Y-Shaped mPEG-PLA Cabazitaxel Conjugates: Well-Controlled

Feb 22, 2013 - Y-Shaped mPEG-PLA Cabazitaxel Conjugates: Well-Controlled ..... Journal of Drug Delivery Science and Technology 2014 24, 12-21 ...
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Y‑Shaped mPEG-PLA Cabazitaxel Conjugates: Well-Controlled Synthesis by Organocatalytic Approach and Self-Assembly into Interface Drug-Loaded Core−Corona Nanoparticles Fethi Bensaid,†,‡ Olivier Thillaye du Boullay,†,‡ Abderrahmane Amgoune,†,‡ Christian Pradel,†,‡ L. Harivardhan Reddy,§ Eric Didier,§ Serge Sablé,§ Guillaume Louit,§ Didier Bazile,§ and Didier Bourissou*,†,‡ †

Université de Toulouse, UPS, LHFA, 118 route de Narbonne, 31062 Toulouse, France CNRS, LHFA, UMR 5069, 31062 Toulouse, France § Sanofi Research and Development, Lead Generation to Candidate Realization Platform, 13 Quai Jules Guesde, 94403 Vitry-sur-Seine, France ‡

S Supporting Information *

ABSTRACT: A well-defined poly(ethylene glycol) methyl ether-b-poly(lactic acid) copolymer (mPEG-PLA) featuring a new, Y-shaped, architecture with a hydroxyl functional group between the two blocks has been prepared and thoroughly characterized. The functional copolymer was then readily coupled to diglycolyl-cabazitaxel. The resulting copolymer conjugates assembled into stable and monodisperse nanoparticles (NPs) in aqueous suspension. The architecture of the copolymer conjugate is shown to impact the spatial distribution of the drug within the nanoparticles. With the Y-shaped architecture, cabazitaxel was found localized at the interface of the hydrophobic PLA core and the hydrophilic mPEG corona of the NPs, as substantiated by variable temperature NMR analysis of the nanoparticles in D2O. Preliminary in vitro release studies reveal dependence on the architecture of the copolymer conjugate. This new approach offers promising perspectives to finely tune the position of the active ingredient in polymeric nanoparticles.

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delivery applications.8 Their ability to form core−corona nanostructures was recognized in the early 1990s,9−11 and the presence of the PEG chains at the surface of the nanoparticles was shown to impart longer systemic circulation time and lower toxicity in vivo as compared to the NPs derived from simple PLA.9,10,12 Since then, PEG-PLA NPs have been extensively studied for the encapsulation of a variety of hydrophobic drugs including taxanes.13a,b Taxanes (paclitaxel, docetaxel, cabazitaxel, etc.) are an important category of cytotoxic chemotherapeutic agents with wide applications in cancer therapy. The poor solubility of taxanes in aqueous medium requires the use of surfactants (Cremophor EL, polysorbate 80) and ethanol which actually results in toxicity issues in clinic13c and necessitates the administration of corticosteroids and premedication prior to the chemotherapy.13d It is thus highly desirable to develop alternative formulations of taxanes that avoid the use of surfactant vehicles to overcome the associated side effects. Taxanes have been encapsulated into PEG-PLA nanoparticles either by noncovalent or by covalent approaches. In the noncovalent encapsulation technique, the drug is

INTRODUCTION

Over the past few decades, polymeric nanoparticles (NPs) have attracted considerable interest for drug delivery, especially in anticancer chemotherapy.1−4 NPs present great therapeutic potential thanks to their ability (i) to solubilize and stabilize hydrophobic drugs through encapsulation, (ii) to passively accumulate within the tumor interstitium, following in vivo injection, through enhanced permeability and retention (EPR) effect,5,6 and (iii) to release the drug in a controlled and sustained fashion. Particularly efficient polymeric carriers are prepared from biodegradable amphiphilic block copolymers, whose physicochemical properties are ideally suited for encapsulation, transport, and delivery of hydrophobic drugs. Thanks to the hydrophilic/hydrophobic nature and immiscibility of their constitutive blocks, these copolymers readily selfassemble in aqueous media to form core−corona structured nanoparticles.7 The hydrophobic and relatively rigid core enables effective drug encapsulation, while the hydrophilic and mobile corona provides electrostatic stabilization and steric protection to the nanoparticles. Poly(ethylene glycol)-b-poly(lactic acid) copolymers PEGPLA are clearly at the forefront position of amphiphilic block copolymers employed to form polymeric nanoparticles for drug © 2013 American Chemical Society

Received: February 1, 2013 Published: February 22, 2013 1189

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Figure 1. Schematic representation of the various types of mPEG-PLA nanoparticules developed from polymer/drug conjugates.

accessible for cleavage, and the drug release kinetic could be controlled using linker chemistry. Our approach relies on mPEG-PLA copolymers featuring a trivalent moiety at the junction of the two blocks so that the therapeutic agent can be efficiently and selectively introduced at this position. Here we report the synthesis of a well-defined Y-shaped mPEG-PLA/cabazitaxel conjugate combining organocatalytic ROP and fine polymer functionalization chemistry such as protection/deprotection and coupling reactions. High attention has been brought to the characterization of the polymer conjugate in order to clearly quantify and assign the position of the cabazitaxel in the macromolecule. Despite the presence of the cabazitaxel at the mPEG-PLA junction, this copolymer selfassembled into stable nanoparticles. These nanoparticles have been characterized by various physicochemical methods, including nuclear magnetic resonance (NMR), transmission electron microscopy, and dynamic light scattering. The mPEGPLA based linear-shaped conjugate in which the cabazitaxel is grafted at the PLA chain end has also been prepared and systematically compared with the Y-shaped conjugate nanoparticles. Accordingly, the position of cabazitaxel within the polymer conjugate is shown to have a noticeable impact on its spatial distribution within the nanoparticles. In addition, preliminary studies in rat plasma revealed the dependence of the drug release profile upon the architecture of the polymer conjugate.

