Precise engineering of multifunctional PE gylated polyester nanoparticles for cancer cell targeting and imaging

Article abstract of DOI:10.1021/cm403822w

Multifunctional poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) nanoparticles for cancer cell targeting and imaging have been designed by a combination of ring-opening polymerization and "click" chemistry. Nanoparticles containing both a targeting ligand and a fluorescent probe were prepared by blending PLA-b-PEG-ligand, PLA-b-PEG-fluorescent probe, and PLA-b-PEG-OMe copolymers at the molar ratios necessary to achieve the desired surface ligand and fluorescent probe densities. This strategy has been illustrated by the preparation of a large library of a variety of nanoparticles, such as ligand-decorated nanoparticles (with biotin, folic acid or anisamide), fluorescent nanoparticles (UV-vis or near-infrared dyes), and multifunctional nanoparticles decorated with a targeting ligand and a fluorescent probe. Successful targeting was demonstrated by surface plasmon resonance and in vitro experiments on different cancer cell lines.

Full text of DOI:10.1021/cm403822w

Article
pubs.acs.org/cm
Precise Engineering of Multifunctional PEGylated Polyester
Nanoparticles for Cancer Cell Targeting and Imaging
Nicolas Mackiewicz,† Julien Nicolas,*,† Nadege Handke,† Magali Noiray,† Julie Mougin,† Cyril Daveu,‡
̀
́
Harivardhan Reddy Lakkireddy,§ Didier Bazile,§ and Patrick Couvreur†
†Institut Galien Paris-Sud, Univ Paris-Sud, UMR CNRS 8612, Faculte
́ ́
de Pharmacie, 5 rue Jean-Baptiste Clement,
̂
F-92296 Chatenay-Malabry cedex, France
‡Sanofi Research and Development, Fibrosis and Wound Repair, 1 avenue Pierre Brosselette, F-91385 Chilly-Mazarin cedex, France
§Sanofi Research and Development, Lead Generation to Candidate Realization Platform, 13 quai Jules Guesde,
F-94403 Vitry-sur-Seine cedex, France
S
* Supporting Information
ABSTRACT: Multifunctional poly(ethylene glycol)-block-
poly(lactic acid) (PEG-b-PLA) nanoparticles for cancer cell
targeting and imaging have been designed by a combination
of ring-opening polymerization and “click” chemistry. Nano-
particles containing both a targeting ligand and a fluorescent
probe were prepared by blending PLA-b-PEG−ligand, PLA-b-
PEG−fluorescent probe, and PLA-b-PEG−OMe copolymers
at the molar ratios necessary to achieve the desired surface
ligand and fluorescent probe densities. This strategy has been
illustrated by the preparation of a large library of a variety of
nanoparticles, such as ligand-decorated nanoparticles (with biotin, folic acid or anisamide), fluorescent nanoparticles (UV−vis or
near-infrared dyes), and multifunctional nanoparticles decorated with a targeting ligand and a fluorescent probe. Successful
targeting was demonstrated by surface plasmon resonance and in vitro experiments on different cancer cell lines.
However, examples of polymeric nanoparticulate systems gath-
ering all prerequisites for targeted drug delivery (i.e., biode-
gradable, stealth, traceable, and targeted) are rather scarce, thus
deserving more research effort in this area.
INTRODUCTION
■
Spurred by the development of advanced nanoscale systems for
drug delivery, the field of nanomedicine has recently received
tremendous attention.1,2 Since many drugs exhibit a low thera-
peutic efficacy due to nonspecific tissue distribution, as well as
to rapid metabolism and/or excretion from the body, their
encapsulation into colloidal nanocarriers, able to ensure a safe
transport from the injection site to the therapeutic target, has
been widely investigated.3−6 The efficacy and safety of thera-
peutic drugs depends on the ability to deliver those to the
target disease sites with a minimal distribution to off-target
organs of the body. When the drugs reach the target organ,
only those drugs possessing the ability to penetrate into the
target cells show efficacy. However, the drugs possessing poor
cell penetration remain inactive because their intracellular
access remains poor, even if they are delivered efficiently to
the extracellular space of the target organ. For such drugs, the
use of intelligent approaches to enhance their intracellular
delivery is a key requisite for enhancing their therapeutic effi-
cacy, for instance through the design of targeted7−9 or stimuli-
responsive nanoparticulate systems.10 Among the different
classes of carrier materials suitable for the development of nano-
medicines, biodegradable polymers perhaps represent the best
candidates due to the flexibility and robustness offered by mac-
romolecular synthesis methods, the great diversity of macro-
molecular architectures, and their ease of functionalization.11
In this context, nanoparticles made of polyesters, such as
poly(lactic acid) (PLA) or poly(lactic-co-glycolic acid) (PLGA),
hold great promise in the field, due to their biocompatibi-
lity and biodegradability,12,13 and are Food and Drug
Administration-approved polymers. The vast majority of
functionalized polyester nanoparticles with surface-installed
recognition ligands, or functional groups for subsequent coup-
ling with biologically active ligands, are generally obtained by
carbodiimide-assisted coupling chemistry or Michael addition,
using naturally occurring terminal groups (e.g., COOH, NH2,
OH) of polyester chains (or PEG for PEGylated nanopar-
ticles).7 Although very convincing examples of active targeting
using polyester nanoparticles have been reported by such
functionalization pathways,14−20 noteworthy is the fact that
most of these systems face substantial lack of both flexibility
regarding the nanoparticle functionalization (i.e., difficulty to
tune/control the amount of exposed ligand when the
functionalization is achieved on preformed nanoparticles) and
Received: November 18, 2013
Revised: February 4, 2014
Published: February 5, 2014
© 2014 American Chemical Society
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Figure 1. Synthetic pathway to prepare tunable targeted and fluorescent PEGylated polylactide nanoparticles by a combination of ring-opening
polymerization (ROP), copper-catalyzed azide−alkyne cycloaddition (CuAAC), and concomitant self-assembly into nanoparticles in aqueous
solution. Targeting ligands: biotin (VB7), anisamide (Am), and folic acid (FA). Fluorescent ligands: FP547 and FP682.
diversity regarding the nature of the exposed ligands (i.e., a
customized synthetic pathway is usually required for each
ligand). In addition, biologically active ligands are likely to bear
multiple functional groups that can be sensitive to carbodiimide
chemistry, leading to side reactions and/or multisite attach-
ments unless additional steps of protection/deprotection are
undertaken. Copper-catalyzed azide−alkyne Huisgen 1,3-
dipolar cycloaddition (CuAAC), often termed “click” chem-
istry, may then offer valuable benefits in the sense of its
exceptional efficiency and selectivity under relatively mild
conditions.21,22 CuAAC has indeed received tremendous
interest as an established synthetic route to obtain tailor-
made complex materials and has been exploited in many
research areas, among them being dendrimers,23−25 bioconju-
gates,26−28 therapeutics,29−31 and functionalized polymers.32−36
However, very few examples of surface-functionalized
PEGylated biodegradable nanoparticles by click chemistry
have been reported so far.37−41 For instance, in the case of
polyester nanoparticles, Alexander and co-workers elegantly
reported biologically active folic acid (FA)-functionalized
PLGA-b-poly(oligoethylene glycol methyl ether methacryate)
block copolymer nanoparticles for gene delivery, whose FA
moiety was “clicked” at the extremity of the polymethacrylate
backbone.40 FA has also been clicked at the surface of azido-
decorated PLA-b-PEG nanoparticles, but no targeting evalua-
tion has been reported.39
Therefore, the nanoparticles comprise (i) a biodegradable core
made of PLA, (ii) a PEG shell for improved colloidal stability
and stealth features,42 and (iii) surface-displayed biologically
active ligands for cancer cell targeting and fluorescent dyes for
imaging/tracing purposes, both tethered by CuAAC at the
distal end of the PEG chains. The idea was to synthesize a
“clickable” PLA-b-PEG−N3 copolymer, which is further reacted
via CuAAC with the desired alkyne derivative (Figure 1).44
Therefore, by adjusting the stoichiometry of a blend comprising
PLA-b-PEG−ligand and other copolymers (functionalized or
not), tunable multifunctional nanoparticles can be readily pre-
pared by concomitant self-assembly in aqueous solution. The
robustness of our approach is illustrated by the derivatization of
the PEGylated nanoparticles with a library of ligands. For
cancer cell targeting, we used anisamide (Am), which possesses
high affinity for σ-receptors,45,46 and two vitamins, folic acid
(FA) and biotin (VB7), that respectively recognize folate and
biotin receptors, overexpressed at the surface of many cancer
cells.38,47,48 For imaging purposes, we used two hemicyanine
dyes, FP547 and FP682, which emit in the UV−vis region and
in the near-infrared (NIR) suitable for in vivo imaging, respec-
tively. This approach is expected to open interesting prospects
for targeted drug delivery and theranostic applications.
