Π-Π Stacking increases the stability and loading capacity of thermosensitive polymeric micelles for chemotherapeutic drugs

Article abstract of DOI:10.1021/bm400234c

Thermosensitive amphiphilic block copolymers self-assemble into micelles above their lower critical solution temperature in water, however, the micelles generally display mediocre physical stability. To stabilize such micelles and increase their loading capacity for chemotherapeutic drugs, block copolymers with novel aromatic monomers were synthesized by free radical polymerization of N-(2-benzoyloxypropyl methacrylamide (HPMAm-Bz) or the corresponding naphthoyl analogue (HPMAm-Nt), with N-(2-hydroxypropyl) methacrylamide monolactate, using a polyethylene glycol based macroinitiator. The critical micelle temperatures and critical micelle concentrations decreased with increasing the HPMAm-Bz/Nt content. The micelles of 30-50 nm were prepared by heating the polymer aqueous solutions from 0 to 50 C and were colloidally stable for at least 48 h at pH 7.4 and 37 C. Paclitaxel and docetaxel encapsulation was performed by mixing drug solutions in ethanol with polymer aqueous solutions and heating from 0 to 50 C. The micelles had a drug loading capacity up to 34 wt % for docetaxel, which is among the highest loadings reported for polymeric micelles, with loaded micelle sizes ranging from 60 to 80 nm. The micelles without aromatic groups almost completely released loaded paclitaxel in 10 days, whereas the HPMAm-Bz/Nt containing micelles released 50% of the paclitaxel at the same time, which showed a better retention for the drug of the latter micelles. ¹H solid-state NMR spectroscopy data are compatible with π-π stacking between aromatic groups. The empty micelles demonstrated good cytocompatibility, and paclitaxel-loaded micelles showed high cytotoxicity to tumor cells. In conclusion, the π-π stacking effect introduced by aromatic groups increases the stability and loading capacity of polymeric micelles.

Full text of DOI:10.1021/bm400234c

Article
pubs.acs.org/Biomac
Π−Π Stacking Increases the Stability and Loading Capacity of
Thermosensitive Polymeric Micelles for Chemotherapeutic Drugs
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Yang Shi, Mies J. van Steenbergen, Erik A. Teunissen, Luıs Novo, Sabine Gradmann, Marc Baldus,
Cornelus F. van Nostrum,† and Wim E. Hennink*,†
†Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Universiteitsweg 99, P.O.
Box 80082, 3508 TB Utrecht, The Netherlands
‡Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
S
* Supporting Information
ABSTRACT: Thermosensitive amphiphilic block copolymers
self-assemble into micelles above their lower critical solution
temperature in water, however, the micelles generally display
mediocre physical stability. To stabilize such micelles and
increase their loading capacity for chemotherapeutic drugs,
block copolymers with novel aromatic monomers were
synthesized by free radical polymerization of N-(2-benzoylox-
ypropyl methacrylamide (HPMAm-Bz) or the corresponding
naphthoyl analogue (HPMAm-Nt), with N-(2-hydroxypropyl)
methacrylamide monolactate, using a polyethylene glycol
based macroinitiator. The critical micelle temperatures and critical micelle concentrations decreased with increasing the
HPMAm-Bz/Nt content. The micelles of 30−50 nm were prepared by heating the polymer aqueous solutions from 0 to 50 °C
and were colloidally stable for at least 48 h at pH 7.4 and 37 °C. Paclitaxel and docetaxel encapsulation was performed by mixing
drug solutions in ethanol with polymer aqueous solutions and heating from 0 to 50 °C. The micelles had a drug loading capacity
up to 34 wt % for docetaxel, which is among the highest loadings reported for polymeric micelles, with loaded micelle sizes
ranging from 60 to 80 nm. The micelles without aromatic groups almost completely released loaded paclitaxel in 10 days,
whereas the HPMAm-Bz/Nt containing micelles released 50% of the paclitaxel at the same time, which showed a better retention
1
for the drug of the latter micelles. H solid-state NMR spectroscopy data are compatible with π−π stacking between aromatic
groups. The empty micelles demonstrated good cytocompatibility, and paclitaxel-loaded micelles showed high cytotoxicity to
tumor cells. In conclusion, the π−π stacking effect introduced by aromatic groups increases the stability and loading capacity of
polymeric micelles.
