Article pubs.acs.org/cm
Simple Synthesis of Cladribine-Based Anticancer Polymer Prodrug Nanoparticles with Tunable Drug Delivery Properties Yinyin Bao, Tanguy Boissenot, Elise Guégain, Didier Desmael̈ e, Simona Mura, Patrick Couvreur, and Julien Nicolas* Institut Galien Paris-Sud, CNRS UMR 8612, Univ Paris-Sud, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92290 Châtenay-Malabry, France S Supporting Information *
ABSTRACT: Polymer prodrugs based on cladribine (CdA) as an anticancer drug have been prepared by growing short, well-defined polyisoprene (PI) chains (with number-average molar mass = 1420−4980 g mol−1 and dispersity = 1.09−1.20) from CdA-bearing alkoxyamine by nitroxide-mediated polymerization. Nanoparticles were formed by nanoprecipitation into water of the resulting CdA-PI conjugates and exhibited long-term colloidal stability in different media, tunable colloidal characteristics, drug release profiles, and anticancer activities in vitro, simply by modulating the drug/polymer linker, the polymer chain length and the formulation parameters. No obvious in vivo toxicity to mice was observed following repeated intravenous injections.
■
INTRODUCTION Cladribine (CdA, 2-chloro-2′-deoxyadenosine), which is an analogue of the endogenous purine nucleoside 2′-deoxyadenosine (dA), inhibits DNA synthesis, causes cell death, and induces apoptosis. CdA is used as a first-line therapy in hairy cell leukemia and B-cell chronic lymphocytic leukemia.1−5 Moreover, CdA also has shown significant in vitro activity against primary brain tumor cells.6 However, because CdA is administered at high doses, because of its rapid clearance from the bloodstream,7 it is cytotoxic to resting and proliferating cells and may cause temporary weakening of the immune system, thus resulting in bone marrow suppression and opportunistic infections related to immune suppression.8,9 Consequently, it is of great importance to find strategies to increase the circulation time and decrease the dosage of CdA to avoid toxicity to healthy cells, as well as to enhance its efficacy. In recent years, polymer nanocarriers have emerged as promising drug delivery systems for the treatment of severe diseases such as cancer, infections, and neurodegenerative disorders.10−14 Drugs are physically encapsulated into these systems, which not only protect them from metabolization and/or rapid clearance from the body, but also protect healthy tissues from the drug’s inherent toxicity. In the case of drugloaded polymer nanoparticles for cancer therapy, anticancer drugs are usually encapsulated during the formulation of preformed polymers in aqueous solution. Although promising results have been witnessed by this preparation method,14,15 important limitations still need to be overcome (e.g., the “burst release”, the achievement of poor drug loadings, etc.) to promote further translation to the clinic and to reach the market. © 2016 American Chemical Society
To circumvent the above-mentioned limitations, a covalent linkage may be established between the drug and the polymer, resulting in polymer prodrugs.16 This can be performed either from preformed polymers (“graf ting to”),17−32 or by synthesizing drug-bearing monomers for subsequent polymerization (“grafting through”).33−40 More recently, the “drug-initiated” method,41 which consists in using (functionalized) drugs as polymerization initiators, has been developed to prepare welldefined polymer prodrug nanoparticles, using either native functional groups for ring-opening polymerization,42−46 or prefunctionalized drugs for reversible deactivation radical polymerization (RDRP) techniques, respectively.47−51 Recently, our group reported the design of a gemcitabine (Gem)-bearing alkoxyamine48 or chain transfer agent47,49 to prepare well-defined amphiphilic polymer prodrug nanoparticles by nitroxide-mediated radical polymerization (NMP) or reversible addition−fragmentation chain transfer (RAFT) polymerization, respectively. To mimic naturally occurring terpenoids, short polyisoprene (PI) or poly(squalenyl methacrylate) (PSqMA) chains were grown from the drug in a controlled fashion. The resulting Gem-PI and Gem-PSqMA conjugates led to self-stabilized nanoparticles (conversely to ROP-derived polymer prodrugs, which necessitated poststabilization by PEG-based surfactants)42 and exhibited significant anticancer activity in vitro and in vivo. However, the robustness and the versatility of the drug-initiated method from RDRPand, therefore, its future potential in drug Received: June 21, 2016 Revised: August 4, 2016 Published: August 22, 2016 6266