EXPERIMENTAL SECTION
■
1. Materials. Methoxypoly(ethylene glycol), stannous octoate
[Sn(Oct)2], anhydrous toluene, dimethylaminopyridine (DMAP), tri-
ethylamine (TEA), methanesulfonyl chloride (MsCl), sodium azide
(NaN3), magnesium sulfate (MgSO4), concentrated hydrochloric acid
(conc HCl), sodium hydroxide (NaOH), N-bromosuccinimide (NBS),
triphenyl phosphine (PPh3), hydrobromic acid (HBr), triethylene
glycol, sodium hydride (NaH), propargyl bromide, potassium
In order to tackle the above-mentioned issues, we report
here a general strategy deriving from well-established long-
circulating PLA-b-PEG amphiphilic block copolymer nano-
particles42,43 to furnish multifunctional nanoparticles with tun-
able dual click functionalization with both imaging probes and
various targeting ligands for theranostic purposes (Figure 1).
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phthalate, sodium iodide, methanesulfonyl chloride, hydrazine hydrate,
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
(EDC), N-hydroxysuccinimide (NHS), folic acid, (benzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP),
p-methoxybenzoic acid, N,N-diisopropylethylamine (DIEA), copper
bromide (CuBr), N,N,N′,N″,N″-pentamethyldiethylenetriamine,
(PMDETA), Pluronic F68, sodium bicarbonate, and DMEM 2429
were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France)
and used as received. D,L-Lactide (Biovalley) was recrystallized from
ethyl acetate twice and then dried under vacuum. FP547−NHS and
Sn(Oct)2 (18.7 mg, 46.1 μmol) in anhydrous toluene (11.2 mL). The
reaction mixture was degassed by bubbling argon for 20 min and then
stirred in a preheated oil bath at 120 °C for 90 min under inert atmo-
sphere. The reaction was stopped at approximately 55% of monomer
conversion. Toluene was removed under reduced pressure, and the
obtained product was dissolved into a minimum volume of DCM and
subsequently precipitated in Et2O. The precipitate was then dissolved
into a minimum amount of THF, further precipitated in water, and sub-
sequently freeze-dried overnight to yield a white powder. (Mn,NMR
=
29 200 g·mol−1, Mn,SEC = 20 100 g·mol−1, Đ = 1.11). 1H NMR (400 MHz,
CDCl3, δ in ppm): 5.41−4.83 (m, 377H), 4.38−4.15 (m, 3H), 3.84−3.40
(m, 222H), 3.36 (t, J = 4.8 Hz, 2H), 1.82−1.21 (m, 1132H).
FP682−NHS were purchased from Interchim (Montluco̧ n, France).
PEG2500-benzyl was purchased from Polymer Source Inc. (Quebec,
Canada). Penicillin, fetal bovine serum (FBS), phosphate-buffered
saline (PBS), and L-glutamine were purchased from Lonza (Levallois,
France). Trypsin EDTA was purchased from Invitrogen Gibco (Saint-
Aubin, France). DMEM and RPMI1640 were purchased from Fisher
Scientific (Illkirch, France). 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), a tetrazo-
lium compound included in the CellTiter 96 AQueous Non-Radioactive
Cell Proliferation Assay, was purchased from Promega (Lyon, France).
All common solvents were used as received without further distillation
and were purchased from Carlo Erba. Alkynated biotin (VB7−alkyne)
was prepared as published elsewhere.38,49
2.3. Synthesis of the Amino−Tri(ethylene glycol)−Alkyne (H2N−
TEG−Alkyne) Linker. Synthesis of Monoalkyne Triethylene Glycol.
Triethylene glycol (5.62 g, 37.4 mmol, 1 equiv) was dissolved in anhy-
drous THF (50 mL) and the resulting solution was cooled to 0 °C
under dry conditions. Sodium hydride (0.99 mg, 1.1 equiv) was added
slowly to the above solution followed by dropwise addition of pro-
pargyl bromide (80 wt % in toluene, 4.36 mL, 1.1 equiv). The reaction
mixture was stirred for 12 h at room temperature under inert atmo-
sphere. THF was removed under reduced pressure, and the residue
was taken into DCM and washed several times with brine. The result-
ing organic layer was dried over MgSO4, filtered, concentrated under
reduced pressure, and dried under vacuum. The crude product was
purified by column chromatography over silica [cyclohexane (cHex)/
ethyl acetate (AcOEt) 8/2] to give a yellow oil (3.31 g, 47% yield).
1H NMR (300 MHz, CDCl3, δ in ppm): 4.15 (d, J = 2.4 Hz, 2H),
3.70−3.60 (m, 10H), 3.57−3.53 (m, 2H), 2.70 (m, 1H), 2.41 (t, J =
2.4 Hz, 1H).
Synthesis of Phthalimide−Monoalkyne Triethylene Glycol. To a
solution of monoalkyne triethylene glycol (4.0 g, 212 mmol, 1 equiv)
in DCM (60 mL) was added, dropwise under inert atmosphere,
a catalytic amount of DMAP, MsCl (3.3 mL, 2 equiv), and TEA
(5.9 mL, 2 equiv). The reaction mixture was stirred for 4 h at room
temperature. The solution was washed with brine (three times with
50 mL), the aqueous phase was then extracted with DCM (50 mL),
and the combined organic layers were dried over MgSO4, filtered, and
concentrated under reduced pressure. To the previous preparation
(5.1 g, 19.2 mmol, 1 equiv) dissolved in DMF (100 mL) was added
potassium phthalate (7.87 g, 2.2 equiv) and a catalytic amount of
sodium iodide (less than 1 equiv, e.g., a spatula tip). The solution was
stirred at 80 °C overnight and the solvent was removed under reduced
pressure. The resulting residue was purified by column chromatog-
raphy over silica (cHex/AcOEt; from 2/8 to 4/6) to give a yellow oil
(5.7 g, 94% yield). 1H NMR (300 MHz, CDCl3, δ in ppm): 7.81 (dd,
J = 5.5, 3.0 Hz, 2H), 7.69 (dd, J = 5.5, 3.0 Hz, 2H), 4.13 (d, J = 2.4 Hz,
2H), 3.80 (dt, J = 11.4, 6.0 Hz, 4H), 3.65 − 3.56 (m, 8H), 2.40 (t, J =
2.4 Hz, 1H).
2. Synthetic Pathways. 2.1. Synthesis of PLA-b-PEG−OMe
Copolymer (C1). To a mixture of methoxypoly(ethylene glycol)
(Mn,NMR = 2010 g·mol−1, 245 mg, 0.12 mmol) and D,L-lactide (7.01 g,
48.62 mmol) was added, under dry conditions, a solution of Sn(Oct)2
(18.7 mg, 46.1 μmol) in anhydrous toluene (11.2 mL). The reaction
mixture was degassed by bubbling argon for 20 min and then stirred in
a preheated oil bath at 120 °C for 30 min under inert atmosphere. The
reaction was stopped at approximately 54% of monomer conversion.