hydrophobic drugs.13 By fast heating ice-cold solutions of
1. INTRODUCTION
thermosensitive polymers above their CMT, they can form
During the past decades, polymeric micelles formed from
micelles with high drug loading and small size.13 The
amphiphilic block copolymers have been extensively inves-
preparation is accomplished in 1 min and avoids the use of
tigated as drug delivery systems, particularly for the targeted
large amounts of organic solvents.17
delivery of hydrophobic drugs.1−3 Polymeric micelles are
characterized by a size normally below 100 nm and a good
Unfortunately, in vivo pharmacokinetic studies of drug-
loaded micelles showed rapid drug release in the circulation,
accommodation for poorly water-soluble drugs. Their hydro-
probably due to a combination of extraction of the drug from
philic corona, mostly consisting of poly(ethylene glycol)
the micelles and micellar destabilization.18 It is hypothesized
(PEG), endows them with a stealth surface, likely as a result
that, for example, albumin and lipoproteins in the circulation
of a low protein binding to the particles in the blood
circulation.4−6 Further, multifunctions can be introduced
are able to bind amphiphilic polymer molecules, which can
disrupt the dynamic equilibrium of micelles and unimers.19−21
including decorating the surface with targeting ligands and
incorporation of imaging agents.7−11
To tackle the instability of polymeric micelles, research has
therefore been done on micelles’ covalent cross-linking,
Among different types of amphiphilic block copolymers,
including shell,21,22 interface,23 and core cross-linking.18,24,25
thermosensitive block copolymers are receiving increased
interest for the preparation of micelles,12,13 hydrogels14,15 and
Both in vitro and in vivo studies have demonstrated a
to coat liposomes.16 Thermosensitive copolymers, which have a
substantially increased stability of cross-linked micelles leading
thermosensitive and a permanently hydrophilic block, form
micelles above the critical micelle temperature (CMT) of the
thermosensitive block. This yields polymeric micelles with
stealth coronas and hydrophobic cores that can accommodate
Received: February 13, 2013
Revised: April 2, 2013
© XXXX American Chemical Society
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to the circulation kinetics of micelles comparable to that of
pegylated liposomes.18,26,27 However, paclitaxel (PTX)-loaded
core-cross-linked micelles still showed fast drug release after i.v.
administration in mice. This can likely be ascribed to the
premature release and extraction of PTX from micellar core by
plasma proteins. Therefore, the drug retention in the micelles
should be improved to benefit from the good circulation time
of cross-linked micelles.28,29 To this end, doxorubicin has been
covalently linked via a pH-sensitive hydrazone linker to the
cross-linked core of mPEG-b-p(HPMAm-monolactate-co-
HPMAm-dilactate) micelles.30,31 A pharmacodynamic study
showed a very promising therapeutic efficacy of these micelles
in mice bearing B16F10 melanoma carcinoma.32 In a recent
study, dexamethasone has been coupled to the core of mPEG-
b-p(HPMAm-monolactate-co-HPMAm-dilactate) micelles via a
hydrolytically sensitive spacer and also showed excellent
therapeutic effects in mice and rats models of rheumatoid
arthritis.33 However, chemical conjugation methods are not
always feasible and might adversely affect the aimed therapeutic
effect of conjugated drugs. Alternatively, physical interactions,
including π−π stacking,34−41 hydrogen bonding,42 stereo-
complex formation,43 and crystallinity44 have been investigated
to enhance the thermodynamic and kinetic stability of
polymeric micelles but not yet in combination with
thermosensitive, biodegradable polymers.
In the present study, aromatic groups modified N-(2-
hydroxypropyl) methacrylamide (HPMAm), that is, N-(2-
benzoyloxypropyl methacrylamide (HPMAm-Bz) and the
corresponding naphthoyl analogue (HPMAm-Nt), that both
can give π−π stacking were copolymerized with HPMAm-
lactate (HPMAm-Lac) to increase the stability, drug loading
capacity, and drug retention of the obtained polymeric micelles.
HPMAm-Bz and HPMAm-Nt were chosen due to the
following attractive features. First, these monomers can easily
be synthesized by modifying the hydroxyl group of HPMAm
with benzoyl/naphthoyl chloride. Second, hydrolysis of the
ester bond between the pendant groups and the hydrophobic
block converts this block into hydrophilic p(HPMAm). The
increased polarity of the corresponding block induces an
increase of the CMT, dissociation of the micelles, and release of
the payload at physiological temperature. Additionally, the
hydrolyzed block, that is, p(HPMAm), has been clinically
tested to be safe and p(HPMAm) with a molecular weight
lower than 70 kDa will undergo renal excretion.45 Two first-line
anticancer drugs paclitaxel (PTX) and docetaxel (DTX) were
chosen as model drugs (Figure 1) and encapsulated in the
micelles.