DOI: 10.1021/acs.chemmater.6b02502 Chem. Mater. 2016, 28, 6266−6275
Article
Chemistry of Materials
Size Exclusion Chromatography (SEC). Size exclusion chromatography (SEC) was performed at 30 °C with two columns from Polymer Laboratories (PL-gel MIXED-D; 300 mm × 7.5 mm; bead diameter = 5 mm; linear part, 400 to 4 × 105 g mol−1) and a differential refractive index detector (SpectraSystem RI-150 from Thermo Electron Corp.). The eluent was chloroform at a flow rate of 1 mL min−1 (Waters 515 pump) and toluene was used as a flow-rate marker. The calibration curve was based on polystyrene (PS) standards (peak molar masses, Mp = 162−523 000 g mol−1) from Polymer Laboratories. A polyisoprene (PI) calibration curve was constructed by converting the PS standard peak molecular weights (MPS) to PI molecular weights (MPI) using Mark− Houwink−Sakurada (MHS) constants determined for both polymers in CCl4 at 25 °C. For PI, the MHS constants used were KPI = 2.44 × 104 and αPI = 0.712. For PS, KPS = 7.1 × 104 and αPS = 0.54 (MW < 16 700 g mol−1) or KPS = 1.44 × 104 and αPS = 0.713 (MW > 16 700 g mol−1).53 This technique allowed the number-average molar mass (Mn), the weight-average molar mass (Mw), and the dispersity (Đ = Mw/Mn) to be determined. Dynamic Light Scattering (DLS) and Zeta Potential. Nanoparticle diameters (Dz) and zeta potentials (ζ) were measured by dynamic light scattering (DLS) with a Nano ZS from Malvern (scattering angle = 173°) at a temperature of 25 °C. The surface charge of the nanoparticles was investigated by ζ-potential (mV) measurement at 25 °C after dilution with 1 mM NaCl, using the Smoluchowski equation. For the long-term colloidal stability study, nanoparticles were stored in the refrigerator at 4 °C between each measurement. Transmission Electron Microscopy (TEM). The morphology of the different nanoassemblies was examined by cryogenic transmission electron microscopy (cryo-TEM). Briefly, 5 μL of the nanoparticle suspension (5 mg mL−1) was deposited on a Lacey Formvar/carbon 300 mesh copper microscopy grid (Ted Pella). Most of the drop was removed with a blotting filter paper and the residual thin film remaining within the holes was vitrified by plunging into liquid ethane. Samples were then observed using a JEOL Model 2100HC microscope. Synthesis Methods. Synthesis of CdA-TBDMS. Cladribine (0.85 g, 3.0 mmol), imidazole (0.30 g, 4.5 mmol), and tert-butyldimethylsilyl chloride (0.58 g, 3.9 mmol) were dissolved in anhydrous DMF (5 mL) under an argon atmosphere. After stirred at room temperature for 20 h, the mixture was poured into 30 mL of EtOAc. The organic phase was washed with NaHCO3 aqueous solution and brine before being dried over MgSO4. The residue was concentrated under reduced pressure and purified by flash chromatography (SiO2, from EtOAc/ Et3N = 200:1 to EtOAc/MeOH = 10/1, v/v) to give 0.9 g CdATBDMS as a white solid. Yield: 75%. 1H NMR (300 MHz, CDCl3): δ = 8.11 (s, 1H), 6.43 (t, 1H, J = 6.6 Hz), 5.71 (s, 2H), 4.67 (m, 1H), 4.07 (q, 1H, J = 3.6 Hz), 3.90 and 3.88 (2 dd, 2H, J = 3.3 Hz), 2.62− 2.73 (m, 1H), 2.49−2.57 (m, 1H), 0.91 (s, 9H), 0.11 and 0.12 (2 s, 6H). 13C NMR (75 MHz, CDCl3 + DMSO): δ = 161.5, 158.5, 155.1, 143.6, 123.3, 92.4, 88.8, 75.7, 68.1, 30.8, 23.1. MS (ESI): m/z = 422.1 (M + Na)+. Synthesis of CdA-AMA-SG1. AMA-SG1 (0.37 g, 1.0 mmol), DMAP (0.18 g, 1.5 mmol), and EDC·HCl (0.28 g, 1.5 mmol) were dissolved in anhydrous CH2Cl2 (5 mL), and mixed in a reaction flask under an argon atmosphere at room temperature. After 15 min, a solution of CdA-TBDMS (0.2 g, 0.5 mmol) in DMF (1 mL) was added dropwise. After stirred at 30 °C for 18 h, the mixture was poured into 30 mL of EtOAc. The organic phase was washed with NaHCO3 aqueous solution and brine before being dried over MgSO4. The residue was concentrated under reduced pressure and purified by flash chromatography (SiO2, petroleum ether/EtOAc = 1/1, v/v) to give 0.21 g CdA-AMA-SG1 as a white solid. Yield = 63%. 1H NMR (300 MHz, CDCl3): δ = 8.19 (s, 1H), 6.46 (m, 1H), 5.77 (s, 2H), 5.37−5.39 (m, 1H), 4.62−4.67 (m, 1H), 3.91−4.36 (m, 7H), 3.39 and 3.35 (2 d, 1H, J = 26.5 Hz), 2.59−2.92 (m, 2H), 1.53 and 1.52 (2 d, 3H, J = 7.2 Hz), 1.26−1.35 (m, 6H), 1.13−1.19 (m, 18H), 0.91 and 0.92 (2 s, 9H), 0.11 and 0.12 (2 s, 6H). 13C NMR (75 MHz, CDCl3): δ = 172.5, 155.9, 154.1, 150.7, 139.0, 118.4, 85.7 (d), 84.3 (d), 78.4, 75.7 (d), 70.1, 63.6, 62.0, 61.7, 61.4, 59.1, 38.9, 35.4 (dd), 30.5 (d), 30.0 (d), 28.1 (d), 25.9, 18.3, 18.0, 16.6, 16.5, 16.2, 16.1, −5.4, −5.5.