The toluene was removed under reduced pressure and the obtained
product was dissolved into a minimum amount of dichloromethane
(DCM) and further precipitated in diethyl ether (Et2O). The pre-
cipitate was then dissolved into a minimum amount of tetrahydrofuran
(THF) and further precipitated in water and subsequently freeze-dried
overnight to yield a white powder (Mn,NMR = 33 200 g·mol−1, Mn,SEC
=
1
26 100 g·mol−1, Đ = 1.14). H NMR (400 MHz, CDCl3, δ in ppm):
5.34−4.85 (m, 434H), 4.40−4.17 (m, 3H), 3.86−3.41 (m, 186H),
3.36 (s, 3H), 1.77−1.19 (m, 1302H).
2.2. Synthesis of PLA-b-PEG−N3 (C2). Synthesis of Bz−PEG−N3.
To a solution of PEG2500-benzyl (Mn,NMR = 2570 g·mol−1, 2.43 g,
0.94 mmol, 1 equiv), DMAP (58 mg, 0.5 equiv), and TEA (747 μL,
5.6 equiv) in DCM (65 mL), cooled to 0 °C, is slowly added MsCl
(326 μL, 4.4 equiv) over 20 min (Caution: Note that MsCl is highly
reactive and corrosive and therefore should be handle with great care). The
reaction was stirred overnight at room temperature and concentrated
under reduced pressure. The residue was dissolved into dimethylforma-
mide (DMF, 20 mL) and to it was added sodium azide (330 mg,
5.3 equiv). The reaction mixture was stirred at 50 °C for 24 h and con-
centrated under reduced pressure, and the residue was dissolved into
50 mL of brine and subsequently washed three times with the same
volume of brine. The organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure to a minimum volume of DCM.
The latter was precipitated into Et2O to give a white powder (2.16 g,
88% yield). 1H NMR (300 MHz, CDCl3, δ in ppm): 7.30−7.05
(m, 5H), 4.43 (s, 2H), 3.93−3.03 (m, 222H), 3.27 (t, 2H).
Synthesis of H2N−TEG−Alkyne. Phthalimide−alkyne triethylene
glycol (2.03 g, 6.4 mmol, 1 equiv) was dissolved in ethanol (EtOH,
200 mL) and to this solution was added hydrazine hydrate (3.1 mL,
10 equiv). The reaction mixture was stirred overnight under reflux
conditions. The reaction was cooled to room temperature and 8 mL
of conc HCl was added to the reaction mixture (pH ∼2−3). The
precipitate was removed by filtration and the pH was raised to above
10 using 2 M NaOH solution. The aqueous phase was extracted
three times with DCM, and the resulting organic layer was dried over
MgSO4, filtered, concentrated under reduced pressure, and dried
Synthesis of HO−PEG−N3. Bz−PEG−N3 (2.16 g, 0.83 mmol,
1 equiv) was solubilized into conc HCl (20 mL) and stirred at room
temperature for 2 days. A solution of concentrated NaOH was added
up to pH 1.0. The aqueous phase was extracted with DCM (four times
with 50 mL), and the combined organic layers were dried over
MgSO4, filtered, and concentrated under reduced pressure to a mini-
mum volume of DCM. The latter was precipitated into Et2O to give a
1
under vacuum; 911 mg of yellow oil was recovered (76% yield). H
NMR (300 MHz, CDCl3, δ in ppm): 4.13 (d, J = 2.4 Hz, 2H), 3.69−
3.50 (m, 8H), 3.43 (t, J = 5.2 Hz, 2H), 2.79 (t, J = 5.2 Hz, 2H), 2.38 (t,
J = 2.4 Hz, 1H), 1.33 (s, 2H). 13C NMR (75 MHz, CDCl3, δ in ppm):
79.64, 74.53, 73.47, 70.59, 70.40, 70.25, 69.09, 58.37, 41.80.
2.4. Synthesis of Alkynated Ligands. Synthesis of Folic Acid−
Alkyne Triethylene Glycol (FA−TEG−Alkyne). To a solution of amino
alkyne triethylene glycol (319 mg, 1.70 mmol, 1 equiv) in DMF
(70 mL) was added, under inert atmosphere, EDC (393 mg, 1.2 equiv),
NHS (236 mg, 1.2 equiv), and few drops of TEA. The reaction was
heated up to 50 °C and folic acid (754 mg, 1 equiv) was added to the
reaction mixture. The reaction was then stirred overnight at 50 °C.
1
white powder (1.89 g, 91% yield). Mn,NMR = 2510 g·mol−1. H NMR
(400 MHz, CDCl3, δ in ppm): 3.73−3.33 (m, 222H), 3.27 (t,
J = 5.0 Hz, 2H), 2.75 (s, 1H).
Synthesis of PLA-b-PEG−N3. A typical synthesis is as follows. To
a mixture of HO−PEG−N3 (293 mg, 0.12 mmol) and D,L-lactide
(7.01 g, 48.62 mmol) was added, under dry conditions, a solution of
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The solution was concentrated under reduced pressure. The residue
was precipitated into a mixture of DCM and acetone, filtered, and
dried under vacuum. The alkyne−FA conjugate γ-isomer (biologically
more active)50,51 was purified by preparative HPLC in order to remove
unreacted FA, dialkyne FA, and the α-isomer (biologically less active).
The HPLC purification consisted of a reverse-phase column (C18,
Kromasil 10 μm, 4.6 × 250 mm) using a 5−95% acetonitrile gradient
in 20 mM ammonium acetate buffer (adjusted to pH 5) for 20 min
followed by 95% acetonitrile for 5 min. The product was then solu-
bilized in 20% DMF in the eluent. Further purification was carried out
on Kromasil C18 10 μm packed in a 100 mm column (1.5 kg of
phase) eluting with a 20 mM 85% ammonium acetate buffer (pH 5)
and 15% acetonitrile; 200 mg of raw product was eluted at the same
time as the DMF injection peak. The final purification was performed
using preparative HPLC with a reverse-phase column (Waters XBridge
30 × 100, 5 μm) by dissolving 200 mg of the raw product in DMSO
(5 mL) and subsequently adding 5 mL of buffer solution (10 mM
ammonium carbonate adjusted to pH 9.3 with a 28% aqueous ammo-
nia solution) to it. Ten injections of 1 mL were performed using
a gradient going from 95:5 (buffer solution ammonium carbonate/
precipitated in Et2O. Finally, the precipitate obtained was dissolved in
a minimum volume of THF, precipitated into water, and dried under
high vacuum until constant weight. By H NMR, the coupling yield
1
was determined to be 90% (integration of the peaks at 6.95 and
7.8 ppm and compared to the signal of PEG at 3.2 ppm). The same
methodology was also applied to alkynated folic acid (16.3 equiv,
50 °C, 15 h, coupling yield = 76%; integration of the peaks at 6.65,
7.65, and 8.65) to obtain PLA-b-PEG−FA (C3), to alkynated FP547
[0.3 equiv, 50 °C, 15 h, coupling yield = not determined (nd)] to obtain
PLA-b-PEG−FP547 (C5), to alkynated FP682 (0.5 equiv,
40 °C, 18 h, coupling yield = nd) to obtain PLA-b-PEG−FP682
(C6), and to alkynated VB7 (18 equiv, 50 °C, 24 h, coupling yield =
52%, integration of the peak relative to the triazole proton at 7.86 ppm)
to obtain PLA-b-PEG−VB7 (C7). Another batch of PLA-b-PEG−VB7
copolymer (C8) pushed on to 76 mol % coupling (reaction twice as
long) was further used for SPR experiments.
3. Analytical Techniques. 3.1. Nuclear Magnetic Resonance
(NMR) Spectroscopy. NMR spectroscopy was performed in 5 mm
1
diameter tubes in CDCl3 or DMSO-d6 at 25 °C. H and 13C NMR
spectroscopy was performed on a Bruker Avance 300 spectrometer at
300 MHz (1H) or 75 MHz (13C) and a Bruker Avance 400 spec-
trometer at 400 MHz. The chemical shift scale was calibrated on the
basis of the solvent peak.