2. EXPERIMENTAL SECTION
2.1. Materials. N-(2-Hydroxypropyl) methacrylamide (HPMAm)
was purchased from Zentiva, Czech Republic. The mPEG2-ABCPA
macroinitiator (Mn of mPEG = 5000 g·mol−1), N-(2-hydroxypropyl)
methacrylamide monolactate, and PEG-b-p(HPMAm-dilactate) were
prepared as described previously.46 Benzoyl chloride, 2-naphthoyl
chloride, 8-anilino-1-naphthalenesulfonic acid hemimagnesium salt
hydrate (8,1-ANS), pyrene, manganese(II) sulfate (MgSO4), 4-
methoxyphenol (4-MP), aluminum oxide (activated, basic), triethyl-
amine (TEA), (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), and
N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxy-propanesulfonic
acid (AMPSO) were purchased from Sigma-Aldrich (Zwijndrecht, The
Netherlands). Docetaxel (DTX) was purchased from Santa Cruz
Biotechnology, Inc. (Heidelberg, Germany) and paclitaxel (PTX) was
purchased from LC Laboratories (MA, U.S.A.). Acetonitrile (ACN),
dichloromethane (DCM), diethyl ether, and N,N-dimethylformamide
(DMF) were supplied by Biosolve Ltd. (Valkenswaard, The
Netherlands).
2.2. Syntheses of N-(2-Benzoyloxypropyl) Methacrylamide
and N-(2-Naphthoyloxypropyl) Methacrylamide (HPMAm-Bz/
Nt). HPMAm-Bz/Nt were synthesized by reaction of the hydroxyl
group of HPMAm with benzoyl chloride or 2-naphthoyl chloride,
respectively, following a procedure described for modification of the
terminal hydroxyl group of mPEG-b-oligocaprolactone oligomers by
benzoylation or naphthoylation.38 In detail, 10 g (0.070 mol) of
HPMAm and 7.1 g (0.070 mol) of TEA were dissolved in 70 mL of
dry DCM and 0.088 g (0.00070 mol) of 4-methoxyphenol (4-MP) was
added as inhibitor. This solution was added dropwise to a solution of
either benzoyl chloride (9.8 g, 0.060 mol) or 2-naphthoyl chloride
(12.1 g, 0.060 mol) in 70 mL of dry DCM and the resulting solution
was stirred for 24 h under a nitrogen atmosphere at room temperature.
Subsequently, the formed and precipitated TEA·HCl was removed by
filtration and the remaining solution was extracted three times with the
same volume of reverse osmosis (RO) water to remove unreacted
HPMAm and other water-soluble impurities. The DCM phase was
dried with MgSO4 and the inhibitor (4-MP) was removed by Al2O3
column chromatography. The column was washed with DCM, and the
eluent was collected and evaporated under reduced pressure.
1H NMR spectra were recorded using a Gemini 300 MHz
spectrometer (Varian Associates Inc. NMR Instruments, Palo Alto,
CA), using DMSO-d6 as the solvent; the DMSO peak at 2.52 ppm was
used as the reference line. Chemical shifts of both monomers (DMSO-
d6): 8.19 (t, CO-NH-CH2), 5.60 and 5.30 (s, CH2C), 5.15 (q,
CH2−CH(CH3)-O), 3.20 (t, NH-CH2-CH), 1.80 (s, CH3-CC),
1.30 (d, H2−CH(CH3)-O). Chemical shifts of the benzoyl group (δ,
ppm): 8.0 (d, 2H, aromatic CH), 7.61 (t, 1H, aromatic CH), 7.51 (t,
2H, aromatic CH). Chemical shifts of the naphthoyl group (δ, ppm):
8.21 (m, 1H, aromatic CH), 8.18 (d, 1H, aromatic CH), 8.05 (m, 3H,
aromatic CH), 7.71 (m, 2H, aromatic CH).