deliveryare mainly dependent on whether it is applicable to other polymer promoieties and drugs. Yet, this technique has only been applied to Gem. Herein, we report the simple design of well-defined amphiphilic CdA-based polymer prodrug nanoparticles using the drug-initiated method to both tackle the important abovementioned limitations observed with CdA and demonstrate the versatility of this approach to other drugs, thus conferring a significant degree of flexibility (Figure 1). By modulating the
Figure 1. Drug-initiated synthesis of polymer prodrug nanoparticles based on cladribine (CdA) with different CdA/polymer linkers.
drug/polymer linker, the polymer chain length, and the formulation parameters, the resulting nanoparticles exhibited tunable size and drug release profiles, high colloidal stability in aqueous solution, and significant anticancer activity. To the best of our knowledge, this is the first example of CdA-conjugated polymer prodrugs, which provides a new pathway toward efficient and safe drug delivery systems for CdA.
■
EXPERIMENTAL SECTION
Materials. Cladribine was purchased from Sequoia Research Products, Limited (U.K.). N-tert-Butyl-N-[1-diethylphosphono-(2,2dimethylpropyl)] nitroxide (SG1, 85%) was obtained from Arkema (France). Diglycolic anhydride and tetrabutylammonium fluoride were purchased from Thermo Fisher Scientific (USA). 2-[N-tert-butyl-N-(1diethoxyphosphoryl-2,2-dimethylpropyl)aminoxy] propionic acid (AMA-SG1) was prepared according to a published method.52 LysoTracker Red was purchased from Life Technologies. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Sigma (France). Penicillin was purchased from Lonza (Verviers, Belgium). All other materials were purchased from Aldrich at the highest available purity and used as received. Analytical Methods. Nuclear Magnetic Resonance Spectroscopy (NMR). NMR spectroscopy was performed in 5-mm-diameter tubes in CDCl3 or DMSO-d6 at 25 °C. 1H and 13C NMR spectroscopy was performed on a Bruker Avance 300 spectrometer at 300 MHz (1H) or 75 MHz (13C). The chemical shift scale was calibrated on the basis of the internal solvent signals. Mass Spectrometry (MS). Mass spectra were recorded with a Bruker Esquire-LC instrument. High-resolution (HR) mass spectra (electron spin ionization, ESI) were recorded on a ESI/TOF (LCT, Waters) LC-spectrometer. Elemental analyses were performed by the Service de microanalyse, Centre d’Etudes Pharmaceutiques, ChâtenayMalabry, France, with a PerkinElmer 2400 analyzer. 6267
DOI: 10.1021/acs.chemmater.6b02502 Chem. Mater. 2016, 28, 6266−6275
Article
Chemistry of Materials HRMS (ESI+): Calc. for C32H58ClN6O8PSiNa: 771.3409; Found: 771.3406. Synthesis of AMA-SG1-glycol. AMA-SG1 (0.11 g, 0.3 mmol), DMAP (0.07 g, 0.6 mmol), and EDC·HCl (0.12 g, 0.6 mmol) were dissolved in anhydrous CH2Cl2 and then mixed in a reaction flask under an argon atmosphere at room temperature. After 15 min, a solution of glycol (0.07 g, 1.2 mmol) in dimethylformamide (DMF) (1 mL) was added into the flask dropwise. After stirring at 30 °C for 24 h, the mixture was poured into 30 mL of EtOAc. The organic phase was washed with NaHCO3 aqueous solution, 1 M HCl, and brine before being dried over MgSO4. The residue was concentrated under reduced pressure and purified by flash chromatography (SiO2, petroleum ether/EtOAc = 1/1, v/v) to give 0.11 g of AMA-SG1-glycol as a white solid. Yield = 85%. 1H NMR (300 MHz, CDCl3): Major diastereomer: δ = 4.51 (q, 1H, J = 6.9 Hz), 3.87−4.40 (m, 6H), 3.71−3.85 (m, 2H), 3.32 (d, 1H, J = 26.4 Hz), 1.46 (d, 3H, J = 6.9 Hz), 1.24−1.31 (m, 6H), 1.16 (s, 9H), 1.08 (s, 9H). Minor diastereomer: δ = 4.63 (q, 1H, J = 6.9 Hz), 3.89−4.29 (m, 6H), 3.83 (m, 2H), 3.30 (d, 1H, J = 25.2 Hz), 1.53 (d, 3H, J = 6.9 Hz), 1.22−1.32 (m, 6H), 1.15 (s, 9H), 1.10 (s, 9H). 