Y-shaped mPEG-PLA cabazitaxel conjugates: Well-controlled synthesis by organocatalytic approach and self-assembly into interface drug-loaded core-corona nanoparticles

Article abstract of DOI:10.1021/bm400161g

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.

Full text of DOI:10.1021/bm400161g

Article
pubs.acs.org/Biomac
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 Sable,§ Guillaume Louit,§ Didier Bazile,§
́
and Didier Bourissou*,†,‡
†Universite
́
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 nano-
particles (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.
delivery applications.8 Their ability to form core−corona
INTRODUCTION
■
nanostructures was recognized in the early 1990s,9−11 and the
Over the past few decades, polymeric nanoparticles (NPs) have
presence of the PEG chains at the surface of the nanoparticles
attracted considerable interest for drug delivery, especially in
anticancer chemotherapy.1−4 NPs present great therapeutic
was shown to impart longer systemic circulation time and lower
toxicity in vivo as compared to the NPs derived from simple
potential thanks to their ability (i) to solubilize and stabilize
hydrophobic drugs through encapsulation, (ii) to passively
PLA.9,10,12 Since then, PEG-PLA NPs have been extensively
studied for the encapsulation of a variety of hydrophobic drugs
accumulate within the tumor interstitium, following in vivo
including taxanes.13a,b Taxanes (paclitaxel, docetaxel, cabazitax-
injection, through enhanced permeability and retention (EPR)
effect,5,6 and (iii) to release the drug in a controlled and
el, etc.) are an important category of cytotoxic chemo-
therapeutic agents with wide applications in cancer therapy.
sustained fashion. Particularly efficient polymeric carriers are
The poor solubility of taxanes in aqueous medium requires the
prepared from biodegradable amphiphilic block copolymers,
use of surfactants (Cremophor EL, polysorbate 80) and ethanol
whose physicochemical properties are ideally suited for
which actually results in toxicity issues in clinic13c and
encapsulation, transport, and delivery of hydrophobic drugs.
necessitates the administration of corticosteroids and premed-
Thanks to the hydrophilic/hydrophobic nature and immisci-
ication prior to the chemotherapy.13d It is thus highly desirable
bility of their constitutive blocks, these copolymers readily self-
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 nano-
particles either by noncovalent or by covalent approaches. In
the noncovalent encapsulation technique, the drug is
assemble 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 PEG-
PLA are clearly at the forefront position of amphiphilic block
copolymers employed to form polymeric nanoparticles for drug
Received: February 1, 2013
Published: February 22, 2013
© 2013 American Chemical Society
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Figure 1. Schematic representation of the various types of mPEG-PLA nanoparticules developed from polymer/drug conjugates.
encapsulated physically into the PEG-PLA nanoparti-
cules,9−11,14 while in the covalent approach, the drug and the
PEG-PLA copolymer are chemically linked before self-
assembling 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 derivatiza-
tion of the carboxyl chain ends, have been self-assembled into
micelles,18,19 and (ii) PLA conjugates, synthesized by metal-
mediated 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 engi-
neering 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
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 self-
assembled 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 mPEG-
PLA 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 nano-
particles. 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.
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 MB-
SPS-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
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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.
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
1
= 0.4 g, yield = 74%). H 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,
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 JEOL-
JEM 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).
H7), 3.85−3.92 (dd, 2H, J = 9.4 and 24.7 Hz, H8), 4.26 (t, 2H, J3
=
H−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 N-
cyclohexyl-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
1
until full consumption of the monomer, as controlled by H NMR (3
h). Acetic anhydride (39 μL, 0.40 mmol) and 4-dimethylaminopyr-
idine (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.
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 dichloro-
methane 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
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
1
(m = 0.85 g, yield = 85%). H 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.
1
(m = 8.7 g, yield =95%). H NMR (CDCl3, 300 MHz, 298 K): δ
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
(ppm) 1.11 (s, 3H, H6), 3.37 (s, 3H, H1), 3.63 (br, 180H, CH2 PEG),
3
3.69 − 3.78 (m, 4H, H7a and H7b), 4.33 (t, 2H, JH−H = 4.8 Hz, H3).