2.3. Syntheses of ω-Methoxy Poly(ethylene glycol)-b-(N-(2-
benzoyloxy/naphthoyloxypropyl) methacrylamide)-co-(N-(2-
lactoyloxypropyl) methacrylamide) (mPEG-b-p(HPMAm-Bz/
Nt-co-HPMAm-Lac)). The block copolymers were synthesized by
free radical polymerization according to Soga et al.47 using HPMAm-
monolactate and HPMAm-Bz or HPMAm-Nt as the monomers and
mPEG2-ABCPA as the macroinitiator. The monomers were dissolved
at a total concentration of 0.3 g/mL in dried ACN and the monomer-
to-macroinitiator molar ratio was 150/1. To obtain block copolymers
with different contents of HPMAm-Lac and HPMAm-Bz or HPMAm-
Nt, the monomer ratios in the feed were varied between 100/0 to 25/
75 (mol/mol), respectively. The solution was degassed by flushing
with nitrogen for 15 min. Reactions were conducted at 70 °C for 18 h
under a nitrogen atmosphere. The polymers were purified by
precipitation in diethyl ether, and then dissolved in cold RO water
and dialyzed against RO water at 4 °C for 24 h. The polymers were
collected as white fluffy powders after freeze-drying.
The block copolymers are further denoted as mPEG-b-p(HPMAm-
Bzx-co-HPMAm-Lacy) and mPEG-b-p(HPMAm-Ntx-co-HPMAm-
Lacy), respectively, where x and y are the percentages of the
Figure 1. Chemical structures of paclitaxel (PTX, left) and docetaxel
(DTX, right).
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incorporated comonomers in the corresponding polymers determined
by H NMR spectroscopy.
distribution of particle sizes). The measurement angle was 90°. The
light scattering intensity was plotted against the temperature and the
onset on the x-axis, obtained by extrapolation of the LSI-temperatures
curves to the baseline, was considered as the CMT.48−51
2.6. Critical Micelle Concentration (CMC). The CMCs of
mPEG-b-p(HPMAm-Bz/Nt-co-HPMAm-Lac) were measured using
two different fluorescent probes, namely, pyrene and 8-anilino-1-
naphthalene-sulfonic acid magnesium salt (8,1-ANS), respectively.
Because the naphthoyl group of the HPMAm-Nt containing polymers
interfered with the excitation spectrum of pyrene (Supporting
Information, Figure S2), pyrene was only used to measure the CMC
of mPEG-b-p(HPMAm-Bz-co-HPMAm-Lac), whereas 8,1-ANS was
used to measure the CMC of both types of polymers.
1
2.4. Characterization of mPEG-b-p(HPMAm-Bz/Nt-co-
HPMAm-Lac) Block Polymers. The 1H NMR spectra were recorded
1
as described in section 2.1. H NMR of mPEG-b-p(HPMAm-Bz-co-
HPMAm-Lac): 8.0 (b, 2H, aromatic CH), 7.55 (b, 1H, aromatic CH),
7.65 (b, 2H, aromatic CH), 7.35 (b, CO-NH-CH2), 5.4 (d, CH(CH3)-
OH), 5.1 (b, NH-CH2-CH(CH3)-O-(Bz)), 4.8 (b, NH-CH2-CH-
(CH3)-O-(Lac)), 4.1 (b, CH(CH3)-OH), 3.40−3.60 (b, mPEG5000
methylene protons, O-CH2-CH2), 3.2 (b, NH-CH2-CH), 0.6−2.2 (b,
the rest of the protons are from the methyl and backbone CH2
protons).
1H NMR of mPEG-b-p(HPMAm-Nt-co-HPMAm-Lac): 8.6 (b, 1H,
aromatic CH), 8.05 (b, 3H, aromatic CH), 7.6 (b, 3H, aromatic CH),
7.35 (b, CO-NH-CH2), 5.4 (b, CH(CH3)-OH), 5.15 (b, NH-CH2-
CH(CH3)-O-(Nt)), 4.8 (b, NH-CH2-CH(CH3)-O-(Lac)), 4.1 (s,
CH(CH3)-OH), 3.40−3.60 (b, mPEG5000 methylene protons, O-CH2-
CH2), 3.2 (b, NH-CH2-CH), 0.6−2.2 (b, the rest of the protons are
from the methyl and backbone CH2 protons).
