractive index detector, and a 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 2,6dibutyl-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) Spectrometry. NMR general characterization analyses were conducted using a Bruker Avance DPX 300 spectrometer (1H, 300.2 MHz). Further characterization was 4179


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resolution. Samples were analyzed after digestion in Aqua Regia solution at 80 °C for 1 h. Syntheses. Synthesis of Dichlororuthenium(II)(p-cymene)(1,3,5triaza-7-phosphaadamantane) (RAPTA-C). RAPTA-C was synthesized following a literature protocol.1,34 RuCl2(p-cymene) dimer (0.2 g, 3.3 × 10−4 mol) and PTA (0.1 g, 6.6 × 10−4 mol) were dissolved in methanol (70 mL). The solution was refluxed under argon for 3 h. Red-orange crystals were grown from DCM/heptane in the glovebox and the compound confirmed by X-ray crystallography to be consistent with literature.1 1H NMR (500.17 MHz, DMSO-d6, 25 °C): δ (ppm) = 5.74 (dd, 4H), 4.46 (s, 6H), 4.10 (s, 6H), 2.55 (m, 1H), 1.87 (s, 3H), 1.05 (d, 6H). 31P NMR (300.17 MHz, DMSO-d6, 25 °C): δ (ppm) = −33.40 (RuCl2(p-cymene)(PTA), −30.80 (RuCl(OH)(p-cymene)(PTA), −29.01 (RuCl(H2O)(p-cymene)(PTA). Synthesis of Polylactide (PLA) MacroRAFT Agent. The synthesis of the PLA MacroRAFT agent was described in detail earlier.31 The specific concentrations and conditions used in this study were lactide (15 g, 1.0 × 10−1 mol), RAFT agent (0.3 g, 1.1 × 10−3 mol), degassed SnOct2 (58 mg, 1.4 × 10−4 mol), dry toluene (1 mL), 140 °C reaction temperature, and 2 h reaction time. Mn,theo = 13000 g mol−1, Mn,UV−vis = 26000 g mol−1, Mn,SEC = 15000 g mol−1, PDI = 1.39. Synthesis of 2-Chloroethyl methacrylate. Methacryloyl chloride (14 g, 1.4 × 10−1 mol), 2-chloroethanol (10 g, 1.2 × 10−1 mol), and NEt3 (21 mL) were dissolved in THF (70 mL) and reacted for 1 h while cooling the solution in an ice-bath. The product was extracted with diethyl ether, to give a pale yellow liquid. 1H NMR (300.17 MHz, CDCl3, 25 °C): δ (ppm) = 5.78 (s, 1H), 5.23 (s, 1H), 4.01 (t, 2H), 3.33 (t, 2H), 1.57 (s, 3H). Polymerization of HEA and CEMA with PLA MacroRAFT. 2Hydroxyethyl acrylate (HEA; 0.6 g, 5.3 × 10−3 mol), 2-chloroethyl methacrylate (CEMA; 89 mg, 6.0 × 10−4 mol), fluorescein Omethacrylate (F; 24 mg, 6.0 × 10−5 mol), PLA MacroRAFT (0.3 g, 1.5 × 10−5 mol), and AIBN (0.5 mg, 3.0 × 10−6 mol) as initiator were combined in DMAc (5.0 mL) to give [HEA]/[CEMA]/[F]/ [MacroRAFT]/[AIBN] = 350:50:5:1:0.2. The solution was sealed with a rubber septum, deoxygenated by purging with argon for 1 h, and placed in an oil bath at 60 °C. Aliquots were removed with a degassed needle at progressive time intervals. The polymer was precipitated into cold diethyl ether, dissolved in DCM, and reprecipitated before being dried under vacuum to give polylactideb-(poly(2-hydroxyethyl acrylate-co-2-chloroethyl methacrylate-co-fluorescein methacrylate)) (PLA-P(HEA-CEMA-F)) as pale yellow solids. 1 H NMR (300.17 MHz, DMSO, 25 °C): δ (ppm) = 5.18 (PLA, 1H), 4.74 (HEA, OH), 4.23 (CEMA, 2H), 4.01 (HEA, 2H), 3.80 (CEMA, 2H), 3.54 (HEA, 2H), 2.26 (HEA, 1H), 2−1.58 (HEA, 2H and CEMA, 2H), 1.45 (PLA, 3H), 1.00 (CEMA, 1H). (Table 1).