13C NMR (75 MHz, CDCl3): δ = 173.5, 81.2, 70.1, 68.3, 67.7, 62.3 (d), 62.0, 60.3, 59.2 (d), 35.5 (d), 29.5 (d), 28.1, 19.1, 16.6 (d), 16.2 (d). MS (ESI+): m/z = 434.1 (M + Na)+. Synthesis of Digly-AMA-SG1. AMA-SG1-glycol (0.41 g, 1.0 mmol) was mixed with diglycolic anhydride (0.29 g, 2.5 mmol) and triethylamine (0.7 mL, 5 mmol) in anhydrous CH2Cl2 (5 mL), and the reaction mixture was stirred at room temperature for 4 h under an argon atmosphere. The mixture was poured into 20 mL of CH2Cl2 and the organic phase was washed with 1 M HCl and brine, before being dried over MgSO4. The residue was concentrated under reduced pressure to give 0.47 g of Digly-AMA-SG1 as a sticky solid. Yield = 92%. 1H NMR (300 MHz, CDCl3): δ = 4.72 and 4.58 (2 q, 1H, J = 7.2 Hz), 4.33−4.54 (m, 3H), 4.26 and 4.27 (2 s, 2H), 4.20 and 4.19 (2 s, 2H), 3.90−4.16 (m, 4H), 3.44 and 3.42 (2 d, 1H, J = 26.7 Hz), 1.52 and 1.50 (2 d, 3H, J = 6.9 Hz), 1.22−1.32 (m, 6H), 1.17 and 1.16 (2 s, 9H), 1.13 and 1.12 (2 s, 9H). 13C NMR (75 MHz, CDCl3): δ = 172.2, 170.9, 170.1, 75.4, 70.5, 69.1, 68.6, 63.0 (d), 62.6, 62.4, 61.6, 59.9 (d), 35.4 (d), 30.3 (d), 27.9, 17.6, 16.4 (d), 16.1 (d). MS (ESI+): m/z = 550.2 (M + Na)+. Synthesis of CdA-digly-AMA-SG1. AMA-SG1-digly (0.63 g, 1.2 mmol), DMAP (0.14 g, 1.2 mmol), and EDC·HCl (0.23 g, 1.2 mmol) were dissolved in 3 mL anhydrous CH2Cl2, and mixed in a reaction flask under an argon atmosphere at room temperature. After 15 min, a solution of CdA-TBDMS (0.16 g, 0.4 mmol) in DMF (1 mL) was added into the flask dropwise. After stirring at 30 °C for 18 h, the mixture was poured into 30 mL of EtOAc. The organic phase was washed with 1 M HCl, NaHCO3 aqueous solution, and brine, before being dried over MgSO4. The residue was concentrated under reduced pressure and purified by flash chromatography (SiO2, from EtOAc to EtOAc/MeOH = 10/1, v/v) to give 0.31 g of CdA-digly-AMA-SG1 as a white solid. Yield = 91%. 1H NMR (300 MHz, CDCl3): Major diastereomer: δ = 8.18 (s, 1H), 6.44 (t, 1H, J = 6.9 Hz), 5.80 (s, 2H), 5.48 (m, 1H), 4.62 (q, 1H, J = 7.2 Hz), 3.86−4.41 (m, 17H), 3.31 (d, 1H, J = 25.5 Hz), 2.65 (m, 2H), 1.52 (d, 3H, J = 6.9 Hz), 1.23−1.31 (m, 6H), 1.15 (s, 9H), 1.09 (s, 9H), 0.92 (s, 9H), 0.12 and 0.11 (2 s, 6H, J = 2.1 Hz). Minor diastereomer: δ = 8.16 (s, 1H), 6.44 (t, 1H, J = 6.9 Hz), 6.00 (s, 2H), 5.49 (m, 1H), 4.65 (q, 1H, J = 7.2 Hz), 3.89− 4.43 (m, 17H), 3.38 (d, 1H, J = 25.5 Hz), 2.64 (m, 2H), 1.49 (d, 3H, J = 6.9 Hz), 1.22−1.30 (m, 6H), 1.15 (s, 9H), 1.11 (s, 9H), 0.91 (s, 9H), 0.13 and 0.12 (2 s, 6H, J = 1.8 Hz). 13C NMR (75 MHz, CDCl3): Major diastereomer: δ = 173.6, 169.2, 169.1, 156.0, 154.2, 150.6, 138.8, 118.5, 85.6, 84.2, 82.3, 76.2, 70.4, 68.6, 68.0, 67.9, 63.5, 62.6, 61.8, 61.7, 61.6, 58.9, 58.8, 39.1, 35.6, 35.5, 29.6, 29.5, 27.9, 25.9, 19.2, 18.3, 16.6, 16.5, 16.2, 16.1, −5.4, −5.5. Minor diastereomer: δ = 172.4, 169.3, 169.2, 156.1, 154.2, 150.6, 138.8, 118.5, 85.6, 84.2, 76.1, 70.1, 68.3, 68.0, 67.9, 63.5, 62.6, 61.9, 61.8, 61.7, 61.5, 59.0, 58.9, 39.1, 35.3, 35.2, 30.2, 30.1, 28.0, 25.9, 18.3, 18.0, 16.6, 16.5, 16.2, 16.1, −5.4, −5.5. MS (ESI+): m/z = 931.5 (M + Na)+. Polymerization of Isoprene from CdA-AMA-SG1. CdA-AMA-SG1 (55 mg, 0.075 mmol) was placed in a 15-mL-capacity pressure tube (Ace Glass, Model 8648-164) that was fitted with plunger valves and