1
acetonitrile) to 5:95 in 12 min at 30 mL·min−1. H NMR (400 MHz,
DMSO-d6, δ in ppm): 8.61 (s, 1H), 8.29 (br, 1H), 7.8 (t, J = 6.1 Hz,
1H), 7.67 (d, J = 8.2 Hz, 2H), 7.03 (br, 2H), 6.85 (t, 1H), 6.62 (d, J =
8.2 Hz, 2H), 4.44 (d, J = 6.5 Hz, 2H), 4.31 (q, J = 5.1, 8.2 Hz, 1H),
4.12 (d, J = 2.2 Hz, 2H), 3.57−3.36 (m, 12H), 3.50 (hidden t, 1H),
3.35−3.1 (hidden m, 2H), 2.23 (t, J = 7.2 Hz, 2H), 1.91 (dquint, 2H).
LCMS: 611 [M + H]+.
Synthesis of Anisamide−Alkyne Triethylene Glycol (Am−TEG−
Alkyne). To a solution of amino alkyne triethylene glycol (200 mg,
1.07 mmol, 1 equiv) in DCM (20 mL) was added, under inert atmo-
sphere, PyBOP (780 mg, 1.4 equiv), p-methoxybenzoic acid (229 mg,
1.4 equiv), and DIEA (260 μL, 1.4 equiv). The reaction mixture was
stirred overnight at room temperature. The solution was washed with
brine (three times with 20 mL) and the aqueous phase was extracted
with DCM (20 mL). The combined organic layers were dried over
MgSO4, filtered, and concentrated under reduced pressure. The crude
product was purified by column chromatography over silica (eluent
cHex/AcOEt 5/5 to 7/3) to give a yellow oil (300 mg, 90% yield). 1H
NMR (300 MHz, CDCl3, δ in ppm): 7.74 (d, J = 8.8 Hz, 2H), 6.87 (d,
J = 8.9 Hz, 2H), 6.84 (br, 1H), 4.14 (d, J = 2.4 Hz, 2H), 3.81 (s, 3H),
3.71−3.53 (m, 12H), 2.42 (t, J = 2.4 Hz, 1H).
Synthesis of FP547−Alkyne Triethylene Glycol (FP547−TEG−
Alkyne) and FP682−Alkyne Triethylene Glycol (FP682−TEG−
Alkyne). To a solution of FP547−NHS (2.5 mg, 2.55 μmol,
1 equiv) in DMSO (356 μL) was added a solution of DMSO (92 μL)
containing EDC (0.49 mg, 1 equiv), NHS (0.29, 1 equiv), TEA (0.35 μL,
1 equiv), and amino alkyne triethylene glycol (1.07 mg, 2.2 equiv).
The mixture was stirred in the dark at room temperature for 12 h. The
reaction mixture was concentrated under reduced pressure, dissolved
into DCM, and extracted with brine. A pink oil was obtained, which
was subsequently characterized using UV/vis, fluorescence spectros-
copy, and 1H NMR. The resulting spectra were found to be similar to
those provided by the supplier of the fluorophore compound. The
same protocol was used for the synthesis of FP682−TEG−alkyne.
2.5. Conjugation of Alkynated Ligands to PLA-b-PEG-N3
Copolymer. A representative synthesis (PLA-b-PEG−Am, C4) was as
follows (note that molar equivalents of reactants are identical from one
conjugation experiment to another). To a degassed solution of PLA-b-
PEG−N3 (C2, 200 mg, 5.52 μmol, 1 equiv) and alkyne anisamide
(32 mg, 0.1 mmol, 18 equiv) in anhydrous DMF (6 mL) was added,
with a syringe, a degassed solution of CuBr (5.5 mg, 6.9 equiv) and
PMDETA (20 μL, 17 equiv) in anhydrous DMF (400 μL). The reac-
tion mixture was stirred for 15 h at 40 °C under nitrogen. The solution
was concentrated under reduced pressure and the residue was dis-
solved into a minimum amount of THF and subsequently precipitated
in water. The precipitate was freeze-dried, dissolved again into a mini-
mum amount of THF, and further precipitated in water. The pre-
cipitate was freeze-dried to yield a white powder. For poorly water-
soluble alkyne derivatives (i.e., FA and Am), the first freeze-dried
precipitate was dissolved into a minimum volume of DCM and then
3.2. Size Exclusion Chromatography (SEC). SEC was performed
at 30 °C with two columns from Polymer Laboratories (PL-gel
MIXED-D; 300 × 7.5 mm; bead diameter 5 mm; linear part 400 to
4 × 105 g·mol−1) and a differential refractive index detector (Spectra-
System RI-150 from Thermo Electron Corp.). Chloroform was used as
an eluent at a flow rate of 1 mL·min−1 and toluene was used as a flow-
rate marker. The calibration curve was based on poly(methyl meth-
acrylate) (PMMA) standards (peak molar masses, Mp = 625−625 500
g·mol−1) from Polymer Laboratories. This technique allowed Mn (the
number-average molar mass), Mw (the weight-average molar mass),
and Mw/Mn (the dispersity, D) to be determined.
3.3. Dynamic Light Scattering (DLS) and Zeta Potential. Mea-
surement of the nanoparticle diameter (Dz) and the surface charge
reported as zeta potential (ζ) was performed using a Nano ZS from
Malvern (173° scattering angle) at 25 °C. The particle size distribution
values, ranging from 0 to 1, are given by the DLS apparatus and cor-
respond to the ratio of the variance over the square of the average
particle diameter. The ζ-potential (mV) measurement was performed
after dilution with 1 mM NaCl, using the Smoluchowski equation.
3.4. Transmission Electron Microscopy (TEM). Five microliters of
NP dispersion at 10 mg·mL−1 was deposed on Formvar/carbon coated
grids (400 mesh). After 5 min at room temperature, a drop of phos-
photungstic acid 2% filtered on 0.22 μm was added for negative
staining and the excess of volume was eliminated. The grids were
then observed with a JEOL 1400 120 kV electron microscope operat-
ing at 80 kV at a nominal magnification of 5000−40 000. Digital
images were directly recorded on a CCD postcolumn high-resolution
(11 MegaPixel) high-speed camera (SC1000 Orius, Gatan Inc.) using
Digital Micrograph image acquisition and processing software
(Gatan Inc.).
4. Nanoparticle Formation. The copolymer or a blend of dif-
ferent copolymers (30 mg in total) was dissolved in AcOEt (1.2 mL).
The above organic phase was added to 3.3 mL of an aqueous phase
containing 1% w/v Pluronic F68. The mixture was then vigorously
shaken using a vortex shaker for 1 min. The resulting emulsion was
ultrasonicated (using an ultrasonic probe) for 3 min and the organic
solvent was removed under reduced pressure using a rotary evapo-
rator. The resulting nanoparticle suspension was ultracentrifuged at
30 000g for 30 min and the pellet was resuspended in 3 mL of pH 7.4
PBS. The nanoparticles were filtered through a 1 μm glass filter disk
(Acrodisc) and stored at 4 °C until use.
5. Cell Culture. KB-3-1 cells originating from human cervix carci-
noma, were obtained from Pr. S. Chevillard. These cells were cultured
in monolayers in two different media in order to induce or not an
overexpression of the folate receptors, supplemented with 1%
penicillin/streptomycin and 10% v/v FBS in a 5% CO2 humidified
atmosphere at 37 °C. The medium used to overexpress the folate
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1
Figure 2. H NMR spectra in the 0−9 ppm range in DMSO-d6 of PLA-b-PEG−N3 (a), PLA-b-PEG−FA (b), and alkyne−FA (c). The asterisk
indicates the presence of PLA.
receptors (KB-3-1*) was DMEM 2429 (medium without folic acid) in
which L-glutamine (200 mM at 0.584 g·L−1) and sodium bicarbonate
(3.7 g·L−1) were added. The medium used to culture the KB-3-1
control cell line was the usual DMEM (medium containing 4 mg·L−1
folic acid). MCF-7 cells, originating from human breast adenocarci-
noma, were obtained from the American Type Culture Collection
(ATCC). These cells were cultured in DMEM medium supplemented
with 1% penicillin/streptomycin and 10% v/v FBS in a 5% CO2
humidified atmosphere at 37 °C. This cell line was used as another
control, as they do not express folate receptors. PC-3 cells, originating
from human prostate adenocarcinoma, were obtained from the Institut
Curie. These cells were cultured in RPMI 1640 supplemented with
1% penicillin/streptomycin and 10% FBS in a 5% CO2 humidified
atmosphere at 37 °C. This cell line has been reported to overexpress
σ-receptors.52 Cultures of 85−90% confluency were used for all of the
experiments. The cells were trypsinized (trypsin−EDTA), counted,
and subcultured into 96-well plates for viability studies as well as into
24-well plates for confocal microscopic studies.