encapsulated physically into the PEG-PLA nanoparticules,9−11,14 while in the covalent approach, the drug and the PEG-PLA copolymer are chemically linked before selfassembling into NPs.15,16 The latter systems offer several advantages, including (i) precise control of the drug loading, (ii) intrinsic elimination of the burst effect, and (iii) fine-tuning of the release kinetics through the nature of the cleavable linker17 between the drug and the copolymer. To date, two types of PEG-PLA nanoparticules deriving from polymer/drug conjugates have been reported: (i) Paclitaxel and doxorubicin conjugates of diblock mPEG-PLA and triblock PLA-PEG-PLA copolymers, synthesized by chemical derivatization of the carboxyl chain ends, have been self-assembled into micelles,18,19 and (ii) PLA conjugates, synthesized by metalmediated ring-opening polymerization of lactide directly initiated by a hydroxyl-containing drug (paclitaxel, docetaxel, doxorubicin, etc.) have been nanoprecipitated, and the ensuing nanoparticles have been subsequently PEGylated by addition of mPEG-PLGA.20 Noteworthy, the above polymer conjugates all feature the therapeutic agent at a PLA chain end, and thus, the drug is embedded and broadly distributed in the hydrophobic PLA core of the ensuing NPs (Figure 1), rendering the access of the linker difficult for cleavage. Thanks to the spectacular progress achieved recently in the synthesis and chemical modification of polymers, it is nowadays conceivable to design and study multifunctional polymers with more elaborated architectures.21 Such macromolecular engineering offers a unique opportunity to vary and control the properties of polymeric NPs.22−24 In this perspective, we reasoned that fine-tuning of the position of the therapeutic agent within the polymer conjugate may have an influence on the distribution of the drug within the ensuing nanoparticles. In particular, we envisioned the possibility to introduce the drug in between the PEG and PLA blocks, hypothesizing that such original Y-shaped conjugates may result in the distribution of the drug mainly at the interface of the PEG corona and PLA core (Figure 1). Accordingly, the linker would be more

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EXPERIMENTAL SECTION

Materials and Methods. All syntheses and polymerizations were carried out under an argon atmosphere using standard Schlenk techniques. All reagents were purchased from Aldrich and used as received, unless otherwise mentioned. All solvents were sparged with argon and purified using a solvent purification system (Mbraun MBSPS-800 system). Dichloromethane (DCM) was further dried over molecular sieves. DL-Lactide was purchased from PURAC and purified by azeotropic distillation and recrystallization from toluene. It was 1190