The block copolymers were dissolved in 4.5 mL of 120 mM
ammonium acetate buffer (concentration ranging from 1 × 10−5 to 1
mg/mL) for 16 h at 0 °C. Next, 15 μL of pyrene dissolved in acetone
(concentration of 1.8 × 10−4 M) was added and the solutions were
subsequently incubated at 50 °C for 1 min. Next, the samples were
cooled down to 37 °C (40 °C for mPEG-b-p(HPMAm-Bz8-co-
HPMAm-Lac92 because its CMT was around 37 °C) and incubated for
20 h to allow evaporation of acetone. Fluorescence excitation spectra
of pyrene were obtained by a Horiba Fluorolog fluorometer (at an
angle of 90°). The excitation spectra were recorded at 37 °C (40 °C
for mPEG-b-p(HPMAm-Bz8-co-HPMAm-Lac92) from 300 to 360 nm
with an emission wavelength at 390 nm. The excitation and emission
band slits were 4 and 2 nm, respectively. The ratio of excitation
intensity at 338 and 333 nm was plotted against polymer
concentration to determine the CMC.47
The CMC was also determined using 8,1-ANS as a fluorescent
probe. The block copolymers were dissolved at different concen-
trations as described above. A total of 200 μL of the micellar
dispersion/polymer solution was pipetted into the wells of a black 96-
well plate (Greiner). Next, to each well, 2 μL of 5 mM 8,1-ANS
solution in DMF/water (1/10, v/v) was added and the solution/
dispersion was then incubated at 37 °C (40 °C for mPEG-b-
p(HPMAm-Bz8-co-HPMAm-Lac92 because its CMT was around 37
°C) for 20 h. Fluorescence was measured using FluoSTAR (OPTIMA
fluorimeter) at 37 °C. The excitation and emission wavelengths were
355 and 520 nm, respectively. The CMC was determined by plotting
the fluorescence intensity against the polymer concentration.38 The
fluorescence of blank polymer solutions was measured as a reference.
2.7. Preparation of Empty and Drug-Loaded Micelles. Empty
micelles were prepared by a fast heating method as described
previously.13 In short, the polymers were dissolved for 16 h at a
concentration of 10 mg/mL in 120 mM AAB at pH 5.0 and 0 °C.
Next, the polymer solutions were heated in a water bath at 50 °C for 1
min with constant shaking to form micelles. For paclitaxel (PTX) and
docetaxel (DTX)-loaded micelles, 0.2 mL of the drug solution in
ethanol (concentration ranging from 40 to 100 mg/mL) was added to
1.8 mL of an ice cold polymer solution and then immediately heated at
50 °C for 1 min. Subsequently, the micellar dispersions were incubated
overnight at room temperature. The free drug was removed by
filtration, a method frequently applied to remove nonentrapped drugs
from micelles.52−54 The size of the micelles was measured by DLS as
described in section 2.5.
The number-average molecular weight (Mn) of the block
1
copolymers was determined by H NMR as follows: (a) the value of
the integral of the mPEG protons divided by 448 (the average number
of protons per one mPEG chain, Mn = 5000) gives the integral value
for one mPEG chain, and (b) the number of HPMAm-Lac and
HPMAm-Bz/Nt units in the polymers was determined from the ratio
of the integral of the hydroxyl proton (5.4 ppm, 1H, CO-CH(CH3)-
OH) of HPMAm-Lac and aromatic protons of HPMAm-Bz (8.0 ppm,
2H, aromatic CH) or HPMAm-Nt (8.6 ppm, 1H, aromatic CH) to the
integral of one mPEG chain. The Mn of the thermosensitive block was
calculated from the resulting number of HPMAm-Lac and HPMAm-
Bz/Nt units. The mol % of HPMAm-Bz/Nt in the thermosensitive
block of the copolymer was determined by the following two
equations:
mol%of HPMAm‐Bz
integral H at 8.0 ppm)/2
(
=
integral (H at 8.0 ppm)/2 + integral (H at 4.1 ppm))
mol%of HPMAm‐Nt
integral (H at 8.6 ppm)
=
integral (H at 8.6 ppm) + integral (H at 4.1 ppm)
GPC was conducted to measure the number average molecular weight
(Mn), weight average molecular weight (Mw), and polydispersity (PDI,
equal to Mw/Mn) using two serial Plgel 5 μm MIXED-D columns
(Polymer Laboratories) and PEGs of narrow molecular weights as
calibration standards. The eluent was DMF containing 10 mM LiCl,
the elution rate was 0.7 mL/min, and the temperature was 40 °C.47
GPC chromatograms of the macroinitiator and the synthesized
polymer were deconvoluted to calculate the weight ratio of different
fractions. The deconvolution was performed using IGOR Pro by the
function of multipeak fitting.
2.5. Critical Micelle Temperature (CMT). The CMTs of the
block copolymers were measured by dynamic light scattering (DLS).
The polymers were dissolved for 16 h at 0 °C and at a concentration
of 10 mg/mL in 120 mM ammonium acetate buffer (AAB) at pH 5.0.