10 μL·min−1, allowed the binding of ∼6.8 ng·mm−2 of FBP per channel.
The first flow channel (Fc1) was blocked only by ethanolamine, so
that it could be used as a reference channel in order to check whether
the dextran is playing a role in the nonspecific adsorption of nanopar-
ticles. The adsorption of PLA-b-PEG−FA nanoparticles (5 mg·mL−1)
onto the immobilized FBP was assessed and compared with that of the
nonfunctionalized PLA-b-PEG−OMe nanoparticles as control. All the
experiments were conducted at a flow rate of 5 μL·min−1 with a
contact time of 500 s.
Interaction of biotinylated nanoparticles was monitored using a
BIAcore 2000 (GE Healthcare, Uppsala, Sweden). In order to be able
to regenerate the sensor chip, a biotin CAPture kit purchased from GE
Healthcare was used to study the biotin/streptavidin interaction. The
chip coating was made of a carboxymethylated dextran matrix, preim-
mobilized with a single-strand DNA molecule. Then a biotin CAPture
reagent, made of the complementary single-strand DNA molecule
conjugated to streptavidin, was used to capture the biotinylated nano-
particles. Concretely, the chip was rehydrated with the running buffer
[HEPES-buffered saline−NaCl (HBS-N), GE Healthcare] overnight
prior to use. Then the chip surface was conditioned with three 1-min
injections of regeneration buffer (6 M guanidine-HCl, 0.25 M
NaOH provided within the kit by GE Healthcare) with a flow rate
of 10 μL·min−1. The coupling process was performed as follows: (i)
biotin CAPture reagent was injected for 5 min at 2 μL·min−1 and (ii)
the biotinylated nanoparticles (0.0125 mg·mL−1) were then injected
for 3 min at 30 μL·min−1 and rinsing buffer injection was performed
for 3 min. The capture level of nanoparticle was monitored; (iii)
finally, the sensor chip surface was regenerated with two injections of
20 s at 5 μL·min−1 of the regeneration solution before a new experi-
mental cycle. The analyses were done using BIAevaluation software,
version 4.1.1 (GE Healthcare).
6. Cytotoxicity Assay. In a 96-well plate, 5 × 102 cells dispersed in
50 μL of culture medium (as described above) were deposited per
well. After 24 h of preincubation, 50 μL of nanoparticle suspension
in PBS at different copolymer concentrations (0.5, 0.1, 0.05, 0.01
mg·mL−1) was added to each well, and the well plates were incubated
(5% CO2, 37 °C) for 48 h. Twenty microliters of a 5 mg·mL−1 MTS
solution in PBS was added to the wells and then incubated for 3 h. The
absorbance of the resulting dye in each well was analyzed at 492 nm
wavelength using a microplate reader (Labsystem Multiscan MS, Type
352). The experiments were performed in triplicate. The percentage of
the surviving cells was calculated as the absorbance ratio of the treated
to the untreated cells.
7. Biological Evaluation of Targeted Nanoparticles. 7.1. As-
sessment of the Ligand−Receptor Interaction Using Surface
Plasmon Resonance (SPR) Spectroscopy. Interaction of folate-
modified nanoparticles was monitored by surface plasmon resonance
spectroscopy using a BIAcore T100 (GE Healthcare Life Sciences,
7.2. Assessment of Nanoparticle Uptake Using Fluorescence-
Activated Cell Sorting (FACS). Uptake of the targeted nanoparticles by
the cells has been assessed by flow cytometry. Nanoparticles con-
taining the fluorescent probe FP547 but without folic acid (N12; see
Table 1) as a control or with folic acid (N11; see Table 1) have been
prepared using 1% w/v Pluronic F68 as a stabilizer. The KB-3-1 cell
line was cultured in folic acid containing medium to facilitate the
overexpression of folate receptors (as mentioned previously) in a
́
Velizy, France) instrument. For our experiment, series S Sensor chip
CM5 (GE Healthcare) was used to immobilize the folate binding
protein (FBP) according to a previously described protocol.53
The immobilization protocol, which was performed at a flow rate of
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24-well plate up to near confluence (∼300 000 cells/well). The culture
medium in the wells was replaced with 1 mL of the medium containing
nanoparticles, at a final copolymer concentration of ∼60 μg·mL−1, and
incubated for different time intervals (from 10 min to 24 h). After-
ward, the culture medium was removed, and the cells were washed
twice with PBS (1 mL) and subsequently isolated by trypsinization
and centrifugation (5 min at 1000g). The cell pellet was resuspended
in PBS (1 mL), centrifuged (5 min at 1000g), and finally fixed by
Table 1. Composition and Colloidal Characteristics of
Multifunctional PLA-b-PEG Nanoparticles
a
b
PLA-b-PEG−X (mol % )
av
zeta
potential
(mV)
c
diam
c
expt OMe FA Am FP547 VB7
(nm)
PSD
N1
97
81
71
68
69
73
79
84
89
100
69
86
47
91
94
88
75
50
25
0
1
1
1
103
104
116
118
116
112
106
111
109
111
110
109
104
107
72
0.15
0.18
0.17
0.23
0.23
0.20
0.18
0.17
0.14
0.16
0.19
0.19
0.13
0.14
0.16
0.19
0.26
0.21
0.20
0.15
−12.9
−15.8
−8.4
−21.7
−21.2
−19.6
−19.0
−16.6
−18.0
−9.6
−17.3
−11.6
−10.1
−7.8
−17.8
−23.1
−15.5
−25.2
−33.6
−35.1
N2
16
N3
17
N4
24
31
20
16
12
8
N5
N6
N7
N8
N9
N10
N11
N12
N13
N14
N15
N16
N17
N18
N19
N20
17
3
3
2
2
28
5
9
74
19
38
57
76
79
74
66
64
a
Nature of the copolymers: OMe = methoxy (C1), FA = folic acid
(C3), Am = anisamide (C4), FP547 = FP547 fluorescent dye (C5),
b
VB7 = biotin (C8). Molar fraction of X in the blend determined by
1H NMR. Note that the total percentages, except for that of N10, are
not 100% due to nonquantitatve coupling for PLA-b-PEG−X.
Figure 3. Size exclusion chromatograms of PLA-b-PEG−N3 and PLA-
b-PEG−FA by DRI (main graphic) and UV detection at 350 nm
(inset).
c
Determined by DLS (see Experimental Section).
1
Figure 4. H NMR spectra in the 0−9 ppm range in DMSO-d6 of PLA-b-PEG−VB7.
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Figure 5. Synthesis of anisamide−tri(ethylene glycol)−alkyne (Am−TEG−alkyne).
1
Figure 6. H NMR spectrum in the 0−8 ppm range of PLA-b-PEG−Am in DMSO-d6.
incubating in 1% w/v paraformaldehyde in PBS. Flow cytometry
analysis of the cell suspensions was carried out using a BD
LSRFortessa cell analyzer with an excitation wavelength of 561 nm
and an emission signal retrieved between 575 and 589 nm. The same
experiment was also performed on PC-3 cells with nanoparticles N13
and N14 (Table 1).
fixed with nail varnish. The glass slides were kept in the dark at 4 °C
prior to observation. The fluorescence measurements were performed
with a long-pass 560 nm emission filter under 543 nm wavelength
excitation. To specifically observe the intracellular nanoparticles
localization, acquisitions were made at the median plane of the cell
monolayer. The pinhole size was set at 1.0 Airy unit (106 μm
diameter), giving an optical section thickness of 0.8 μm. Each sample
was observed with a zoom factor of 1.5, a master gain of 757, a digital
gain of 1, and a digital offset of 0.05. Prior to observations, the
autofluorescence of PC-3 cells was checked under the acquisition
settings.