and precipitated in diethyl ether and dried under vacuum to give a pale yellow solid: polylactide-b-(poly(2-hydroxyethyl acrylate-2-iodoethyl methacrylate-fluorescein O-methacrylate)) (PLA-P(HEA-IEMA-F)). 1 H NMR (300.17 MHz, DMSO, 25 °C): δ (ppm) = 5.18 (PLA, 1H), 4.74 (HEA, OH), 4.23 (IEMA, 2H), 4.01 (HEA, 2H), 3.40 (IEMA, 2H), 3.54 (HEA, 2H), 2.26 (HEA, 1H), 2−1.58 (HEA, 2H and IEMA, 2H), 1.45 (PLA, 3H), 1.00 (IEMA, 1H). Attachment of PTA to Copolymer. The polymer and PTA (8.0 mg, 5.1 × 10−5 mol) were combined in an NMR tube, sealed with a rubber septum, evacuated and filled with Argon. Degassed DMSO-d6 (1 mL) was transferred to the NMR tube using a degassed gastight needle. The clear solution was analyzed via NMR until conversion from PTA to PTA-polymer was stable. RAPTA-C Complexation. The ruthenium dimer (13 mg, 2.0 × 10−5 mol) was subsequently dissolved in the DMSO solution and the orange solution was analyzed via NMR without further purification. Enzymatic Degradation. Aspergillus niger solution (3.5 mL, c = 0.01 g·mL−1 PBS buffer) was added to the micelle solution (2 mL 1.4 mg of polymer in 2 mL PBS buffer). The solutions were vigorously stirred at 37 °C for 2 days at which point both solutions were brown and cloudy. The solutions were subsequently freeze-dried. DMAc was added to the resulting solid to dissolve the polymer only while the salt and the brown lipase remained undissolved. The precipitate was removed by filtration and DMAc was evaporated using a centrifugal evaporator. The resulting solid (with DMAc traces) was dissolved in DMSO-d6 for NMR analysis. Micellisation of Drug-Loaded Copolymers. Typically, 0.5 mL of the DMSO solution, where [polymer] = 10 mg mL−1, was diluted with 1.5 mL DMF. A total of 3.5 mL of Milli-Q water was then added using a syringe pump, at a rate of 0.2 mL hr−1. The deep red-orange solution was subsequently dialyzed against Milli-Q water (MWCO = 3500 g mol−1) to give a pale orange solution, which was diluted to 10 mL. Samples were analyzed using DLS, TEM, ICPOES, and TGA and used for subsequent cell studies. In Vitro Cell Culture Assays. Human ovarian carcinoma A2780, A2780cis and Ovcar-3 cells were cultured in 75 cm2 tissue culture flasks with RPMI 1640 medium supplemented with 10% fetal bovine serum, 4 mM glutamine, 100 U/mL penicillin, 100 μg mL−1 streptomycin, and 1 mM sodium pyruvate at 37 °C under an atmosphere of 5% CO2. After reaching 70% confluence, the cells were washed with phosphate buffered saline (PBS) and collected by trypsin/EDTA treatment. The cell suspension was used for the evaluation of cellular responses. Cytotoxicity Evaluation. A2780, A2780cis, and Ovcar-3 cells were used for cytotoxicity analyses. The cells were seeded in 96-well cell culture plates at 4000 cells/well and cultured at 37 °C for 1 day. Solutions were sterilized by UV irradiation for one hour in a biosafety cabinet and then serially diluted (2× dilution) with sterile water and incubated for 2 h. The medium in the cell culture plate was discarded and 100 μL of fresh 2× concentrated RPMI 1640 serum medium was added. The samples were added into the plate at 100 μL per well for 72 h. The cell viability was measured using a WST-1 assay (Roche Diagnostics). This is a colorimetric assay for the quantification of cell viability and proliferation that is based on the cleavage of a tetrazolium salt (WST-1) by mitochondrial dehydrogenases in viable cells. Increased enzyme activity leads to an increase in the amount of formazan dye, which is measured with a microplate reader. After incubation for three days, the culture medium was removed and 100 μL fresh medium was added along with 10 μL of WST-1. The plates were then incubated for an additional 4 h at 37 °C. After incubation, the absorbance of the samples against the background control on a Benchmark Microplate Reader (Bio-Rad) was obtained at a wavelength of 440 nm with a reference wavelength of 650 nm. Four wells under each condition were used for the measurement to calculate the means and standard deviations. 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 assay was performed as described by Franken et al.35 with some modifications. A total of 40000 cells were seeded to each well of a 24-well tissue culture plate