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diethylether at 0 °C. The white precipitate was then filtered, washed, and dried under vacuum. The product was then dried by azeotropic distillation from toluene. After removal of the solvent, the residue was dried under vacuum overnight and stored in a glovebox prior to use (m = 0.4 g, yield = 74%). 1H NMR (CDCl3, 500 MHz, 298 K): δ (ppm) 0.98−1.03 (bm, 21H, H9 and H10), 1.16 (s, 3H, H6), 3.37 (s, 3H, H1), 3.63 (m, 180H, CH2 PEG), 3.60−3.80 (dd, 2H, J = 11.1 and 55.8 Hz, H7), 3.85−3.92 (dd, 2H, J = 9.4 and 24.7 Hz, H8), 4.26 (t, 2H, J3H−H = 4.8 Hz, H3). 13C NMR (CDCl3, 125.7 MHz, 298 K): δ (ppm) 11.7 (C9), 17.3 (C6), 17.8 (C10), 50.3 (C5), 58.9 (C1), 63.1 (C3), 66.1 (C7), 66.7 (C8), 68.7 (C2′), 70.4 (C2), 71.8 (C2″), 174.8 (C4). 29Si NMR (CDCl3, 99.3 MHz, 29 K): δ (ppm) −22.0 (-O-Si-). SEC: Mn = 2100 g/mol, Mw/Mn = 1.09. Synthesis of mPEG-PLA Copolymer 3. The macroinitiator 2 (0.18 g, 79.6 μmol) and DL-lactide (0.8 g, 5.6 mmol, 70 equiv) were dissolved in 7 mL of anhydrous dichloromethane. A solution of Ncyclohexyl-N′-3,5-bis(trifluoromethyl)phenyl thiourea (92 mg, 224 μmol) and (+)-sparteine (28 mg, 112 μmol) in dichloromethane (1 mL) was added. The reaction mixture was stirred at 35 °C under argon until full consumption of the monomer, as controlled by 1H NMR (3 h). Acetic anhydride (39 μL, 0.40 mmol) and 4-dimethylaminopyridine (DMAP; 10 mg, 82 μmol) were then added and the reaction mixture was allowed to stir for 1 h. The solution was concentrated under vacuum and the copolymer was precipitated in 50 mL diethylether at 0 °C. After filtration, the polymer was washed with 20 mL of methanol and dried under vacuum overnight (m = 1 g, yield =75%). 1H NMR (CDCl3, 500 MHz, 298 K): δ (ppm) 0.98−1.03 (br, 21H, H9 and H10), 1.16 (s, 3H, H6), 1.58 (m, 423H, CH3 PLA), 2.12 (s, 3H, H15), 3.37 (s, 3H, H1), 3.63 (m, 180H, CH2 PEG), 3.73−3.80 (m, 2H, H8), 4.20−4.40 (m, 4H, H3 and H7), 5.16 (m, 141H, CH PLA). 13C NMR (CDCl3, 125.7 MHz, 298 K): δ (ppm) 11.8 (C9), 16.5 (C13 and C6), 17.8 (C10), 48.5 (C5), 58.8 (C1), 63.5 (C3), 65.0 (C7), 66.7 (C8), 68.1 (C2′), 68.8 (C12), 70.1 (C2), 71.8 (C2‴), 72.2 (C2″), 169.50 (C11 and C14), 174.9 (C4). SEC: Mn = 11850 g/mol, Mw/Mn = 1.15. Removal of the Silyl Group to Give mPEG-PLA Copolymer 4. The protected copolymer 3 (1 g, 71 μmol) was dissolved in 10 mL of anhydrous dichloromethane. BF3−Et2O (0.5 g, 3.56 mmol) was then added dropwise at 0 °C. The reaction mixture was stirred at 0 °C under argon for 1 h and then at 30 °C for 19 h. Dichloromethane was evaporated under vacuum. The residue was solubilized in 5 mL of dichloromethane and then precipitated in 50 mL of diethylether at 0 °C, washed with 20 mL of methanol, and then with 20 mL of pentane. The white precipitate was filtered and dried under vacuum overnight (m = 0.85 g, yield = 85%). 1H NMR (CDCl3, 500 MHz, 298 K): δ (ppm) 1.09 (s, 3H, H6), 1.58 (m, 426H, CH3 PLA), 2.12 (s, 3H, H13), 3.37 (s, 3H, H1), 3.63 (m, 180H, CH2 PEG), 4.33 (m, 4H, H3 and H7), 5.16 (m, 142H, CH PLA). 13C NMR (CDCl3, 125.7 MHz): δ (ppm) 16.5 (C11), 48.5 (C5), 58.7 (C1), 63.4 (C3), 64.2 (C7), 66.5 (C8), 68.2 (C2′), 68.8 (C10), 70.1 (C2), 71.7 (C2‴), 72.2 (C2″), 169.5 (C9), 174.8 (C4). SEC: Mn = 11800 g/mol, Mw/Mn = 1.18. Elem. Anal. Calcd for C520H762O332: C, 50.67%; H, 6.23%. Found: C, 50.54%; H, 6.19%. Traces: Pd < 2 ppm, S < 10 ppm, F 149 ppm. Synthesis of Diglycolyl-cabazitaxel. Cabazitaxel acetone solvate (5 g, 5.62 mmol) was dissolved in 100 mL of dichloromethane in a 250 mL flask, then diglycolic anhydride (6.53 g, 56.22 mmol) and DMAP (0.068 g, 0.56 mmol) were added under nitrogen. The reaction was allowed to stir at room temperature overnight. Then water (2 × 50 mL) was added to the solution to extract the salts. After drying of the organic solution with MgSO4, the solution was concentrated to dryness at 40 °C under reduced pressure. The dry extract was treated with 4 volumes of diisopropylether and the suspension was stirred for 30 min and then filtrated. The solid was washed again twice with 2 volumes of diisopropylether. After drying at 40 °C under reduced pressure, a white powder was isolated (m = 5.04 g, yield = 96%). 1H NMR (CDCl3, 500 MHz, 298 K): δ (ppm) 0.96 (s, 3 H, H16), 0.98 (s, 3 H, H17), 1.37 (m, 9 H, H7′), 1.44−1.58 (m, 2 H, H14a and H14b), 1.51 (s, 3 H, H19), 1.80 (brs, 4 H, H18 and H6a), 2.23 (s, 3 H, C4OCOCH3), 2.67 (m, 1 H, H6b), 3.21 (s, 3 H, C7OCH3), 3.28 (s, 3 H, C10OCH3), 3.58 (d, J = 7.3 Hz, 1 H, H3), 3.75 (dd, J = 6.8 and 10.5 Hz, 1 H, H7),