7.3. Confocal Microscopy. In vitro imaging of the nanoparticle
uptake without or with anisamide as targeting ligand into PC-3
human prostate adenocarcinoma cells overexpressing σ-receptors was
performed using confocal microscopy. Nanoparticles containing the
fluorescent probe FP547 but without anisamide (N14; see Table 1) as
a control or with anisamide (N13; see Table 1) have been prepared
using 1% w/v Pluronic F68 as a stabilizer. Briefly, PC-3 cells were
seeded on a type-I collagen-coated glass disk (25 mm in diameter) at a
concentration of 25 000 cells·cm−2 (or 50 000 cells/mL/well) in a 24-
well plate. After 24 h of preincubation, the cells were incubated with an
aqueous suspension of nanoparticles (200 μg·mL−1, diluted in RPMI
1640). At 1, 6, and 10 h postincubation, the cells monolayers were
washed with fresh medium and then imaged using a confocal laser
scanning microscope (LSM 510 META (Zeiss) equipped with a 1 mW
helium neon laser and a Plan-Apochromat 63× objective lens
(numerical aperture/1.4, oil immersion). Prior to the imaging, the
glass disks were submerged into a freshly prepared paraformaldehyde
solution (4%) for 10 min. The latter was then neutralized by the
addition of ammonium chloride (50 mM for 10 min), and finally,
the glass disks were washed three times with PBS. The glass disks were
mounted on a glass slide using Vectashield (Vector Laboratories) and
RESULTS AND DISCUSSION
■
1. Synthesis and Characterization of PLA-b-PEG−
Ligand. In order to ensure an efficient ligand display at the sur-
face of the nanoparticles, the strategy was to prepare amphi-
philic block copolymers comprising a biodegradable PLA block
to which is attached a linear PEG chain bearing a terminal azide
group for further click reaction. Performing the coupling on
the PLA-b-PEG−N3 (C2) copolymer, prior to the formation of
nanoparticles, allows one to well-characterize the resulting
conjugate. Importantly, its blending with nonfunctionalized
PLA-b-PEG (C1) and/or PLA-b-PEG functionalized with other
ligands can conduct to multifunctional PLA-b-PEG nanopar-
ticles with tunable surface functionalization, which is of high
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Figure 8. Emission (solid line) and excitation (dashed line) spectra of
PLA-b-PEG−FP547 (a) and PLA-b-PEG−FP682 (b) copolymer.
Figure 7. Size exclusion chromatograms of PLA-b-PEG−N3 and PLA-
b-PEG−Am by DRI (main graphic) and UV detection at 375 nm
(inset).
importance for the rational design of efficient drug delivery
systems. Noteworthy is the fact that PLA-b-PEG (C1) was
obtained from a 2100 g·mol−1 PEG, whereas all functionalized
PLA-b-PEG−ligands were synthesized with a PEG of 2500 g·
mol−1 in order to promote the ligand display at the surface of
the nanoparticles.
Azidopoly(ethylene glycol) (N3−PEG−OH) was first ob-
tained from commercially available benzylpoly(ethylene glycol)
and used as macroinitiator for the ring-opening polymerization
(ROP) of D,L-lactide to furnish well-defined PLA-b-PEG−N3
(C2, Mn,NMR = 29 200 g·mol−1, Mn,SEC = 20100 g·mol−1, Đ =
1.11). Biologically active ligands and fluorescent probes were
then derivatized with alkyne moieties and clicked with PLA-b-
PEG−N3 under CuAAC conditions. The orthogonality of
CuAAC, in combination with the robustness of the PLA-b-PEG
nanoparticulate system, make this synthetic pathway virtually
applicable to any kind of small- to average-sized biologically
active ligand and fluorescent probe for the design of multi-
functional, biodegradable, and PEGylated nanoparticles. This
synthetic flexibility was demonstrated by the use of a variety of
ligands, either for targeting cancer cells or for imaging purposes.
The first selected targeting ligands were two vitamins, namely
FA and VB7, which are reported to be overexpressed at the
surface of a variety of cancer cells.47,54 For the synthesis of folic
acid-containing construct, FA was modified by an alkyne−
amino tri(ethylene glycol) linker via EDC/NHS coupling
chemistry and subsequently clicked to PLA-b-PEG−N3 (see
Experimental Section), followed by an extensive purification to
yield the γ-isomer of alkyne−FA, which is considered to be the
more active form of FA.50,51 Successful syntheses of alkyne−FA
Figure 9. Evolution of the average diameters and the particle size
distributions of nanoparticles N1−N3 (Table 1) in PBS stored at 4 °C.
which is likely to be assigned to the triazole proton resulting
from the click chemistry (Figure 2b). On the basis of the peaks
at 6.65, 7.65, and 8.65 ppm, a coupling yield of 76% has been
calculated.
From SEC analysis using differential refractive index (DRI)
and UV (at 350 nm to follow FA) detections, it was observed
that the clicked copolymer exhibited a strong UV absorbance
that matched with the DRI signal, in contrast to the starting
PLA-b-PEG−N3 copolymer, for which no signal was detected
1
(Figure 3). This data, in addition to that revealed by H NMR
analysis, confirmed the conjugation of FA to the copolymer.
PLA-b-PEG−VB7 (C7) was successfully synthesized under
similar CuAAC conditions from VB7 previously derivatized
with propargyl amine (Figure 4). 1H NMR analysis revealed all
characteristic signals of the VB7 moiety together with the
triazole proton at δ 7.86 ppm and the CH2 in the α-position of
the triazole at δ 4.48 ppm, from which a coupling yield of 52%
has been calculated.
and PLA-b-PEG−FA (Mn,NMR = 30 700 g·mol−1, Mn,SEC
=
1
19 500 g·mol−1, Đ = 1.16) were confirmed by H NMR spec-
troscopy (Figure 2). All the peaks corresponding to alkyne−FA
were assigned, among which were the alkyne proton at 2.5 ppm
and the CH2 in the α-position of the alkyne at 4.1 ppm
(Figure 2c). For the purified PLA-b-PEG−FA, all the char-
acteristic signals of FA and PLA-b-PEG were retrieved in the
spectrum, as well as a signal at δ 7.9 ppm (partially overlaid),
For the synthesis of Am-containing construct, the Am moiety
was formed in situ during the amidation reaction between
p-methoxybenzoic acid and the H2N−TEG−alkyne derivative,
1
leading to the desired Am−TEG−alkyne (Figure 5). H NMR
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Figure 10. Normalized SPR sensorgrams (RUNPs/RUFBP or RUNPs/RUbiotin CAPture reagent) obtained by injections of PLA-b-PEG−FA at 5 mg·mL−1
(N4−N10, Table 1) (a) or PLA-b-PEG−VB7 at 0.0125 mg·mL−1 (N10, N15−N20, Table 1) (c) nanoparticles over folate binding protein (FBP) or
streptavidin (SAv), respectively, immobilized on sensor chips. Evolution of the normalized maximum SPR signal as a function of the percentage of
FA (b) or VB7 (d) at the surface of the nanoparticles (dashed lines are guides for the eyes).
spectroscopy (Figure 6) revealed all expected proton signals
(among which was the triazole proton at δ 7.7 ppm), thus
confirming the successful coupling, with a 90% yield.
The SEC profile of PLA-b-PEG−Am, with UV detection at
375 nm, which is characteristic of Am (although UV absor-
bance of the triazole cannot be ruled out at this wavelength),
combined with the DRI trace, confirmed the successful linkage
of the Am group homogeneously on the polymer chains
(Figure 7).