Table 1. Polymerization of HEA and CEMA with PLA MacroRAFT Agent in N,N-Dimethyl Acetamide at 60 °Ca polymer

time (h)

XHEA (%)

XCEMAb (%)

A

3

21

49

B

5

40

90

PLAxP(HEAy-CEMAz) (NMR) PLA347-HEA74CEMA25 PLA347-HEA140CEMA45

Mn,SEC (g mol−1)

PDI

28000

1.34

30500

1.38

a

[HEA]/[CEMA]/[F]/[MacroRAFT]/[AIBN] = 350:50:5:1:0.2. bX = monomer conversion.

Conjugation of RAPTA-C to (PLA-P(HEA-CEMA-F)) Copolymer. Following the procedure detailed in Blunden et al.,10 RAPTA-C was attached via a three-step procedure: Finkelstein Reaction. Polymers were transferred to a 50 or 100 mL round-bottomed flask, fitted with a reflux condenser, and dissolved in dry acetone (A, 3 mL; B, 5 mL) with vigorous stirring and heat (60 °C). Sodium iodide (A, 59 mg; B, 75 mg) was subsequently added and the solution turned cloudy. The solution was refluxed at 70 °C for 3−5 days, at which time the solution was cloudy and yellow. It was cooled 4180


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and incubated overnight. Samples of micelles A, micelles B, and RAPTA-C were added to the cells such that the Ru concentration of each well was 25 μM. After a 4 h incubation, the cells were washed thrice with PBS and collected by trypsin treatment. Single cell suspension was then plated in six-well plates with fresh PRMI 1640 medium (400 cells per well), and the medium was changed every three days. After seven days of incubation, the cells were washed thrice with PBS and incubated with a staining solution for 1 h. The staining solution is an aqueous solution with 0.5% crystal violet and 2.5% glutaraldehyde. The plates were washed five times with water and airdried. The colony over 50 cells was counted using Image J. 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

(1)

Figure 1. SEC traces of the polymerization of HEA and CEMA with PLA MacroRAFT agent in N,N-dimethyl acetamide at 60 °C; [HEA]/ [CEMA]/[F]/[MacroRAFT]/[AIBN] = 350:50:5:1:0.2.

(2) Laser Scanning Confocal Microscopy. A2780 cells were seeded in 35 mm Fluorodishes (World Precision Instruments) at a density of 60000 per dish and cultured for three days with RPMI 1640 medium supplemented with 10% fetal bovine serum. Micelle solution was loaded to the cells at a working concentration of 100 μg mL−1 polymer and incubated at 37 °C for 3 h. After incubation, the cells were washed thrice with PBS, stained with 2.0 μg mL−1 Hoechst 33342 (Invitrogen) for 5 min, and washed thrice with PBS. Finally, 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 rinsed with PBS. The dishes 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. Fluorescence Intensity. The fluorescence intensities of the micelles were measured under a Cary Eclipse Fluorescence Spectrophotometer (Aglient Technologies). The excitation and emission wavelengths were 490 and 512 nm, respectively. The experiments were carried out in triplicate. The standard deviation was calculated from three independent measurements. Cellular Uptake of Ruthenium. A2780 cells were seeded in six-well plates and incubated at 37 °C with 5% CO2 for one day prior to micelle treatment. During treatment, the medium was replaced with incubation media containing either (a) micelles A, (b) micelles B, or (c) RAPTA-C in RPMI-1640 at 37 °C for 2 h. The cell monolayer was washed five times with cold PBS and treated with trypsin/EDTA to detach the cells. The cells were digested with Aqua Regia at 80 °C for 1 h and the Ru concentration determined using ICPMS. A one-way analysis of variance (ANOVA) was performed on the data to reveal statistical differences. A Tukey’s post hoc test was followed for pairwise comparison if there was a significant difference in ANOVA.