The solutions were heated in a water bath at 50 °C for 1 min with
constant shaking to form micelles. Subsequently, the light scattering
intensity (LSI) of the samples was continuously measured using DLS
while the samples were cooled from 50 to 0.1 °C in 2 h. DLS was
performed using a Malvern 4700 system (Malvern Ltd., Malvern,
U.K.) consisting of an Autosizer 4700 spectrometer, a pump/filter
unit, a model 2013 air-cooler argon ion laser (75 mW, 488 nm,
equipped with a model 2500 remote interface controller, Uniphase), a
water bath, and a computer with DLS software (PCS, version 3.15,
Malvern). Autocorrelation functions were analyzed by the cumulants
method (fitting a single exponential to the correlation function to
obtain the mean size and the polydispersity) and the CONtIN routine
(fitting a multiple exponential to the correlation function to obtain the
2.8. Drug Content Assay. The concentrations of the drugs loaded
in the micelles were determined by UPLC analysis using Waters
Acquity system consisting of a binary solvent manager, a sample
manager and a UV detector. An Acquity HSS T3 1.8 μm column (2 ×
50 mm) was used with a gradient eluent method at a flow rate of 1
mL/min, and a column temperature of 50 °C. Dispersions of drug-
loaded micelles (0.1 mL) were diluted with 0.9 mL of ACN and
subsequently vortexed to destabilize the micelles and dissolve the drug,
and then centrifuged at 12.000 g for 10 min. A total of 7 μL of the
supernatant was injected and the drug was measured at a wavelength
of 227 nm. Samples of the drugs in ACN in a concentration range of
0.2 to 500 μg/mL were used for calibration. Encapsulation efficiency
(EE) and loading capacity (LC) were calculated from the UPLC
analysis results as follows:
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Scheme 1. Synthesis of N-(2-Benzoyloxypropyl) Methacrylamide (HPMAm-Bz) and N-(2-Naphthoyloxypropyl)
Methacrylamide (HPMAm-Nt)
Scheme 2. Synthesis of mPEG-b-p(HPMAm-Bz/Nt-co-HPMAm-Lac)
concentration of drug measured
concentration of drug added
2.10. In Vitro Stability and Drug Retention Study. The in vitro
EE =
LC =
× 100%
colloidal stability of drug-loaded and empty micelles based on mPEG-
b-p(HPMAm-Nt28-co-HPMAm-Lac72) in pH 7.4 and 10.0 buffers at 37
°C was studied by monitoring the size and the light scattering intensity
of the micelles by DLS. Empty micelles at a polymer concentration of
10 mg/mL were prepared in water as described in section 2.7. The pH
of the micellar dispersion was adjusted to pH 10.0 or 7.4 by diluting 5-
fold with 500 mM Na2CO3/NaHCO3 pH 10.0 buffer or 500 mM
NaH2PO4 pH 7.4 buffer. The samples were incubated at 37 °C and the
DLS measurements were conducted continuously for 48 h.
Drug retention in the micelles in pH 7.4 phosphate buffer at 37 °C
was evaluated by measuring the solubilized drug concentration in the
micellar dispersion. PTX-loaded micelles were prepared as described
in section 2.7 in pH 5.0 120 mM AAB and the pH was adjusted to 7.4
by diluting 5-fold with 500 mM phosphate pH 7.4 buffer. The released
PTX crystallized and precipitated due to its low water solubility (0.3
μg/mL38). The samples were incubated at 37 °C with constant
shaking, and aliquots were taken at different time points and
centrifuged at 5000 g for 10 min to spin down the precipitated
drug. Next, 0.1 mL of the supernatant was mixed with 0.3 mL of ACN
and vortexed for 1 min, followed by centrifugation at 12.000 g for 10
min, and the drug concentration was determined by UPLC, as
described in section 2.8.
concentration of drug measured
concentration of drug measured + polymer added)
(
× 100%
2.9. Transmission Electron Microscopy (TEM). TEM was
performed using a Philips Tecnai12 equipped with a Biotwin lens and
a LaB6 filament, operated at 120 kV acceleration voltage. In detail,
micellar dispersions were diluted 10 times with 120 mM ammonium
acetate buffer and droplets of the micellar dispersions (around 10 μL)
were put on a piece of Parafilm. A glow discharged grid (copper 200
mesh grid with a carbon coated thin polymer film, Formvar or
Pioloform on top) with the film side pointing down was placed on this
droplet. After 3 min, the grid was taken off and the excess liquid was
removed using a filter paper. Immediately, the grid with the film was
put upside down on a 5 μL droplet of uranyl acetate 2%. After 1 min,
the grid was taken off and the excess uranyl acetate was removed using
filter paper. The grid was put on a filter paper and left for 5 min. Next,
the grid was loaded into a TEM sample holder and Images were
captured with a SIS Megaview II CCD-camera and processed with
AnalySIS software.