subsequently sublimed and then stored under argon in a glovebox. Methoxy-polyethylene glycol (mPEG-OH) was azeotropically distilled from toluene and dried under vacuum. N-(3,5-Trifluoromethyl)phenyl-N′-cyclohexylthiourea (TU) and benzyl-protected 2,3-bis(methylol) propionic acid were prepared as previously reported.25,26 (+)-Sparteine was twice distilled from CaH2 under argon and stored in the glovebox. NMR spectra were recorded at room temperature on Bruker Avance 300 MHz, Bruker Avance 400 MHz and Bruker Avance 500 MHz devices equipped with a cryoprobe. The number-average molar masses Mn, the weight-average molar masses Mw and the polydispersity index (Mw/Mn) were measured by size exclusion chromatography (SEC) at 35 °C with a triple detection line composed of an Alliance Waters e2695, a MALS miniDAWN (Wyatt) light scattering detector, a Viscostar-II (Wyatt) viscometer and a Waters 2414 refractometer. THF was used as eluent at a flow rate of 1.0 mL/min. A Styragel (WAT054405) precolumn and two Shodex (KF-802.5 and KF-804) columns were used. Matrix-Assisted Laser Desorption/Ionization (MALDI-TOF-MS) analyses were performed on a MALDI MicroMX from Waters equipped with a 337 nm nitrogen laser. An accelerating voltage of 20 kV was applied. Mass spectra of 1000 shots were accumulated. Ultra Performance Liquid Chromatography (UPLC) analyses were performed on a UPLC chain equipped with a pump, an automatic injector and an UV PDA detector (Acquity UPLC, Waters). Microanalyses (elementary and traces) C, H, N, Pd, S and F were performed by the Central Analytic Center (SCA, CNRS-Solaize). The morphology of the nanoparticles was observed by TEM using a JEOLJEM 2100F microscope with an acceleration field of 200 kV. The size (hydrodynamic diameter) of the nanoparticles was measured by DLS using a Zetasizer Nano ZS (Malvern). Synthesis of Methoxy-polyethylene Glycol-diol (mPEG(OH)2) 1. Methoxy-polyethylene glycol (Mn = 2300 g/mol, Mw/Mn = 1.09; 10 g, 5 mmol) and the protected bis-HMPA (1.35 g, 6.1 mmol) were dissolved in 45 mL of anhydrous dichloromethane in a 250 mL Schlenk vessel. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 0.96 g, 5 mmol) and 4-(dimethylamino)pyridinium p-toluenesulfonate (DPTS; 0.6 g, 2 mmol) were subsequently added to the medium. The reaction medium was stirred at 40 °C under argon for 48 h. The reaction medium was subsequently extracted with 20 mL of a 1 M HCl solution, 20 mL of a 10% NaHCO3 solution and 20 mL of H2O. After drying the organic phase over Na2SO4, filtering and evaporating the solvent, the residue was precipitated from ether at 0 °C. The precipitate was then filtered off and dried under vacuum overnight to give a white solid (m = 9.68 g, yield =88%). To that solid, palladium-on-charcoal (10% Pd/C; 0.95 g, 10% w/w) was added in a 250 mL, two-necked, round-bottomed, Schlenk flask equipped with a balloon filled with hydrogen (H2), followed by a vacuum-argon cycle. A total of 40 mL of dichloromethane and 40 mL of methanol (MeOH) were subsequently added. A vacuum-H2 cycle was carried out. The reaction medium was stirred under static H2 at room temperature for 4 h. The mixture was then filtered through Celite, the solvents were removed under vacuum, and the residue was dried under vacuum overnight to give a yellowish solid (m = 8.7 g, yield =95%). 1H NMR (CDCl3, 300 MHz, 298 K): δ (ppm) 1.11 (s, 3H, H6), 3.37 (s, 3H, H1), 3.63 (br, 180H, CH2 PEG), 3.69 − 3.78 (m, 4H, H7a and H7b), 4.33 (t, 2H, 3JH−H = 4.8 Hz, H3). 13 C NMR (CDCl3, 125.7 MHz, 298 K): δ (ppm) 16.9 (C6), 49.5 (C5), 58.8 (C1), 63.2 (C3), 67.1 (C7), 68.7 (C2′), 70.4 (C2), 71.8 (C2‴), 72.6 (C2″), 175.5 (C4). SEC: Mn = 2080 g/mol, Mw/Mn = 1.08. Elem. Anal. Calcd for C96H192O51: C, 53.32%; H, 8.95%. Found: C, 53.53%; H, 9.13%. Traces: Pd < 2 ppm, S < 10 ppm, F 35 ppm. Selective Protection of 1. mPEG-diol 1 (0.5 g, 0.24 mmol) was dissolved in 5 mL of anhydrous dichloromethane. Triehylamine (TEA; distilled over KOH; 0.2 g, 2.01 mmol) was then added, followed by dropwise addition of tris(isopropyl)silyl chloride (TIPSCl; 0.40 g, 2 mmol) at 0 °C. The reaction mixture was stirred at 35 °C under argon. After 24 h, the formed salts were filtered and the organic phase was washed with a HCl 1 M solution (5 mL), a NaHCO3 solution (5 mL) and H2O (5 mL). The organic phase was dried on Na2SO4 and concentrated under vacuum; the residue was precipitated in 1191