thermowells. Isoprene (0.75 mL, 7.5 mmol) and dioxane (0.75 mL) were added and the tube was subjected to three cycles of freeze−thaw degassing, then backfilled with argon. The tube was placed in an oil bath at 115 °C for 2 h (P1), 4 and 8 h (P2), or 16 h (P3) and then cooled to room temperature by placing in a bath of cold water. The residue was concentrated under reduced pressure to give CdA-PI as a colorless product. Another polymerization was performed for 16 h with [isoprene]0/[CdA-digly-AMA]0 = 200/1 (P4). Polymerization of Isoprene from CdA-digly-AMA-SG1. CdAdigly-AMA-SG1 (54 mg, 0.06 mmol) was placed in a 15-mL-capacity pressure tube (Ace Glass, Model 8648-164) that was fitted with plunger valves and thermowells. Isoprene (0.6 mL, 6.0 mmol) and dioxane (0.6 mL) were added, and the tubes were subjected to three cycles of freeze−thaw degassing, then backfilled with argon. The tubes were placed in an oil bath at 115 °C for 2 and 4 h (P1d) or 16 h (P2d) and then cooled to room temperature by placing them in a bath of cold water. The residue was concentrated under reduced pressure to give CdA-digly-PI as a colorless product. Two other polymerizations were performed for 16 h with [isoprene]0/[CdA-digly-AMA]0 = 200/ 1 (P3d) and 400/1 (P4d). Deprotection of CdA-PI and CdA-digly-PI. TBDMS-protected CdA-PI or CdA-digly-PI (100 mg) was dissolved in 0.5 mL of THF, then tetrabutylammonium fluoride (1 M in THF, 50 μL) was added and the solution continued to be stirred for 15 min before pouring it into 10 mL of MeOH. The polyisoprene was stored in the refrigerator to settle out over 36 h. Polymers were analyzed by 1H NMR and SEC. NMR analysis showed a complete disappearance of TBDMS protecting groups. Polymerization of Isoprene from AMA-SG1-digly. AMA-SG1digly (32 mg, 0.06 mmol) was placed in a 15-mL-capacity pressure tube (Ace Glass, Model 8648-164) fitted with a plunger valve and thermowell. Isoprene (1.21 mL, 12.0 mmol) and dioxane (1.21 mL) were added and the tube was subjected to three cycles of freeze−thaw degassing, then backfilled with argon. The tube was placed in an oil bath at 115 °C for 20 h and then cooled to room temperature by placing in a bath of cold water. The residue was concentrated under reduced pressure and precipitated in MeOH to give AMA-digly-PI (P0d) as a colorless product. Mn = 2650 g mol−1, Đ = 1.15. Nanoparticle Preparation from CdA-PI and CdA-digly-PI. Nanoparticles were prepared via the nanoprecipitation technique.54 Unless noted otherwise, a final nanoparticle concentration of 2.5 mg mL−1 was targeted. Briefly, 2.5 mg of the corresponding polymer was dissolved in 0.5 mL of THF, and then was added dropwise to 1 mL of Milli-Q water (Millipore, Bedford, MA, USA) under stirring. THF was then evaporated at room temperature using a Rotavapor (Buchi) until constant weight. Average diameter (Dz) and zeta potential measurements were performed in triplicate. P2d nanoparticles with a diameter of 180 nm were obtained using a CdA-PI concentration of 5 mg mL−1, whereas those with a diameter of 130 nm were obtained using a CdAPI concentration of 2.5 mg mL−1. Drug Release. To determine the kinetics of CdA release from CdA-PI nanoparticles, 100 μL of nanoparticles P3d and P4 (2.5 mg mL−1) were added to 900 μL of human serum or PBS solution.55 The mixture was incubated at 37 °C, and aliquots (100 μL) of incubation medium were removed at different time points, spiked with 4 μL of 500 μM Gemcitabine (Internal Standard) before addition of 1 mL of a mixture of acetonitrile/methanol (90/10, v/v) and ultracentrifugated (15 000g, 20 min, 4 °C). The supernatant was then evaporated to dryness under a nitrogen flow at 30 °C. The released drug was quantified by reverse-phase high-performance liquid chromatography (HPLC) (Waters, Milford, MA, USA) with a C18 column. Briefly, the chromatographic system consisted of a Waters 1525 Binary LC pump, a Waters 2707 Autosampler, a C18 Uptisphere column (3 μm, 150 mm × 4.6 mm; Interchim), HPLC column temperature controllers (Model 7950 column heater and chiller; Jones Chromatography, Lakewood, CO, USA), and a Waters 2998 programmable photodiodearray detector. The HPLC column was maintained at 30 °C. Detection was monitored at 270 nm. The HPLC mobile phase was 0.05 M sodium acetate in water (pH 5.0, solvent A) and methanol (solvent B). The residues were dissolved in 100 μL of solvent A, and elution was 6268