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2.11. In Vitro Cytotoxicity of Empty and PTX-Loaded
Micelles. The in vitro cytotoxicity of empty and PTX-loaded
mPEG-b-p(HPMAm-Bz27-co-HPMAm-Lac73) and mPEG-b-p-
(HPMAm-Nt28-co-HPMAm-Lac72) micelles was evaluated using
B16F10 melanoma cells. mPEG-b-p(HPMAm-dilactate), which
showed a good cytocompatibility, was used as a negative control,
whereas Cremophor EL/ethanol (1/1, v/v; Taxol) was used as a
positive control. B16F10 cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with fetal bovine serum
(FBS) (final concentration 10% v/v) and 1% of antibiotics. The cells
were cultured at 37 °C and in a 5% CO2 humidified atmosphere. Cells
were seeded into 96-well plates at a density of (5 × 103 cells/well) and
incubated for 24 h at 37 °C in a 5% CO2 humidified atmosphere.
Stock solutions of empty micelles (polymer = 9 mg/mL) and PTX-
loaded micelles (PTX = 1 mg/mL, polymer = 9 mg/mL) were
prepared in 5 mM HEPES buffer (pH 7.4), as described in section 2.7.
PTX solubilized in Cremophor EL (Taxol) was prepared as follows: 12
mg of PTX was dissolved in 1.0 mL ethanol, and to this solution 1.0
mL Cremophor EL was added and the mixture was sonicated for 30
min.10 The obtained Taxol formulation (PTX = 6 mg/mL) was diluted
6-fold with 5 mM HEPES buffer of pH 7.4. A formulation without
PTX was prepared by mixing Cremophor EL and ethanol (1/1, v/v)
followed by diluting with 5 mM HEPES buffer. The empty micelles
and the Cremophor EL/ethanol solution were diluted with 5 mM
HEPES pH 7.4 buffer to yield polymer concentrations ranging from 1
ng/mL to 1 mg/mL. The stock solutions of the drug formulations
were diluted with 5 mM HEPES pH 7.4 buffer to yield PTX
concentrations in the incubation medium ranging from 0.01 ng to 10
μg/mL. To evaluate the cytotoxicity, 100 μL of the different
formulations and 100 μL of culture medium (DMEM + 10% FBS)
were added to the cells. A total of 100 μL of 5 mM HEPES pH 7.4
buffer and 100 μL culture medium (DMEM + 10% FBS) were used as
a control. The cells were incubated at 37 °C in a humidified
atmosphere with 5% CO2 and after 72 h the cell viability was
determined using a XTT colorimetric assay.55
molecular weight shoulders (AUC lower than 10−15%) that
had the same retention times as PEG 5 and 10 kDa (Figure S6),
which indicates that small amounts of PEG 5 and 10 kDa
homopolymers were present in the final polymer. The PEG 5
kDa likely originates from the macroinitiator synthesis, that is,
PEG of 5 kDa that was not coupled to the azo initiator. The
PEG 10 kDa is most likely formed due to the combination of
two 5 kDa PEG free radicals during the polymerizations
(Supporting Information, section 2). Furthermore, Figure S5
shows the presence of a double peak (at 17.7 and 18.5 min)
apart from the PEG shoulders (at 20.0 and 21.4 min) in the
GPC chromatogram of the synthesized polymer. Chain
termination by recombination during free radical polymer-
ization results in the formation of 10 kDa PEG chain and
triblock copolymers of mPEG-p(HPMAm-Bz/Ntx-co-HPMAm-
Lacy)-mPEG. The triblock copolymer due to its higher
molecular weight has a lower retention time than the diblock
mPEG-p(HPMAm-Bz/Ntx-co-HPMAm-Lacy), which could ex-
plain the bimodality of the main distribution of the GPC
chromatogram. The weight ratio between these two popula-
tions, calculated by deconvoluting the GPC chromatogram, is
around 1.5/1 (w/w; Figure S5). Chain termination during
polymerizations can be suppressed by applying controlled
radical polymerizations, including atom transfer radical
polymerization (ATRP) and reversible addition−fragmentation
chain transfer (RAFT) polymerization,57,58 which is the subject
of present investigations. Besides, the macroinitiator contained
a small amount of ABCPA not coupled to two but to one
methoxy PEG-OH chain (Supporting Information, Figure S6).