Figure 2. NMR spectra of the polymerization of HEA and CEMA with PLA MacroRAFT agent in DMSO-d6 at 25 °C; [HEA]/[CEMA]/[F]/ [MacroRAFT]/[AIBN] = 350:50:5:1:0.2; A, 1 h; B, 3 h; C, 5 h.

incorporated into the polymer slower than the methacrylate, the hydrophilic block has a slight gradient structure with CEMA slightly enriched at the periphery of the resulting micelle. The higher reactivity of HEA compared to methacrylate is in good agreement with the literature although MMA was used as the comonomer in the reported case.36 Reactivity ratios of r1 = 0.328 and r2 = 1.781 for HEA and MMA were reported. However, CEMA may have a slightly different effect on the polymerization and it also needs to be considered that the PLA macroRAFT agent could influence the consumption of the two monomers.37 The molecular weight of the polymer increased linearly with monomer conversion. The molecular weight distribution (PDI) remained narrow, although slight broadening due to low molecular weight tailing can be observed at higher conversions (Table 1 and Figure 1). The broadening is typical for RAFT polymerizations where

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RESULTS AND DISCUSSION Polymer Synthesis. A polylactide macroRAFT agent (PLA) was polymerized via ring-opening polymerization using a reactive RAFT agent as initiator, following a modified literature procedure.31 The structure was confirmed by 1H NMR and the molecular weight of PLA was determined using the signals of the RAFT functionality compared to the PLA signals. Subsequently, the PLA was chain extended with a mixture of HEA, CEMA and 1% fluorescein O-methacrylate (Figure 1). Incorporation of the fluorescing monomer was deemed necessary to monitor the cell uptake. The consumption of both monomers was monitored over time using 1H NMR analyses (Figure 2 and Table 1). Because the acrylate was 4181


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These values were consistent with the conjugation to the homopolymer in Blunden et al.10 As found previously, the polymer−PTA and polymer−RAPTA-C peaks were broad, due to the inherent rigidity of polymers. Furthermore, the residual unreacted PTA underwent a complexation reaction producing RAPTA-C on addition of the RuCl2(p-cymene) dimer, shown by the 31P chemical shift at −103 ppm moving to −33 ppm. A highly complex 1H NMR spectra with multiple spin systems results from the N-alkylation of PTA due to the decrease in molecular symmetry.40 Due to the combined characteristic broadening from polymer entities, the 1H spectrum is difficult and tedious to interpret. However, the pcymene region for the reactions provided further evidence for the conjugation reaction as the peaks for the residual unreacted RuCl2(p-cymene) dimer, RAPTA-C and the broad peak for the conjugated RAPTA-C are evident (Figure 5). Also, consistent with Blunden et al.,10 the first step was found to be the ratelimiting step in the conjugation. Peak “e” in Figure 3 no longer appears at 4.23 ppm after the addition of PTA, indicating the complete conversion of all iodide units to PTA. All conjugated and unconjugated PTA was subsequently consumed during the RAPTA-C complexation, giving both free RAPTA-C and conjugated RAPTA-C. Free RAPTA-C was removed by dialysis during micelle formation and dialysis. Both polymers A and B led to similar results. The resulting structures had a polymer composition of PLA347-b-P(HEA74(RAPTA-C-EMA)25) (polymer A) and PLA347-b-P(HEA140(RAPTA-C-EMA)45) (polymer B). Micellization of Drug-Loaded Amphiphilic Polymers. After drug conjugation, the DMSO solutions were diluted with DMF and then water was slowly added and the mixture dialyzed to remove DMSO, DMF, and unreacted RAPTA-C. It was necessary to dilute the initial DMSO solution to facilitate the formation of well-defined micelles. High polymerization concentrations in DMSO can often result in particle sizes of broad distribution. The DMSO solution was diluted with DMF, but it is possible to use DMSO instead. The dialyzed pale orange solutions were analyzed using DLS (Figure 6 and Table 2) and TEM (Figures 7 and 8) to determine micelle size and homogeneity. The DLS PDI indicates that the micelle size is not fully uniform, consistent with TEM images that show the presence of smaller micelles. The size distribution found by DLS was larger than the images obtained by TEM, because micelles swell in solution and contract when dried. However, the large differences between diameters obtained from TEM and DLS cannot be explained by hydration alone and may be indicative of substantial aggregation in solution (Table 2). The tailing in the DLS size distribution can be attributed to aggregation of micelles, as shown in Figure 7b and Figure 8a. This aggregation was found to be more prominent in micelle B samples, clearly indicated by the Z-average and PDI (Table 2). This may influence the cytotoxic effect and cell uptake displayed by each of the micelle samples. Interestingly, the average micelle size increases after RAPTA-C conjugation, but the PDI decreases indicating better and more homogeneous micelle formation. TEM images clearly show the core−shell structure of the micelles, for both stained and unstained samples. This provides further evidence that RAPTA-C is indeed conjugated and located in the shell of the micelle. The shell appears darker than the core in the stained samples, due to the higher electron density of ruthenium. However, the converse is evident for the unstained samples, where the core appears darker.