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Article

Synthesis of the Linear-Shaped mPEG-PLA/Cabazitaxel Conjugate 7. mPEG-PLA-OH copolymer 6 (0.2 g, 16.3 μmol) and diglycolyl-cabazitaxel (34 mg, 35 μmol) were dissolved in 4 mL of dichloromethane under nitrogen. Then, DMAP (5 mg, 40.7 μmol) and DIPC (4.4 mg, 34.23 μmol) were added. The solution was stirred for 24 h at 35 °C. The organic phase was concentrated to dryness and the extract was treated with 40 mL of methanol and 2 drops of dichloromethane at 0 °C. The suspension was stirred for 2 h at room temperature, then filtrated, and the solid was dried at room temperature under reduced pressure to obtain the expected compound (m = 0.17 g, yield = 85%). 1H NMR (CDCl3, 500 MHz, 298 K): δ (ppm) 1.21 (s, 3H, H16), 1.22 (s, 3H, H17), 1.35 (s, 9H, H7′), 1.40− 1.70 (m, 414H, H5 CH3 PLA), 1.72 (s, 3H, H19), 1.80 (m, 1H, H6a), 2.01 (s, 3H, H18), 2.20 (m, 1H, H14a), 2.32 (m, 1H, H14b), 2.45 (br s, 3H, C4OCOCH3), 2.71 (m, 1H, H6b), 3.30 (s, 3H, C7OCH3), 3.36 (s, 3H, H21 CH3O mPEG), 3.44 (s, 3H, C10OCH3), 3.48−3.81 (m, 174H, CH2 PEG), 3.80 (d, J = 7.3 Hz, 1H, H3), 3.86 (dd, J = 6.6 and 11.0 Hz, 1H, H7), 4.08−4.40 (m, 8H, CH2 diglycolyl and H cabazitaxel), 4.83 (s, 1H, H10), 5.01 (d, J = 10.7 Hz, 1H, H5), 5.03−5.33 (m, 138H, CH PLA), 5.40−5.55 (m, 3H, H cabazitaxel), 5.68 (d, J = 7.3 Hz, 1H, H2), 6.29 (br t, 1H, H13), 7.30 (m, 3H, HAr), 7.40 (t, J = 7.7 Hz, 2H, HAr), 7.50 (t, J = 7.7 Hz, 2H, HAr), 7.60 (t, J = 7.7 Hz, 1H, HAr), 8.12 (d, J = 7.7 Hz, 2H, HAr). 13C NMR (CDCl3, 75.5 MHz, 298 K): δ (ppm) 10.3 (C19), 14.4 (C18), 16.3 (C25, PLA), 20.9 (C16), 22.7 (C4-OCOCH3), 26.7 (C17), 28.1 (C7′), 31.9 (C6), 34.9 (C14), 43.3 (C15), 47.3 (C3), 56.8 (C8), 57.0−57.2 (C3′, C7OCH3 and C10OCH3), 58.3 (C21), 63.8 (C23), 67.5 (-CH2OCH2- diglycolyl), 69.1 (C24, PLA), 70.1 (C22, PEG), 72.4 (C13), 74.7 (C2′), 74.9 (C2), 76.4 (C20), 78.8 (C1), 80.6 (C7), 81.5 (C6′ and C4), 82.4 (C10), 84.1 (C5), 126.3−133.6 (CAr), 135.1 (C11), 139.2 (C12), 155.1 (C5′), 167.0 (C26 PLA and CO benzoate), 168.0 (C4COCH3), 169.2 (-COO- diglycolyl), 176.9 (C1′), 205.0 (C9). SEC: Mn = 11800 g/mol, Mw/Mn = 1.12. Nanoparticles Formulation. The copolymer/cabazitaxel conjugate 5 or 7 (30 mg) was dissolved in 1.5 mL of acetone (organic phase). The organic phase was added dropwise into 3 mL of water for injection (WFI), under magnetic stirring (500 rpm during 20 min). Acetone was then evaporated at 37 °C under vacuum using Rotavapor (from 300 to 45 mbar during 30 min). The final volume of nanodispersion was then adjusted to 3 mL with WFI to compensate for any loss of water during the evaporation step. Thus, the final concentration of the nanodispersion was 10 mg/mL. Characterization of Nanoparticles. Transmission electron microscopy (TEM): The morphology of the nanoparticles was observed by TEM using a JEOL-JEM 2100F microscope with an acceleration field of 200 kV. Preparation of the samples: few drops of the nanoparticles dispersion, diluted 10-fold (0.5 or 1 mg/mL), were incubated with 0.2% (w/v) of phosphotungstic acid for 30 min. The sample was subsequently placed on a copper grid and dried at ambient temperature. Dynamic light scattering (DLS): the size (hydrodynamic diameter) of the nanoparticles was measured by DLS using a Zetasizer Nano ZS (Malvern). The sample (0.5 or 1 mg/mL) was placed in a capillary cell after filtration (1.2 μm PVDF filter). Measurements were carried out at 20 °C with a detection angle of 173 °C in triplicate. The wavelength used was 633 nm. Nanoparticles were suspended in 0.22 μm filtered NaCl (10−3 M) solution (NPs concentration = 0.2−0.4 mg/mL), and zeta potential was measured on Malvern Zetasizer Nano ZS in triplicate. Determination of the Critical Micelle Concentration (CMC) of Y-Shaped and Linear mPEG-PLA Cabazitaxel Conjugates. CMC of Y-shaped and linear mPEG-PLA cabazitaxel conjugates were determined using pyrene as an extrinsic probe. Serial solutions with fixed Pyrene concentration of 6.15 × 10−7 M and various concentrations of polymer conjugates comprised between 1 × 10−2 and 10 mg/mL were prepared. Fluorescence spectra were obtained at 20 °C. Fluorescence measurements were taken at an excitation wavelength of 400 nm and the emission monitored from 300 to 350 nm. Preparation of Nanoparticles for 1H NMR Analysis in D2O. Nanoparticle formulations were prepared as described above using D2O and deuterated acetone as solvent. Acetone was evaporated by