DOI: 10.1021/acs.chemmater.6b02502 Chem. Mater. 2016, 28, 6266−6275
Article
Chemistry of Materials
Figure 2. Synthesis of (a) CdA-AMA-SG1 and (b) CdA-digly-AMA-SG1 alkoxyamine initiators for the preparation of CdA-PI and CdA-digly-PI polymer prodrugs by NMP, respectively. (P4d) at a CdA-equivalent dose of 11 mg kg−1 (20 mgCdA‑PI mL−1), (iv) AMA-digly-PI nanoparticles (P0d) at an equivalent PI concentration to P3d (8.4 mgPI mL−1), and (v) glucose 5%. The injected volume was 10 μL per gram of body weight. Mice were regularly monitored for changes in weight and behavior, and were humanely sacrificed 72 h after the last injection.
carried out at a flow rate of 0.8 mL/min isocratically for 7 min with solvent A/solvent B = 0.6/0.2, followed by a 1 min linear gradient to 100% solvent B. This was followed by a 15 min hold at solvent B, and a 1 min linear gradient back to solvent A/solvent B = 0.6/0.2. The system was held for 6 min for equilibration back to initial conditions. Biological Evaluations. Cell Lines and Cell Culture. Murine leukemia cell line L1210 was kindly provided by Dr. Lars Petter Jordheim (Université Claude Bernard Lyon I, Lyon, France) and maintained as recommended. Briefly, L1210 cells were cultured in Dulbecco’s minimal essential medium (DMEM). All media were supplemented with 10% heat-inactivated FBS (56 °C, 30 min) and penicillin (100 U mL−1). Cells were maintained in a humid atmosphere at 37 °C with 5% CO2. In Vitro Anticancer Activity. MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide] was used to test cytotoxicity of polymer prodrug nanoparticles and cell viability. Briefly, cells (5 × 103/well) were seeded in 96-well plates. After 1 h of incubation, the cells were then exposed to a series of concentrations of polymer prodrug nanoparticles (concentration is expressed in μM of drug but note that when one drug molecule is linked to one polymer chain, as it is the case here, it is equal to the molar concentration of the polymer prodrug), control polyisoprene nanoparticles, or free Cladribine for 72 h. After drug exposure, the medium was added with 20 μL of MTT solution (5 mg mL−1 in PBS) for each well. The plates were incubated for 1 h at 37 °C and the medium was removed after centrifugation (200g, 5 min, 25 °C). Dimethyl sulfoxide (DMSO, 200 μL) was then added to each well to dissolve the formazan crystals. Absorbance was measured at 570 nm, using a plate reader (Metertech Σ 960, Fisher Bioblock, Illkirch, France). The percentage of surviving cells was calculated as the absorbance ratio of treated to untreated cells. The inhibitory concentration 50% (IC50) of the treatments was determined from the dose−response curve. All experiments were set up in sextuplicate to determine means and SDs. Intravenous Injections and Preliminary Toxicity to Mice. Female athymic nude mice 6−8 weeks old were purchased from Harlan Laboratory. All animals were housed in appropriate animal care facilities during the experimental period, and were handled according to the principles of laboratory animal care and legislation in force in France (Authorization No. 00247.02). The intravenous injections of CdA-based prodrugs were performed at equimolar doses comparatively to free CdA. Mice were randomly divided into 5 groups of 8; each and all groups received five intravenous injections on days 0, 1, 2, 3, and 4 in the lateral tail vein with either (i) CdA at a dosage of 11 mg kg−1, (ii) CdA-digly-PI nanoparticles (P3d) at a CdA-equivalent dose of 11 mg kg−1 (12 mgCdA‑PI mL−1), (iii) CdA-digly-PI nanoparticles