termination reactions can be suppressed, but cannot be fully avoided.27,28 As a result, many RAFT polymerizations result in molecular weight distributions with a tail of terminated (“dead”) polymer. In addition, it is also possible that some PLA chains are inactive and do not undergo extension during the RAFT process. As discussed in the earlier literature, not all RAFT agents initiate the polymerization.31 RAPTA-C Conjugation. Two polymers were isolated for further modification (labeled polymers A and B in Table 1). The chloride end groups of the CEMA monomer were converted to iodide end groups using the Finkelstein reaction.38 The 1H NMR signal corresponding to CH2−Cl at 3.80 ppm shifted upfield to 3.40 ppm when the chloride was substituted with iodide. This signal could be monitored and integrated relative to the adjoining CH2 signal at 4.23 ppm, which did not move (Figure 3). The reaction was stopped when complete halogen substitution had been achieved.

Figure 3. 1H spectra showing the attachment of RAPTA-C to PLAP(HEA-IEMA-F) in DMSO-d6 at 25 °C; A: polymer before Finkelstein, B: polymer after Finkelstein, C: PTA added to polymer in solution, D: dimer added to solution. See Figure 2 for the chemical structure of the full polymer and the assignment of each number.

RAPTA-C was then attached to the polymer using the method detailed in Blunden et al.10 PTA was initially bound via a substitution reaction of iodide with nitrogen. Ruthenium dimer was subsequently added, resulting in the complexation formation of RAPTA-C. The reaction was followed using 1H (Figure 3) and 31P NMR (Figure 4 and Supporting Information, Figure S1). The 31P chemical shift of the initial PTA peak at −103 ppm shifted to −84 ppm (Figure 4 and Supporting Information, Figure S1, bottom spectra) as the alkylation of PTA induces a deshielding of the 31P signal.39 The subsequent complexation induces a very large deshielding of the corresponding signal, shown by the peak shift to −18 ppm (Figure 4 and Supporting Information, Figure S1, top spectra). 4182


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Figure 4. 31P spectra showing the attachment of RAPTA-C to PLA-P(HEA-IEMA-F) polymer A via a two-step, one-pot synthesis in DMSO-d6 at 25 °C; bottom: PTA added to polymer in solution, top: dimer added to solution.

Figure 5. p-Cymene region of the 1H spectra showing the attachment of RAPTA-C to PLA-P(HEA-IEMA-F), in DMSO-d6 at 25 °C. The broad polymer, residual unreacted RuCl2(p-cymene) dimer and RAPTA-C p-cymene peaks are at 5.95, 5.86, and 5.75 ppm, respectively; bottom: polymer A, top: polymer B.

Table 2. Micelles A and B Analyzed by DLS and TEM at 25°C, with and without Conjugated RAPTA-C

micelles A (without RAPTA-C) micelles B (without RAPTA-C) micelles A (with RAPTA-C) micelles B (with RAPTA-C)

Figure 6. Average number distribution for micelles A and B analyzed by DLS at 25 °C.