For fluorescence imaging purposes, the fluorescent probes
FP547 and FP682 were clicked on PLA-b-PEG−N3. These
fluorescent probes were purchased as NHS derivatives, which
were subsequently reacted with the H2N−TEG−alkyne linker.
PLA-b-PEG−FP547 and PLA-b-PEG−FP682 were successfully
prepared and characterized by 1H NMR spectroscopy and SEC
after extensive purification to remove the unreacted fluorescent
probe residues. All characteristic peaks from the fluores-
cent probes were retrieved in the NMR spectra of the final
constructs (see Figure S1, Supporting Information). It is
noteworthy to point out that the coupling of the fluorescent
probes onto PLA-b-PEG−N3 under CuAAC conditions did
not alter their fluorescent properties, as observed from the
fluorescence spectra of PLA-b-PEG−FP547 and PLA-b-PEG−
FP682 (Figure 8).
2. Preparation and Characterization of Multifunc-
tional Nanoparticles. Multifunctional nanoparticles contain-
ing both a targeting ligand and a fluorescent probe were pre-
pared by the emulsion/solvent evaporation technique. In order
to achieve the desired surface ligand and fluorescent probe den-
sities, the different copolymers (i.e., PLA-b-PEG−ligand, PLA-
b-PEG−fluorescent probe, and PLA-b-PEG−OMe) were
blended at the desired molar ratios. This has been illustrated
by the preparation of a large library of a variety of nanoparticles,
such as ligand-decorated nanoparticles, fluorescent nanopar-
ticles, and multifunctional nanoparticles decorated with
targeting ligands and fluorescent probes (see Table 1).
The nanoparticles were prepared at a concentration of
10 mg·mL−1 in PBS and exhibited mean diameters ranging
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Figure 11. Cell viability (assessed using MTS assay) after a 48 h incubation of KB-3-1 (a), KB-3-1* (b), MCF-7 (c), and PC-3 (d) cells with
nonfunctionalized (N12 and N14, Table 1), FA-functionalized (N11, Table 1), and Am-functionalized (N13, Table 1) nanoparticles, as a function of
the nanoparticle concentration. All the experiments were repeated three times, and the results are expressed as the ratio of absorbance of the treated
cells ( SD) to that of the untreated control cells.
between 64 and 118 nm (DLS data) with relatively narrow
particle size distributions and negative surface charges. TEM
experiments were also performed and showed spherical
nanoparticles with average diameters in good agreement with
DLS measurements (Figure S2, Supporting Information).
These nanoparticles exhibited excellent colloidal stability upon
storage, as shown with the case of representative fluorescent nano-
particles, without ligands (N1) or with ∼16 mol % of either FA
(N2) or Am (N3) ligands, stored for at least 15 days (Figure 9).
4. Assessment of the Recognition of PLA-b-PEG−
Ligand Nanoparticles by the Corresponding Receptors
in Vitro. The ability of the targeting ligands positioned at the
distal end of the PEG chains of the nanoparticles to recognize
the corresponding receptors was investigated by SPR spectros-
copy, which is a mimic of cell surface receptors. SPR analysis
was performed for PLA-b-PEG−VB7 nanoparticles and PLA-b-
PEG−FA nanoparticles using streptavidin (SAv)- and folate
binding protein (FBP)-coated sensor chips, respectively. The
SPR analysis was not performed for PLA-b-PEG−Am be-
cause σ-receptors are transmembrane proteins that could hardly
be coated onto the SPR sensor chips.
sensor chips, whereas strong interactions were observed only
between the PLA-b-PEG−OMe/PLA-b-PEG−FA nanoparticles
(N4−N9) and the FBP-coated sensor chips (Figure 10a),
thereby confirming both the suitable positioning of FA on the
nanoparticle surface and its ability to specifically recognize
the folate receptors. SPR measurements of the nanoparticles
prepared with different molar ratios of FA allowed determining
the FA concentration needed for optimum binding efficiency of
the nanoparticles. Indeed, by plotting the final SPR signal value
for each sample as a function of the FA mole percent, the maxi-
mum specific binding was found at ∼14 mol % FA (Figure 10b),
where the specific signal represented ∼70% of the total signal
(the nonspecific signal is roughly estimated by the RU value for
the control nanoparticles without FA on their surface, N10).
Beyond ∼14 mol % FA, the specific signal decreased, suggesting
a loss of binding, which is likely due to mild stacking between
FA moieties and/or receptor overbinding (i.e., too many FA on
the surface of the nanoparticles may facilitate the binding of a
single nanoparticle to multiple receptors, thus reducing the
number of bonded nanoparticles).55 In our experiments, the FA
content in the nanoparticles could not be increased beyond
31 mol %, due to the limited aqueous solubility of the FA and
consequent impact on the colloidal stability of the nanopar-
ticles. Therefore, increasing the local concentration of FA
may also result in nonsolvated areas, which would also alter the
ligand/receptor recognition. All nanoparticles injected in the
reference channel (i.e., dextran functionalized with ethanolamine)
resulted in a low binding signal, suggesting that the dextran does
To assess the affinity of folic acid from PLA-b-PEG−FA
nanoparticles toward the folate binding protein, a library of
PLA-b-PEG−FA nanoparticles was prepared by blending
different molar ratios of PLA-b-PEG−OMe/PLA-b-PEG−FA,
leading to FA amounts ranging from 0 to 31 mol % (N4−N10,
Table 1). As expected, the SPR experiments revealed no bind-
ing of the nanoparticles of whatever nature with the uncoated
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not induce nanoparticle adsorption. This also supports the idea
that the nonspecific signal previously obtained is likely the result
of interactions between the PEG chains and the FBP.
Different batches of VB7-functionalized nanoparticles were
prepared resulting from copolymer blends of variable PLA-b-
PEG−OMe/PLA-b-PEG−VB7 ratios, with VB7 amounts
ranging from 0 to 76 mol % (N10 and N15−N20, Table 1).
The fine-tuning of the surface-displayed VB7 allowed a dose−
response pattern to be obtained. Similarly to the case of FA-
functionalized nanoparticles, a maximum of binding of VB7-
functionalized nanoparticles to SAv was also noticed, but at
∼20 mol % of VB7. Above this threshold, the binding intensity
decreased. This decrease could be attributed again to the hydro-
phobic interactions between VB7 moieties due to their moder-
ate solubility in water at room temperature and/or receptor
overbinding.
Interestingly, at increasing concentration of the VB7-
functionalized nanoparticles, the SPR sensorgrams reached a
plateau (Figure S3, Supporting Information). This allowed ap-
parent dissociation rate constant (Kd,app) values of the inter-
action between biotinylated nanoparticles and SAv to be deter-
mined according to the Boltzmann sigmoid method. Kd,app
ranged from 5 × 10−11 to 5× 10−10 M (Figure S4, Supporting
Information), which is higher than typical biotin/streptavidin
Kd values (∼10−15 M). This could be explained by the two rea-
sons previously mentioned and also by the fact that nano-
particles are spherical systems for which only a small part of the
ligands is accessible for receptor recognition.
In summary, the surface functionalization of the nano-
particles with a high ligand density does not guarantee the best
ligand−receptor interaction efficiency, and only moderate sur-
face functionalization yields (∼10−20 mol %) may be enough
to reach an optimal binding efficacy. Actually, since the tethered
ligands interact with protein receptor, it is assumed that the
steric hindrance naturally provokes limited ligand−protein
accessibility. However, although optimum amounts of FA and
VB7 moieties have been clearly shown, these values must be
taken with caution, as they have been obtained under specific
experimental conditions (e.g., a given receptor density coated
on the chip, a given temperature, a given buffer solution), which
may not accurately reflects in vitro cell culture or in vivo con-
ditions and therefore may have been subjected to variations.