4.02 (s, 2 H, CH2 diglycolyl), 4.13 (s, 2 H, CH2 diglycolyl), 4.31 (d, J = 17.0 Hz, 1 H, H20a), 4.38 (d, J = 17.0 Hz, 1 H, H20b), 4.51 (s, 1 H, H2′), 4.70 (s, 1 H, H10), 4.95 (d, J = 10.5 Hz, 1 H, H5), 5.06 (m, 1 H, H3′), 5.16 (d, J = 8.5 Hz, 1 H, H4′), 5.37 (d, J = 7.3 Hz, 1 H, H2), 5.82 (br t, J = 9.4 Hz, 1 H, H13), 7.19 (t, J = 7.8 Hz, 1 H, HAr), 7.36 (d, J = 7.8 Hz, 2 H, HAr), 7.43 (t, J = 7.8 Hz, 2 H, HAr), 7.66 (t, J = 7.8 Hz, 2 H, HAr), 7.73 (t, J = 7.8 Hz, 1 H, HAr), 7.88 (d, J = 9.3 Hz, 1 H, HAr), 7.97 (d, J = 7.8 Hz, 2 H, HAr). 13C NMR (CDCl3, 75.5 MHz, 298 K): δ (ppm) 10.3 (C19), 14.4 (C18), 20.9 (C16), 22.7 (OCOCH3), 26.7 (C17), 28.1 (C7′), 31.9 (C6), 34.9 (C14), 43.3 (C15), 47.3 (C3), 56.8 (C8), 57.0 to 57.2 (C3′, C7-OCH3 and C10-OCH3), 67.7 (-CH2OCH2diglycolyl), 72.4 (C13), 74.7 (C2′), 74.9 (C2), 76.4 (C20), 78.8 (C1), 80.6 (C7), 81.5 (C6′ and C4), 82.4 (C10), 84.1 (C5), 126.3−133.6 (CHAr), 135.1 (C11), 139.2 (C12), 155.1 (C5′), 167.0 (CO benzoate), 168.0 (C4-OCOCH3), 169.5 (-COO- diglycolyl), 176.9 (C1′), 205.0 (C9). Synthesis of the Y-Shaped mPEG-PLA/Cabazitaxel Conjugate 5. In a 25 mL flask, the mPEG-PLA-Y-OH copolymer 4 (0.2 g, 0.0157 mmol) and diglycolyl-cabazitaxel (32.3 mg, 0.0345 mmol) were dissolved in 4 mL of dichloromethane with 100 mg activated molecular sieves 4 Å (powder). After stirring for 10 min, DMAP (4.4 mg, 0.0345 mmol) and N,N′-diisopropylcarbodiimide (DIPC; 4.3 mg, 0.0345 mmol) were added to the solution. The suspension was stirred for 24 h at 35 °C and then filtered over PTFE filter (0.22 μm). The organic phase was concentrated to dryness and the residue was treated with 40 mL methanol and 2 drops of dichloromethane. The suspension was stirred for 2 h at RT, then filtered, and the solid was dried at room temperature under reduced pressure to give the desired conjugate 5 (m = 0.184 g, yield = 92%). 1H NMR (CDCl3, 500 MHz, 298 K): δ (ppm) 1.21 (s, 3 H, H16), 1.22 (s, 3 H, H17), 1.26 (m, 3 H, H26), 1.35 (s, 9 H, H7′), 1.40−1.70 (m, 486 H, CH3 PLA and protons from cabazitaxel), 1.72 (s, 3 H, H19), 1.80 (m, 1 H, H6a), 2.01 (br s, 3 H, H18), 2.13 (s, 3 H, H33), 2.21 (m, 1 H, H14a), 2.32 (m, 1 H, H14b), 2.45 (br s, 3 H, C4OCOCH3), 2.71 (m, 1 H, H6b), 3.31 (s, 3 H, C7OCH3), 3.38 (s, 3 H, H21 CH3O mPEG), 3.45 (s, 3 H, C10OCH3), 3.48−3.81 (m, 180 H, CH2 PEG), 3.86 (d, J = 7.3 Hz, 1 H, H3), 3.91 (dd, J = 6.6 and 11.0 Hz, 1 H, H7), 4.08−4.40 (m, 7 H, CH2 diglycolyl and H cabazitaxel), 4.83 (s, 1 H, H10), 5.02 (d, J = 10.7 Hz, 1 H, H5), 5.17 (m, 162 H, CH PLA), 5.50 (m, 2 H, H cabazitaxel), 5.67 (d, J = 7.3 Hz, 1 H, H2), 6.29 (br t, J = 9.0 Hz, 1 H, H13), 7.31 (m, 3 H, HAr), 7.40 (t, J = 7.7 Hz, 2 H, HAr), 7.50 (t, J = 7.7 Hz, 2 H, HAr), 7.60 (t, J = 7.7 Hz, 1 H, HAr), 8.11 (d, J = 7.7 Hz, 2 H, HAr). 13C NMR (CDCl3, 75.5 MHz, 298 K): δ (ppm) 8.4 (C19), 15.2 (C18), 16.6 (C31 PLA), 19.4 (C16), 24.9 (C4-OCOCH3), 28.1 (C17), 28.7 (C7′), 32.6 (C6), 35.9 (C14), 41.5 (C15), 47.8 (C3), 48.3 (C25), 56.7 (C3′), 57.4 (C8), 57.5 (C7OCH3), 57.8 (C10OCH3), 58.7 (C21), 63.1 (C23), 63.3 (C27 and C28), 68.5 (-CH2OCH2- diglycolyl), 69.1 (C30, PLA), 70.1 (C22, PEG), 71.9 (C13), 74.6 (C2′), 75.0 (C2), 78.9 (C1), 80.11 (C7), 81.6 (C6′), 82.5 (C4), 83.2 (C10), 84.1 (C5), 126.3−130.1 (CAr), 135.5 (C11), 140.8 (C12), 157.5 (C5′), 163.0 (CO benzoate), 168.5 (C4OCOCH3), 169.5 (-C29OO- PLA and -COO- diglycolyl), 173.9 (C24, PEG), 176.9 (C1′), 207.2 (C9). SEC: Mn = 11920 g/mol, Mw/Mn = 1.24. Synthesis of the linear-shaped copolymer mPEG-PLA-OH 6. The macroinitiator mPEG-OH (0.35 g, 175 μmol) and DL-lactide (1.75 g, 12.2 mmol, 70 equiv) were dissolved in 10 mL of anhydrous dichloromethane. A solution of N-cyclohexyl-N′-3,5-bistrifluoromethylphenyl thiourea (184 mg, 488 μmol) and (+)-sparteine (56.2 mg, 244 μmol) in dichloromethane (2.2 mL) was then added. The reaction mixture was stirred at 35 °C under argon until full consumption of DLlactide, as monitored by 1H NMR. After 1 h, the mixture was concentrated under vacuum, and then the polymer was precipitated in 100 mL of diethylether at 0 °C. The white precipitate was then filtered, washed with 40 mL of methanol, and dried under vacuum overnight (m = 1.8 g, yield = 85%). 1H NMR (CDCl3, 500 MHz, 298 K): δ (ppm) 1.57 (m, 411H, H5 CH3 PLA), 3.33 (s, 3H, H1), 3.63 (m, 175H, CH2 PEG), 4.20−4.30 (m, 2H, H3), 4.4 (q, 1H, H6), 5.16 (m, 137H, H4, CH PLA). 13C NMR (CDCl3, 125.7 MHz, 298 K): δ (ppm) 16.5 (C5, PLA), 58.8 (C1), 64.2 (C3), 69.1 (C4, PLA), 70.1 (C2), 167.0 (C7, PLA). SEC: Mn = 11700 g/mol, Mw/Mn = 1.15. 1192

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Scheme 1. Preparation of the Hydroxyl Functionalized mPEG-PLA Copolymer 4

Figure 2. MALDI-TOF spectra of the functionalized mPEG 2. blowing nitrogen gas over the sample to avoid water vapor. 1H NMR spectra were recorded at different temperatures (20, 40, 60, 70, 80, 90 °C) on a Bruker Advance (500 MHz).