■
RESULTS AND DISCUSSION Design and Synthesis of CdA-Based Polymer Prodrugs. Given its numerous advantages, in terms of simplicity and innocuousness, NMP was chosen to grow well-defined, short polymer chains from CdA, previously derivatized by an alkoxyamine initiator. Among the different functional groups of CdA, the C-6 amino group of the purine base was first selected as a chemical handle to link an alkoxyamine initiator to conduct NMP, either from AMA-SG152 or AMA-SG1-NHS56 under amidation conditions. However, despite successful protection of the two hydroxyl groups (see sections 1.1 and 1.2 in the Supporting Information), no coupling was obtained with the usual amidation reagents, such as ethylchloroformate, N,N′dicyclohexylcarbodiimide (DCC), 2-(7-aza-1H-benzotriazole-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), likely because of the low reactivity of the aromatic amine group (see section 1.3 in the Supporting Information). However, moderate yield (∼16%) was obtained by using propylphosphonic anhydride (T3P).57 Considering that CdA is not subjected to enzymatic deamination in vivo by deaminases,58 conversely to other nucleoside analogues such as Gem or adenosine,59,60 coupling to CdA by esterification was then attempted on the secondary hydroxyl group after protection of the primary hydroxyl group by TBDMSCl (Figure 2a). Protected CdA was reacted with AMA-SG1 using carbodiimide-assisted coupling chemistry, giving CdA-AMASG1 with 63% yield (see Figure 2a, as well as Figure S1 in the Supporting Information). To tune the lability of the linkage between CdA and the alkoxyamine, and therefore influence the drug release profile, a diglycolate linker61−63 was inserted between CdA and the alkoxyamine. Compared to a single ester bond, the diglycolate group is likely to endow the drug− polymer conjugates with more rapid drug release kinetics, 6269
DOI: 10.1021/acs.chemmater.6b02502 Chem. Mater. 2016, 28, 6266−6275
Article
Chemistry of Materials
Figure 3. Synthesis of CdA-PI and CdA-digly-PI polymer prodrugs by NMP in dioxane at 115 °C from CdA-AMA-SG1 or CdA-digly-AMA-SG1 alkoxyamine initiators, respectively: (a) plot of ln[1/(1 − conv)] versus time (where conv is the isoprene conversion), and (b) plot of numberaverage molar mass (Mn), determined by SEC (plain symbols) or 1H NMR (empty symbols) and dispersity (Đ = Mw/Mn) versus conversion. Dashed lines connecting data points are present only as guides for the eye. The solid black line represents the theoretical Mn.
Table 1. Characterization of CdA-PI and CdA-digly-PI Polymer Prodrugs, and the Resulting Nanoparticles expt
Mna (g mol−1)
DPn,SECb
DPn,NMRc
Đa
Dzd (nm)
P1 P2 P3 P4g P1d P2d P3dg P4dh
1420 1560 2270 2520 1470 1710 2960 4980
12 14 24 28 10 16 34 64
14 15 26 31 16 17 55 95
1.17 1.09 1.15 1.14 1.10 1.13 1.17 1.20
167 201 152 137 143 128 127 111
± ± ± ± ± ± ± ±
1 2 2 2 2 2 2 1
PSDd
ζe (mV)
0.06 0.12 0.08 0.10 0.07 0.10 0.08 0.11
−69 −70 −64 −71 −66 −68 −70 −66
± ± ± ± ± ± ± ±
1 1 2 3 1 2 1 2
%CdAf (wt %) 20.1 18.3 12.6 11.3 19.4 16.7 9.6 5.7
a Determined by SEC, calibrated with PS standards and converted into PI by using Mark−Houwink−Sakurada (MHS) parameters.53 bCalculated according to DPn,SEC = (Mn,SEC − MWalkoxyamine + MWTBDMS)/MWisoprene. cCalculated from ratio of areas under the peak at 7.8−8.2 ppm (proton next to the nitrogen on the heterocycle) and 5.0−5.5 ppm (vinylic H in isoprene repeat unit (1,4-addition), corresponding to ∼81% of total isoprene units).53 dDetermined by DLS. eZeta potential. f%CdA = MWCdA/Mn,SEC. g[isoprene]0/[alkoxyamine]0 = 200/1. h[isoprene]0/[alkoxyamine]0 = 400/1.