Z-average (d· nm) from DLS

PDI

160.7

0.439

100

235.5

0.601

150

251.8

0.332

20.8 ± 0.65

90

554.5

0.370

18.0 ± 0.45

100

zeta potential (mV)

diameter (d· nm) from TEM

digestion using Aqua Regia. TGA analyses were conducted by pouring a sample of the micelle solution used for cell studies into the TGA pan and heated until dry. It was subsequently analyzed via TGA to determine the Ru concentration in the sample. The remaining solid was then recalculated to obtain information about the original concentration. Prior to the TGA analysis, the RAPTA-C drug was analyzed. From the TGA analysis of RAPTA-C, it was found that RuCl3 is the final residue since Ru in RAPTA-C is only 21%, while the weight

ICPOES and TGA were used to determine the final ruthenium content in each sample. The solutions with a known amount of polymer in DMSO were diluted and then dialyzed against water similar to the solution preparation for the in vitro studies. The aqueous solutions had to be further diluted and treated for ICPOES analysis in two ways: with and without 4183


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Figure 7. Polymer A (a) and (b) sample drop-loaded onto grid and air-dried. (c, d) Sample drop-loaded onto grid, air-dried and stained with phosphotungstic acid.

residue of 44% equates to RuCl3 (107.43 g mol−1). The measured amounts using all techniques are within the same order of magnitude although some errors are due to the multitude of dilution steps. Interestingly, the theoretical content of micelle A is close to the values measured indicating the complete reaction of all iodine functionalities. In contrast, the measured Ru-content in micelle B is slightly higher than the theoretical amount (Table 3). This may either be to errors during the sample preparation or maybe some free RAPTA-C is still in the polymer and was not fully removed by dialysis. Degradation of Micelles. According to our hypothesis depicted in Scheme 1, the micelle should disassemble once the PLA block underwent hydrolytic cleavage in the digestive environment of the cells. To simulate this environment the micelle were stirred with hydrolases, here Aspergillus niger, for 2 days at 37 °C. The polymer was separated from the enzyme and analyzed using 1H NMR (Supporting Information, Figure S2). The enzymatic degradation of the block copolymer could be confirmed by the disappearance of the PLA signals. In addition, traces of the resulting lactic acid and lactate were still visible, although most of the degradation products were most likely removed during the drying step under high vacuum. The 1 H NMR signals belonging to PHEA were still visible revealing that the ester bond of this repeating unit remains largely untouched. The resulting polymer was now fully water-soluble. As a result, DLS did not expose any particles with sizes >10 nm confirming the absence of micelles.

In Vitro Cell Studies of Drug-Loaded Micelles. Micelles self-assembled from polymers A and B were tested against ovarian A2780, cisplatin-resistant ovarian A2780cis and ovarian OVCAR-3 cell lines, and compared with the drug RAPTA-C. The toxicity of the polymer prior to drug conjugation was in addition investigated and found to be nontoxic at concentrations up to 250 μg mL−1 (Supporting Information, Figure S3). Figure S3 (Supporting Information) shows the survival rate of both polymers before RAPTA-C attachment, which is close to 100% rendering the polymer itself nontoxic. A significant decrease (∼10×) in the IC50 value for micelles was found for all cell lines, compared with RAPTA-C alone (Table 4, Supporting Information, Figures S4−6). Micelles A displayed the highest toxicity against all cell lines, with a marked difference between the two micelle samples on A2780cis (Table 4). Micelles assembled from polymer A had the highest toxicity. This increased toxicity of micelles A compared to micelles B cannot be immediately explained. Higher toxicities are often associated with higher cell uptake of the drug delivery system, as it has recently been demonstrated with a drug delivery system for cisplatin.42 The uptake of the drug carrier was therefore monitored using confocal fluorescence microscopy. The micelles composed from polymers A and B were incubated with A2780 cells for three hours. The cell nuclei (blue) and lysosomes (red) were subsequently stained to help identify the location of the green fluorescent micelles (Figure 9). The 4184


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Figure 8. Polymer B (a) and (b) Sample drop-loaded onto grid and air-dried. (c) and (d) Sample drop-loaded onto grid, air-dried and stained with Phosphotungstic acid.