■

Preparation of the Y- and Linear-Shaped Copolymer/ Cabazitaxel Conjugates. For this work, we have chosen the taxane cabazitaxel as case study. Cabazitaxel (commercialized by Sanofi under the brand name Jevtana) has been approved by the U.S. Food and Drug Administration in 201026 and by the European Medicines Agency in 2011, in combination with prednisone, for the treatment of hormone refractory prostate cancer of patients previously treated with docetaxel containing treatment regimen. To obtain Y-shaped conjugates, a grafting point must be introduced at the junction of the PEG and PLA blocks. To this end, we turned to 2,2-bis(hydroxymethyl)propionic acid (bis-HMPA) as trivalent motif. Bis-HMPA is commonly used to prepare branched macromolecules (such as

RESULTS AND DISCUSSION

The aim of this study was to design a biodegradable PEG-PLA copolymer with a functional group between the two blocks, allowing for the incorporation of the therapeutic agent at specific position in the copolymer, that is, between the two blocks. We were intrigued to see if this new macromolecular architecture has an influence on the accessibility of the drug in the nanoparticle. 1193

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Scheme 2. Preparation of the Y-Shaped mPEG-PLA/Cabazitaxel Conjugate 5

dendrimers,27,28 comb-shaped block copolymers,29 Y-shaped mitkoarm copolymers.30). In our case, the carboxylic acid and one of the hydroxyl groups of bis-HMPA were intended to append the PEG and PLA blocks, leaving a hydroxyl group for cabazitaxel conjugation. Scheme 1 depicts the detailed synthetic sequence of the hydroxyl functionalized mPEG-PLA copolymer. The benzylidene acetal derived from bis-HMPA was first coupled with a monomethyl ether poly(ethylene glycol) mPEG−OH (Mn = 2300, Mw/Mn = 1.09) in the presence of ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and 4(dimethylamino)-pyridinium p-toluenesulfonate (DPTS). The benzylidene acetal was then deprotected by hydrogenolysis using Pd/C as catalyst, and after workup, the 1,3-diol functionalized mPEG 1 was obtained in high yield (84% over two steps). Its steric exclusion chromatography (SEC) characteristics are virtually unchanged (Mn = 2080 g/mol, Mw/Mn = 1.08). In a second step, one hydroxyl group of the mPEG diol 1 was selectively protected using iPr3SiCl and triethylamine. After aqueous washings and precipitation in diethyl ether, the functionalized mPEG 2 was obtained in good yield (74%). The dissymmetrization of the 1,3-diol moiety is clearly apparent by NMR spectroscopy. Two distinct 13C NMR signals are observed for the terminal CH2O groups (at δ 66.1 and 66.7 ppm) and these are associated with two different AB systems in the 1H NMR spectrum (the two protons of each CH2 become diastereotopic as the adjacent quaternary carbon atom is now stereogenic). In addition, the MALDI-TOF spectrum (Figure 2) shows a single polymer population c o r r es p o n d in g t o t h e m o l ec u l a r f o r m u l a M e O(CH2CH2O)nCOC(Me)(CH2OH)CH2OSi(iPr)3 Na+, confirming the complete and selective protection of one hydroxyl group. The use of a sterically demanding chlorosilane is found to be critical to achieve selective monoprotection. Untractable mixtures of mono- and diprotected mPEG diols were obtained with triethylchlorosilane and dimethyltertbutylchlorosilane. The functionalized mPEG 2 was then used as macroinitiator for the ring-opening polymerization (ROP) of lactide, and to avoid undesirable initiation with traces of water, 2 was dried by azeotropic distillation of toluene. Here we took advantage of organocatalyzed ROP31 to obtain the desired functionalized mPEG-PLA copolymer 3 under mild conditions and with good control. The combination of N-cyclohexyl-N′-3,5-bis[trifluoromethyl]phenyl thiourea (TU) and (+)-sparteine was selected as catalyst. This bifunctional system was shown to provide a very good compromise between efficiency and functional-group tolerance in the ROP of lactones.25,32 Despite its macromolecular nature and the associated steric shielding, 2 efficiently initiates the ROP of D,L-lactide (70 equiv) and the polymerization was complete within 3 h at 35 °C in