Interestingly, obtaining a maximum binding/uptake from
FA-56−59 or VB7-decorated60 nanoparticulate systems has been
rarely reported, perhaps because of the difficulty in accurately
tuning the amount of surface-exposed ligand. For instance,
Poon et al.56 determined an optimum number of FA moieties at
the surface of PEGylated polyester nanoparticles to be ∼2200,
which is in excellent agreement with our data (∼2000 FA for
N8 containing 12% FA). Similarly, Bae et al. showed an optimal
binding in the ∼10−25% FA range for PEGylated polymeric
micelles.61
5. Targeting Cancer Cells. Multifunctional nanoparticles
(i.e., fluorescent and targeted) were then tested for their ability
to target cancer cells in vitro using confocal microscopy and
flow cytometry. Fluorescence, which is necessary to track the
nanoparticles by each of these techniques, was readily achieved
by the addition of a small amount (∼2−3 mol %) of PLA-b-
PEG−FP547 in the copolymer blends. PC-3 cells, originating
from the human prostate adenocarcinoma and exhibiting
σ-receptors, were incubated with Am-containing nanoparticles
(28 mol % Am, N13, Table 1), whereas FA-containing nano-
particles (17 mol % FA, N11, Table 1) were tested on both
Figure 12. Mean fluorescence intensity evolution with incubation time
of PC-3 cells with Am-functionalized nanoparticles N13 (Table 1) and
the nonfunctionalized nanoparticles N14 (Table 1) (a). Representative
Nomarski (top) and confocal microscopy (bottom) images of the
PC-3 cells incubated for 1 and 6 h with nonfunctionalized nano-
particles (N14, Table 1) and Am-functionalized nanoparticles (N13,
Table 1) (b). Lines connecting data points are guides for the eye only.
the epidermoid carcinoma cell line KB-3-1, for which folate
receptors (FRs) are normally expressed, and its FR over-
expressed counterpart KB-3-1* cell line, as well as the MCF-7
breast cancer cell line exhibiting low FR density. In all cases,
nonfunctionalized nanoparticles (N14 and N12, Table 1) with
identical fluorescence intensity were used as controls. Prior to
biological evaluations, the absence of cytotoxicity of each kind
of nanoparticles was confirmed by performing MTS assay on
each cell line (48 h of incubation with nanoparticles at concen-
trations up to 0.5 mg·mL−1). MTS assay was used instead of
MTT because its absorbance wavelength at 492 nm (570 nm
for MTT) does not overlap with the excitation wavelength of
the fluorophore FP542. In all cases, whatever the concentration
and the nature of the nanoparticles, no noticeable cytotoxicity
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Figure 13. Mean fluorescence intensity evolution with incubation time of KB-3-1* (a), KB-3-1 (b), and MCF-7 (c) cells with FA-functionalized
nanoparticles N11 and the nonfunctionalized nanoparticles N12 (Table 1). Mean fluorescence evolution with incubation time of KB-3-1* cells (d)
with the FA-functionalized nanoparticles N11 after 1 or 35 days after manufacture. Lines connecting data points are guides for the eye only.
was observed (Figure 11), thus allowing the safe biological
evaluations to be performed.
greater cellular uptake than that of the nonfunctionalized nano-
particles (N12) in KB-3-1 cells (Figure 13b), while a massive
uptake of these nanoparticles by the KB-3-1* cell line over-
expressing FR (Figure 13a), which plateaued after 6 h of
incubation, has been witnessed and accounted for a ∼115-fold
higher cellular uptake as compared to that of the nonfunction-
alized nanoparticles over the same period of time.
Another important question that needed to be addressed was
the potential surface reorganization of the FA-functionalized
nanoparticles with time due to the hydrophobic nature of FA
and its possible impact to decrease or even hamper the cell
uptake due to buried FA groups inside the hydrophobic nano-
particle core. Uptake of the nanoparticles by KB-3-1* cells was
therefore monitored with the FA-functionalized nanoparticles
N11 stored for 35 days and compared with similar nano-
particles freshly prepared. Flow cytometry results revealed no
significant difference of the cell uptake between the two kinds
of nanoparticles (Figure 13d), thus suggesting the presence of
same amount of FA at the surface of nanoparticles even after
storage for more than 1 month.
Taken together, these results clearly demonstrated the effi-
cient uptake of targeted Am- functionalized and FA-function-
alized nanoparticles into the cancer cells. Thus, the PLA-b-
PEG−ligand nanoparticles designed using click chemistry were
shown to be efficient in cancer cell targeting, and the multi-
functional nanoparticles bearing targeting ligand and the fluo-
rescence probe allowed to target cancer cells follow their in
vitro fate due to the fluorescent labeling and could simul-
taneously monitor the response to treatment. This general
Flow cytometry experiments showed that incubation of PC-3
cells with fluorescent, Am-functionalized nanoparticles N13
resulted in improved cell uptake from 6 h postincubation com-
pared to that obtained from nonfunctionalized nanoparticles
N14 (Figure 12a). For instance, after 24 h, the uptake of N13
accounted for an increase of ∼50% compared to that of N14.
This enhanced uptake was confirmed by confocal microscopy
observations, during which PC-3 cells were treated with both
kinds of nanoparticles. After 1 h postinjection, N14 led to a
faint fluorescence signal, whereas after 6 h, cells treated with
nanoparticles N13 gave a significantly higher fluorescence
signal inside the cells than that obtained with nonfunctionalized
nanoparticles (Figure 12b).
In order to assess the FR-mediated targeting, FA-function-
alized nanoparticles with 17 mol % FA (N11) were incubated
with three cancer cell lines (KB-3-1, KB-3-1*, and MCF-7)
exhibiting different levels of FR expressions and analyzed by
flow cytometry for the measurement of FP547 fluorescence.
The results revealed a poor uptake of the nonfunctionalized
nanoparticles (N12) by all the cell lines tested, while the
FA-functionalized nanoparticles showed degrees of binding in
good agreement with the levels of FR expression at the sur-
face of the different cell lines (Figure 13). Indeed, both types
of nanoparticles (nonfunctionalized nanoparticles and FA-
functionalized nanoparticles) were uptaken similarly by the
MCF-7 cells, which poorly express FR (Figure 13c). However,
FA-functionalized nanoparticles (N11) exhibited a ∼5-fold
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CONCLUSION
■
In this study, precise macromolecular engineering was per-
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order to prepare targeted and fluorescently labeled PLA-b-PEG
nanoparticles for cancer cell targeting and imaging. Well-
defined PLA-b-PEG−N3 diblock copolymers were successfully
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectrum of PLA-b-PEG−FP547 copolymer, TEM
images of the nanoparticles, and characterization of PLA-b-
PEG−VB7 nanoparticles by SPR. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Wang, S. L. Biomaterials 2010, 31, 908.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: julien.nicolas@u-psud.fr. Tel: +33 1 46 83 58 53. Fax:
+33 1 46 83 55 11.
■
Notes
(22) Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8,
1128.
The authors declare no competing financial interest.
(23) Wu, P.; Feldman, A. K.; Nugent, A. K.; Hawker, C. J.; Scheel, A.;
Voit, B.; Pyun, J.; Frechet, J. M. J.; Sharpless, K. B.; Fokin, V. V. Angew.
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Christianen, P. C. M.; Svendsen, A.; Patkar, S. A.; Vind, J.; Maan, J. C.;
Rowan, A. E.; Nolte, R. J. M. Chem. Commun. 2006, 2012.
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ACKNOWLEDGMENTS
The authors are thankful to Sanofi colleagues Odile
Angouillant-Boniface and Dr. Eric Brohan for the preparative
HPLC experiments of alkyne−FA, to Danielle Le Met
her help with the SPR experiments, to Valerie Nicolas
(Plateforme Imagerie Cellulaire, IFR 141) and Sophie Grives
for their help with the confocal microscopy exeriments, and to
Dr. Dominique Lesuisse from Sanofi for the postdoc funding of
N.H. N.M. is also grateful to Sanofi for financial support. This
work has benefited from the facilities and expertise of the
platform for Transmission Electron Microscopy of IMAGIF
■
́
ayer for
́
̀
́
(Centre de recherche de Gif). The CNRS and the French
Ministry of Research are also warmly acknowledged for
financial support.
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