micelles were colocalized with the lysosomes, indicative of an endocytic pathway. Upon initial inspection, the green fluorescence seems to be more intense inside the cells when employing polymer A as the drug carrier. Since the fluorescence of the micelle samples at the concentrations used for confocal imaging is statistically similar (Table 5), it can be inferred that micelles prepared from polymer A induce a better cell uptake. Although the confocal fluorescent studies give a first indication of a better uptake of micelles A, a more quantitative approach is necessary. Toxicity can be related directly to the amount of drug that enters the cells. The final amount of ruthenium internalized in A2780 cells was analyzed by ICPMS (Figure 10). Micelles had a significantly higher uptake than RAPTA-C. Also consistent with the increased cytotoxicity, micelles prepared from polymer A had a significantly higher uptake than micelles prepared from polymer B. A colony formation assay was used to evaluate the effects of cell regrowth after exposing them to ruthenium. Cells were incubated with a solution of 25 μM RAPTA-C and the equivalent amount of drug in the micelle for four hours. The cells were subsequently washed to remove the micelles and the drug and plated in six well plates at a very low density for seven days to allow colony formation (Figure 11). Fewer colonies were formed by both cell lines, after treatment with micelles A at a Ru concentration of 25 μM. In contrast, the regrowth of colonies was visibly higher after treatment with micelles B and RAPTA-C. The colony formation assay was also assessed via

Table 3. Ruthenium Content in Polymeric Micelles was Determined Using ICPOES and TGA Analyses TGA

theoretical

sample

[Ru] (mmol L−1)a

ICPOES [Ru] (mmol L−1)b

[Ru] (mmol L−1)c

[Ru] (mmol L−1)d

polymer A polymer B

5.0 × 10−2 1.2 × 10−1

6.7 × 10−2 1.2 × 10−1

3.4 × 10−2 1.0 × 10−1

5.1 × 10−2 0.8 × 10−1

ICPMOES without digestion. ICPMOES results in mg L−1 were converted to mmol L−1 using the molecular weight of Ru = 101.07 g mol−1. bICPMOES with Aqua Regia digestion. ICPMOES results in mg L−1 were converted to mmol L−1 using the molecular weight of Ru = 101.07 g mol−1. cTGA solution analysis gave Ru weight in mg and was converted to mmol L−1 using the molecular weight of RuCl3 = 207.42 g mol−1. dCalculated using the theoretical composition of the polymer. a

Table 4. IC50 (μM) Values of RAPTA-C and Micelles SelfAssembled from Polymers A and B, against Ovarian A2780, Ciplatin-Resistant Ovarian A2780cis, and Ovarian OVCAR-3 Cancer Cell Lines IC50 (μM)

A2780

A2780cis

OVCAR-3

RAPTA-C literature RAPTA-C micelle A micelle B

353 ± 1441 271 15 51

>20023 266 24 101

300 46 61

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Figure 9. Confocal microphotographs of A2780 cells after incubation with micelles at 37 °C for 3 h. Polymers (green) were labeled with fluorescein. Cell nuclei (blue) were stained with Hoechst 33342. Lysosomes (red) were stained with LysoTracker Red DND-99. Scale bar = 5 μm.

It is evident that both micelles enhance the performance of RAPTA-C due to better cell uptake. Micelles A, prepared from PLA347-b-P(HEA74-(RAPTA-C-EMA)25), enhance this effect significantly compared to micelles B, prepared from PLA347-bP(HEA140-(RAPTA-C-EMA)45). The key is the better uptake of micelles A compared to micelles B. Micelles B are slightly larger, but this size increase is not pronounced enough to explain the difference. However, a remarkable difference between both micelles is the strong tendency of micelles B to aggregate. This is potentially due to the longer hydrophilic water-soluble blocks which result in more star-like micelles, which are able to promote entanglements between micelles. These entanglements may then be held together by the hydrophobic nature of the drug, which is in higher abundance in micelle B. Comparison of Micelles A and B. There is a clear difference in the behavior of micelle A and B. Cell uptake of micelle A is more efficient resulting in a higher concentration of Ru inside the cell (Figure 10). The better uptake has a direct effect on the toxicity and the long-term effect of both micelles. Better uptake of the drug carrier translates directly into lower IC50 values. It can even be argued that there is a linear relationship between uptake and toxicity and the subsequent inhibition of further cell growth even when the cells are washed with cell growth media only. The origin of this difference is not immediately evident. Micelle B has a larger hydrophilic block and higher drug loading. However, this seems to have only little effect at first sight on the physical properties on the micelle. According to TEM results (Table 2), both micelles have similar sizes (90 and 100 nm, respectively). In theory, micelles of similar sizes should have comparable cell uptakes. However, the larger amount of hydrophobic drugs in micelle B does probably result in a more collapsed shell and even more to a much higher tendency to aggregate. The measure diameter in the hydrated state is well above 500 nm (Table 2), which coincides with the formation of a cloudy solution. Sonication of the solution can slightly reduce the hydrodynamic diameter since the aggregates