dichloromethane (using 4 mol % of thiourea and 2 mol % of sparteine). In the same pot, the hydroxyl end group of the PLA block was readily acetylated using acetic anhydride (Ac2O) and 4-dimethylamino-pyridine (DMAP).33 After standard workup, the functionalized mPEG-PLA copolymer 3 was obtained in 75% yield. SEC analysis indicates the presence of a unique polymer population. The exact molar mass as determined by triple detection (Mn = 11850 g/mol) fits nicely with that expected from the lactide/macroinitiator ratio (Mntheo = 12511 g/mol) and the molar mass distribution is fairly narrow (Mw/ Mn = 1.15). In addition, the 1H NMR spectrum shows identical integrations for the signals associated with the MeO chain-end of the PEG block (3.37 ppm), the OAc chain-end of the PLA block (2.12 ppm), and the Me group of bis-HMPA moiety (1.16 ppm), indicating very good control of the initiation and end-capping. Finally, the protected copolymer 3 was treated with boron trifluoride diethyl etherate (BF3·Et2O)30 to release the hydroxyl group in between the PEG and PLA blocks. NMR spectroscopy indicates complete removal of the triisopropylsilyl protecting group, and the integrity of the polymer backbone was ascertained by SEC analysis, both Mn and Mw/Mn remaining fairly constant (11800 g/mol and 1.18, respectively, for the deprotected copolymer 4). The diglycolyl moiety was chosen as covalent link between the mPEG-PLA copolymer and cabazitaxel. The hydroxyl group in the 2′ position of taxanes is known to be fairly reactive and its chemical derivatization has been largely explored, including the formation of ester prodrugs.34 Consistently, reaction of cabazitaxel with diglycolic anhydride in dichloromethane under DMAP catalysis readily afforded its diglycolyl conjugate (96% yield). Coupling with the functionalized copolymer 4 was then achieved with N,N′-diisopropylcarbodiimide (DIPC) and DMAP in dichloromethane (Scheme 2). After workup, the Yshaped copolymer/cabazitaxel conjugate 5 was isolated in high yield (92%). SEC analysis shows the presence of a single polymer population with essentially the same characteristics (Mn = 11920 g/mol and Mw/Mn = 1.24) than before cabazitaxel conjugation. High-field NMR spectroscopy (500 MHz, CDCl3, 298 K) enables to precisely assign most 1H and 13C signals of the conjugate 5. In particular, the relative integrations of the 1H resonance signals associated with the MeO chain-end of the PEG block (3.38 ppm), the OAc chain-end of the PLA block (2.13 ppm), the Me group of the bis-HMPA moiety (1.26 ppm), and the OMe groups in positions 7 and 10 of cabazitaxel (3.31 and 3.45 ppm, respectively) are identical within the margin of error. This indicates that the covalent conjugation is quantitative, and accordingly, the loading in cabazitaxel can be estimated to 8 wt %. In addition, UPLC analyses have been carried out on the isolated sample to confirm that the 1194

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Scheme 3. Preparation of the Linear-Shaped mPEG-PLA/Cabazitaxel Conjugate 7

Figure 3. TEM images (top) and DLS traces (bottom) of the linear-shaped (7, L) and Y-shaped (5, Y) cabazitaxel conjugates.

Figure 4. Variable temperature 1H NMR spectra (500 MHz) of nanoparticles deriving from the Y-shaped (5, right) and linear-shaped (7, left) mPEG-PLA/cabazitaxel conjugates in D2O.

cabazitaxel has been covalently coupled to the copolymer and not simply coprecipitated, and indeed less than 0.7 mol % of cabazitaxel-diglycolyl and free cabazitaxel was detected. To evaluate the precise influence of the position at which cabazitaxel is introduced, a related linear-shaped copolymer conjugate 7 featuring the cabazitaxel at the PLA chain end was prepared. The same synthetic methodology was followed (Scheme 3). The monomethyl ether poly(ethylene glycol) mPEG-OH was used as macroinitiator for the ROP of lactide in the presence of the thiourea/sparteine bifunctional catalyst.

The resulting mPEG-PLA-OH copolymer 6 (Mn = 11700 g/ mol and Mw/Mn = 1.15) was then coupled with cabazitaxeldiglycolyl under the same conditions than used previously. The linear-shaped copolymer/cabazitaxel conjugate 7 was characterized by SEC and NMR to ascertain the integrity of the polymer backbone and quantitative coupling of cabazitaxel. Preparation and Characterization of the Nanoparticles. The formation of nanoparticles from the Y- and linearshaped conjugates was then investigated using nanoprecipitation technique, in which acetone solutions of 5 or 7 were slowly 1195

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Figure 5. Release profiles of the nanoparticles deriving from the Y-shaped (5, plasma. The values are the mean ± SD, n = 3.

▲)

and linear-shaped (7) mPEG-PLA/cabazitaxel conjugates in rat

PLA core becomes more mobile and thus the corresponding CH and CH3 are clearly visible above 60 °C. For the nanoparticles derived from the Y-shaped conjugate 5, additional signals assigned to cabazitaxel were observed in the aromatic region (δ ∼ 8.5 ppm) above 60 °C. This indicates a higher degree of solvation of the cabazitaxel in the nanoparticles derived from the Y- versus linear-shaped conjugate, and thus, a different spatial distribution of cabazitaxel occurred depending on the copolymer architecture. When coupled at the PLA chain-end (linear-shaped conjugate), the cabazitaxel is embedded in the PLA core, but when it is introduced at the mPEG-PLA junction (Y-shaped conjugate), the cabazitaxel is rather located at the periphery of the hydrophobic core and at the interface with the hydrophilic corona. Finally, in vitro release studies were performed to assess the influence of the Y- versus linear-shaped architecture of the conjugate on the cabazitaxel release profile. Nanoparticle suspensions were incubated in rat plasma at 37 °C for 24 h. Aliquots were harvested at various time intervals and analyzed by high-performance liquid chromatography (HPLC with UV detection, 230 nm; Figure 5). For both samples, the initial release was negligible (no burst effect), as generally expected for covalent conjugates. Noteworthy, the release kinetic of cabazitaxel was about two times faster with the nanoparticles made from Y- versus linear-shaped conjugate (47 vs 24% at 24 h). No significant quantity of cabazitaxel-diglycolyl was detected in the release medium, compared to cabazitaxel (