13C 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
1
pressure, a white powder was isolated (m = 5.04 g, yield = 96%). H
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),
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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 (-CH2OCH2-
diglycolyl), 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 Con-
jugate 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 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
1
(m = 0.17 g, yield = 85%). H 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 con-
jugate 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.
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-bistrifluorome-
thylphenyl 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 DL-
lactide, 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
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.
1
(m = 1.8 g, yield = 85%). H 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.
1
Preparation of Nanoparticles for H NMR Analysis in D2O.
Nanoparticle formulations were prepared as described above using
D2O and deuterated acetone as solvent. Acetone was evaporated by
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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.
1
blowing nitrogen gas over the sample to avoid water vapor. H 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.
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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 copoly-
mer. 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
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 Y-
shaped 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,
1
systems in the H 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
corresponding to the molecular formula MeO-
(CH2CH2O)nCOC(Me)(CH2OH)CH2OSi(iPr)3 Na+, con-
firming 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
1
298 K) enables to precisely assign most H 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
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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.
1
Figure 4. Variable temperature H 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 cabazitaxel-
diglycolyl under the same conditions than used previously. The
linear-shaped copolymer/cabazitaxel conjugate 7 was charac-
terized by SEC and NMR to ascertain the integrity of the
polymer backbone and quantitative coupling of cabazitaxel.
Preparation and Characterization of the Nanopar-
ticles. The formation of nanoparticles from the Y- and linear-
shaped conjugates was then investigated using nanoprecipita-
tion technique, in which acetone solutions of 5 or 7 were slowly
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▲
Figure 5. Release profiles of the nanoparticles deriving from the Y-shaped (5, ) and linear-shaped (7) mPEG-PLA/cabazitaxel conjugates in rat
plasma. The values are the mean SD, n = 3.
added to water under vigorous stirring. The organic solvent was
then removed under vacuum and the final concentration was
adjusted to 10 mg/mL by addition of water. TEM and DLS
analyses (Figure 3) revealed the formation of well-defined
spherical nanoparticles (mean DLS diameter D ∼ 27 nm for
linear-shaped and D ∼ 35 nm for Y-shaped polymer conjugates,
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.
and mean TEM diameter Dm(200 particles) = 20
3.0 nm for
linear-shaped and Dm(200 particles) = 25 3.0 nm for Y-shaped
polymer conjugates) with monomodal particle size distribution
and low polydispersities (PDI ∼ 0.17) for both types of
copolymer/cabazitaxel conjugates. This demonstrates that the
Y-shaped architecture of the conjugate and the introduction of
a large drug at the mPEG-PLA junction do not perturb the self-
assembling process. In addition, the polymeric nanodispersions
for both architectures showed good stability over time, no
modification being detected by TEM and DLS after several
weeks at room temperature (Figure S7).
The critical micelle concentrations (CMC) of Y-shaped and
linear mPEG-PLA/cabazitaxel nanoconjugates were determined
to evaluate the influence of the position of cabazitaxel on the
self-assembly properties of the macromolecules (Figure S8).
The CMC values clearly indicate that the presence of the large
hydrophobic cabazitaxel at the interface between the PLA core
and the mPEG corona do not significantly disturb the self-
assembly of the polymer conjugates. Indeed, the CMC of the Y-
shaped mPEG-PLA/cabazitaxel nanoparticles was found to be
only 25% higher (1 mg/L) than that of the linear analogue
(0.75 mg/L).