Table 5. Fluorescence Intensity of Polymer and Micelle Samples at Concentrations Used for Microscopy Imaging fluorescence intensity

micelle A

micelle B

polymer A

polymer B

avg std dev (n = 3)

90.49 0.63

92.14 0.38

48.65 1.34

56.94 0.84

Figure 10. Concentration of ruthenium per million A2780 cells measured by ICPMS; mean ± SD, n = 3; ***P < 0.001.

the surviving fraction, which is the ratio of colonies formed after treatment and the number of cells seeded that can become colonies. The surviving fraction in Table 6 reveals that 25 μM Ru from micelles A inhibit the colony formation. There is no obvious inhibition effect of micelles B and RAPTA-C (Ru at 25 μM) on the colony formation ability. These results, thus, suggest Micelle A treatment is highly effective in suppressing the colony-forming ability of A2780 and A2780cis cells. This result can be understood considering the retention time of a micelle inside the cell. While drugs leave the cell often very quickly, micelles have been observed to reside inside the cell at an extended period of time with only little evidence of exocytosis. Cells that may not have succumbed to apoptosis yet still carry the toxic load, which eventually leads to cell death affecting the formation of colonies.41 4186


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Figure 11. Colony formation of A2780 (A) and A2780cis (B) cells; scale bar = 10 mm.

cytotoxicity, the best cell uptake of ruthenium and were highly effective in suppressing the colony-forming ability of cells. This may due to better and increased homogeneity of micelle formation and decreased aggregation of these micelles. Further work will entail a detailed assessment of the metastatic effects of the macromolecular RAPTA-C chemotherapeutics.

Table 6. Surviving Fraction of Cancer Cells after a 4 h Exposure to Micelles A, B, and RAPTA-C micelle A micelle B RAPTA-C

Ru (μM)

A2780

A2780cis

OVCAR-3

25 25 25

0.36 1.04 1.08

0.33 0.91 0.90

0.88 1.15 1.31

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could be broken up for a short time. However, these aggregates quickly form again in water as well as buffer or cell growth media. Particles of sizes well above 500 nm, however, are not efficiently taken up by cells. This highlights the importance of high stability of micelles against aggregation to ensure the effective delivery of the drug into the cell.

ASSOCIATED CONTENT

S Supporting Information *

31

P NMR of the conjugation of RAPTA-C to the polymer, 1H NMR of the polymer before and after enzymatic degradation, and cytotoxicity data of the polymer before and after RAPTA-C conjugation vs concentration using various ovarian cancer cell lines (A2870, A2870cis, OVCAR-3). This material is available free of charge via the Internet at http://pubs.acs.org.

■

CONCLUSIONS An amphiphilic block copolymer capable of self-assembling into polymeric micelles was identified as an appropriate drug carrier for RAPTA-C. Following from previous work, it was known that RAPTA-C could be attached to a polymer moiety via nucleophilic substitution of an available halogen with an amide in the PTA ligand. A series of water-soluble biodegradable block copolymers were designed incorporating the CEMA monomer to allow for conjugation of RAPTA-C. The chloride functionality could be successfully converted to iodide using Finkelstein reaction. This detour was necessary since the direct polymerization of 2-iodo ethyl methacrylate is not possible due to the high chain transfer constant of iodides, which in fact utilized in iodine-transfer polymerization.43 Two of these polymers were used to test the RAPTA-C conjugation, micellization, and subsequent cytotoxicity and cell uptake of these moieties. Confocal microscopy images confirmed cell uptake of the micelles into the lysosome of the cells, indicative of an endocytic pathway. On average, across the tested cell lines, a 10-fold increase in toxicity was found for the macromolecular drugs when compared to the RAPTA-C molecule. Furthermore, the cell uptake of ruthenium was analyzed and a significant increase was found for the micelles compared to RAPTA-C. Notably, micelles prepared from the polymer containing fewer HEA units had the highest

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +61-2-93856250. Tel.: +61-2-93854344. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS

■

REFERENCES

M.H.S. thanks the ARC (Australian Research Council) for funding in the form of a Future Fellowship (FT0991273). The authors would like to thank the UNSW Mark Wainwright Analytical Centre for support.

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