of Mn with conversion, together with low dispersities, were obtained from both alkoxyamines (see Figure 3). By varying the polymerization time from 2 h to 16 h and the [isoprene]0/[alkoxyamine]0 ratio from 100/1 to 400/1 (see the Experimental Section and Tables S1 and S2 in the Supporting Information), it was possible to prepare two distinct libraries of well-defined prodrugs of each type with variable chain lengths. Mn of deprotected CdA-based polymer prodrugs (Table 1 and Figures S3−S8 in the Supporting Information) ranged from 1420 g mol−1 to 2520 g mol−1 for Cla-PI (P1−P4) and from 1470 g mol−1 to 4980 g mol−1 for Cla-digly-PI (P1d−P4d), with low dispersity (Đ = 1.09−1.20), as demonstrated by 1H NMR spectroscopy and SEC analyses. By tuning the PI chain length, the drug loading was varied from 11.3 wt % to 20.1 wt % for CdA-PI and from 5.7 wt % to 19.4 wt % for CdA-digly-PI. Note that these values are significantly higher, compared to traditional drug-loaded polymer nanoparticles for which drug loadings usually reach just a few percent. Formulation and Colloidal Characteristics of the Nanoparticles. CdA-based polymer prodrug nanoparticles were obtained by nanoprecipitation into water of a solution of the different CdA-PI and CdA-digly-PI prodrugs in THF, to
resulting in a faster anticancer activity. Diglycolic anhydride was first reacted with a hydroxyl group-bearing AMA-SG1 before the resulting product (digly-AMA-SG1) was reacted with the protected CdA to give CdA-digly-AMA-SG1 with a coupling yield as high as 91% (see Figure 2b, as well as Figure S2 in the Supporting Information). Given its chemical degradability,64 as well as its biocompatibility65 and its structural similarity with natural terpenoids, PI was selected for the design of CdA-based polymer prodrugs by NMP (Figure 2). In addition, its hydrophobicity combined with the low targeted isoprene conversion (to achieve low Mn values and, therefore, high drug loadings) and the hydrophilicity of CdA (solubility in water ∼5−6 mg mL−1) are expected to give amphiphilic CdA-PI polymer prodrugs and stable nanoparticles after their formulation in aqueous solution. NMP of isoprene was initiated by either CdA-AMA-SG1 or CdA-digly-AMASG1 and performed at 115 °C in dioxane. It was followed by deprotection of TBDMS groups by tetra-n-butylammonium fluoride (TBAF) to give the CdA-PI (Px) and CdA-digly-PI (Pxd) conjugates, respectively (Figure 2). Because of the ability of NMP to control the polymerization of isoprene from AMASG1-based alkoxyamines,53 first-order kinetics, linear evolutions 6270
DOI: 10.1021/acs.chemmater.6b02502 Chem. Mater. 2016, 28, 6266−6275
Article
Chemistry of Materials
Figure 4. Representative cryogenic transmission electron microscopy (cryo-TEM) images of (a) CdA-PI P3, (b) CdA-digly-PI P3d, and (c) CdAdigly-PI P4d nanoparticles.
target a final concentration of 2.5 mg mL−1 after removal of THF under reduced pressure. As shown in Table 1, welldispersed polymer nanoparticles were formed with average diameters in the 110−160 nm range and narrow particle size distributions (PSD = ca. 0.1) as measured by dynamic light scattering. Representative cryo-TEM images showed spherical morphologies and colloidal characteristics (i.e., mean diameter, size distribution) that are in good agreement with dynamic light scattering (DLS) data for both series of CdA-based nanoparticles (see Figure 4). Colloidal characteristics of the different polymer prodrug nanoparticles were then monitored with time at room temperature. They exhibited very high colloidal stability in water for at least 2−3 months (Figure 5). Importantly, targeting a polymer prodrug concentration as high as 20 mg mL−1 did not affect the colloidal stability, as constant average diameter and particle size distribution also were observed in a one month-period (see Figure S9 in the Supporting Information). Colloidal stability was also shown in cell culture medium and in phosphate-buffered saline (PBS) (Figure S10 in the Supporting Information). In PBS, only a slight increase of the average diameter was observed, while particle size distribution still remained low (Figure S10b). The high colloidal stability is likely the result of an efficient electrostatic stabilization provided by the strongly negative surface charges (typically approximately −70 mV) of the different CdA-PI and CdAdigly-PI nanoparticles, as shown by zeta potential measurements (Table 1). Interestingly, the average diameter can be accurately tuned by either of two options: (i) Varying the polymer chain length; the longer the chain length, the smaller the diameter (likely because of a higher compaction of the longer polymer chains, which is due to increased hydrophobic interactions/lower solubility). For instance, increasing the Mn of Gem-digly-PI from 1470 g mol−1 to 4980 g mol−1 (P1d−P4d) decreased the average diameter from 143 nm to 111 nm (see Figure 5a and Table 1) (ii) Varying the nanoparticle concentration during the nanoprecipitation process; the higher the concentration, the higher the average diameter. For instance, for CdA-digly-PI nanoparticles (P4d), when the polymer concentration was varied from 1 mg mL−1 to 10 mg mL−1 at a fixed THF/water ratio. A linear increase in the average diameter was obtained, from 93 nm to 178 nm (see Figure 5b). Drug Release in Different Media. Prior to performing biological evaluations, the release of CdA from CdA-PI and CdA-digly-PI nanoparticles was measured by HPLC in different media (PBS and human serum) to investigate the potential influence of the linker’s nature between CdA and the
Figure 5. (a) Evolution with time of the average diameter (solid symbols) and the particle size distribution (open symbols) determined by DLS of CdA-PI (P1 and P3) and CdA-digly-PI (P1d−P4d) nanoparticles. (b) Concentration-size dependence of CdA-digly-PI nanoparticles P4d prepared by nanoprecipitation.
PI moiety over the release kinetics (Figure 6). Although incubation of CdA-PI nanoparticles P4 (Mn = 2520 g mol−1) in PBS or in human serum gave almost no release of CdA (