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
(<0.8 μg/mL at 24 h, that is, 1.5% vs 15−55 μg/mL for
cabazitaxel at the same time). The faster release kinetic of
cabazitaxel in the case of the Y-shaped conjugate versus linear-
shaped conjugate is consistent with the distribution of
cabazitaxel at the core−corona interface of the nanoparticles,
To observe if the macromolecular architecture of the
copolymer conjugates impacts the position of cabazitaxel
1
within the nanoparticles, H NMR studies were carried out in
D2O at variable temperature on nanoparticles based on linear-
shaped and Y-shaped cabazitaxel conjugates (Figure 4). At 25
°C, similar spectra were obtained whatever the Y- or linear-
shaped architecture of the conjugate. In agreement with the
core−corona structure of the nanoparticles, the PLA block was
not visible at 25 °C as it forms a solid-like core.35 None of the
characteristic signals of cabazitaxel was detected either,
indicating as well its reduced mobility. In fact, only the CH2
groups of the PEG block were seen, indicating the presence of a
hydrophilic corona with substantial mobility. Upon heating, the
1
as shown by H NMR. Note that faster release kinetic of
cabazitaxel in tumor may be expected to have positive
implication for cancer treatment, because once the nano-
particles distribute to the tumor, a faster drug release kinetic
may be beneficial to achieve a rapid inhibition of the tumor.36
These preliminary results illustrate that the architecture of the
copolymer/cabazitaxel conjugate may indeed influence the
release kinetic of cabazitaxel from the nanoparticles.
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CONCLUSIONS
■
In conclusion, we have designed and prepared via organo-
catalytic polymerization a well-defined mPEG-PLA copolymer
conjugate featuring a new, Y-shaped architecture. The drug,
cabazitaxel in this work, has been introduced at the interface of
the PEG and PLA blocks, thanks to a trivalent motif (bis-
HMPA). The Y-shaped architecture of the conjugate did not
prevent self-assembling of the amphiphilic copolymer, and
stable spherical nanoparticles were readily obtained using
nanoprecipitation technique. Noteworthingly, the architecture
of the conjugate impacts the spatial distribution of the drug
within the nanoparticles. With the Y-shaped conjugate,
cabazitaxel was found preferentially located at the interface of
the hydrophobic PLA core and hydrophilic PEG corona of the
NPs, as substantiated by variable-temperature 1H NMR
analyses in D2O. The Y-shaped architecture of the conjugate
was also shown to offer better accessibility for hydrolysis, and
the release kinetic of cabazitaxel was about two times faster with
the nanoparticles made from Y- versus linear-shaped conjugate.
From these results, the modification of the drug position within
the polymer−drug conjugates appears as an interesting and
promising approach to finely tune the structure and properties
of the derived NPs. Ongoing work in our laboratory aims at
exploring further such Y-shaped conjugates.
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ASSOCIATED CONTENT
* Supporting Information
Detailed materials and methods and atom labeling for NMR
assignments. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
(20) (a) Tong, R.; Cheng, J. Angew. Chem., Int. Ed. 2008, 47, 4830−
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AUTHOR INFORMATION
Corresponding Author
*Fax: (+33) 5 61 55 82 04. E-mail: dbouriss@chimie.ups-tlse.fr.
Notes
■
(21) For selected references, see: (a) Khandare, J. J.; Jayant, S.;
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(22) To enhance and direct the delivery of NPs, targeting moieties
have been introduced at the PEG chain end of PEG-PLA block
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Mann, N.; Wang, A.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad,
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Retnarajan, B.; Sabnis, A.; Schnipper, E.; Song, J. J.; Song, Y. H.;
Summa, J.; Tompsett, D.; Troiano, G.; Van Geen Hoven, T.; Wright,
J.; LoRusso, P.; Kantoff, P. W.; Bander, N. H.; Sweeney, C.;
Farokhzad, O. C.; Langer, R.; Zale, S. Sci. Transl. Med. 2012, 4,
128ra39.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the CNRS, UPS, and ANRT (CIFRE
Fellowship to F.B.) for financial support of this work. Michel
Veillard (Sanofi) and Bertrand Castro (Sanofi) are acknowl-
edged for their scientific support in initiating this work. We also
thank Yannick Coppel (LCC Toulouse) for his assistance with
the NMR experiments.
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