This contamination yields, beside the diblock copolymer of
mPEG-p(HPMAm-Bz/Ntx-co-HPMAm-Lacy), also a small
fraction of homopolymer of p(HPMAm-Bz/Ntx-co-HPMAm-
Lacy). This can be another reason for the multimodality of the
GPC distribution. Overall, the mixtures of different polymers
including di/triblock copolymers and homopolymers can form
mixed micelles in aqueous solutions, which was demonstrated
by DLS analysis of the micelles showing that there was only one
size population with a low polydispersity (<1.1).
2.12. Detection of π−π Stacking in the Micelles by Solid
State NMR Spectroscopy. Empty micelles of mPEG-b-p(HPMAm-
Nt18-co-HPMAm-Lac82) were prepared in D2O according to section
2.7. Solid-state NMR experiments were performed using a Bruker
Avance III spectrometer equipped with a 4 mm double resonance (1H,
13C) probehead at 11.7 T static magnetic field. A MAS rate of 1 kHz
1
and a H radio frequency field strength of 66 kHz were used. 2D
1H−1H NOESY56 experiments were conducted using a mixing time of
The ratios of HPMAm-Bz/Nt and HPMAm-Lac in the
30 ms. Spectral referencing was done using adamantane.
1
copolymers, determined by H NMR spectroscopy, matched
those of the feed (Supporting Information, Table S1). Four
polymerization with 25 or 75% feed of HPMAm-Bz or
HPMAm-Nt were quenched at an early stage and the
3. RESULTS AND DISCUSSION
3.1. Syntheses of Monomers. N-(2-Benzoyloxypropyl)
methacrylamide and N-(2-naphthoyloxypropyl) methacryla-
mide (HPMAm-Bz/Nt) were synthesized by the reaction of
benzoyl chloride or 2-naphthoyl chloride with the hydroxyl
group of HPMAm (Scheme 1). After purification, the products
were obtained in a high yield (88% for HPMAm-Bz and 84%
for HPMAm-Nt) as pale-yellow and yellow solid with a melting
point of 53 and 89 °C, respectively. Their structures were
1
composition of the formed polymers was examined by H
NMR spectroscopy. The results showed that copolymer
compositions were close to that of feed (Supporting
Information, Table S2), which points out that HPMAm-Bz/
Nt and HPMAm-Lac have similar relativities, which ensures
that random copolymers of the different monomers were
formed.
3.3. Critical Micelle Temperature (CMT). Figure 2 shows
the CMTs of the mPEG-b-p(HPMAm-Bz/Nt-co-HPMAm-Lac)
with different HPMAm-Bz/Nt content, measured by DLS as
the disappearance of light scattering upon cooling. This figure
shows that the CMT of mPEG5000-p(HPMAm-Lac) was 55 °C.
The CMT decreased with increasing HPMAm-Bz/Nt content
in the block copolymers which can be explained by hydro-
phobic interaction and π−π stacking between the aromatic
groups of the thermosensitive block. The lowest CMTs (2.5
and 0.7 °C) of mPEG-b-p(HPMAm-Bz/Nt-co-HPMAm-Lac)
were obtained for polymers containing 26.9 mol % HPMAm-Bz
and 28.4 mol % HPMAm-Nt, respectively. In other words, the
polymers with the HPMAm-Bz/Nt content between 11.8% and
1
confirmed by H NMR spectroscopy.
3.2. Syntheses of Polymers. Block copolymers of mPEG-
b-p(HPMAm-Bz/Nt-co-HPMAm-Lac) were synthesized via
free radical polymerization using a previously published
macroinitiator route (Scheme 2). mPEG with a number
average molecular weight (Mn) of 5 kDa was the hydrophilic
block, and the thermosensitive block was composed of
HPMAm-Bz/Nt and HPMAm-Lac. The polymers were
obtained in high yields after purification (72−90%). GPC
analysis showed that the Mn of the synthesized polymers was
1
between 14 and 22 kDa, which is close to that based on H
NMR analysis, and the PDIs (Mw/Mn) were around 1.7. GPC
analysis (Figure S5) showed the presence of two small
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28.4%. This suggests a slightly stronger interaction between the
polymer chains in micelles of the latter copolymers.
3.5. Preparation and Characterization of Empty and
Drug-Loaded Micelles. The Z-average hydrodynamic diam-
eters of empty micelles, prepared by the fast heating method13
and determined by DLS ranged from 30 to 50 nm, with very
low polydispersities (<0.1, Figure 4). The lower sizes of those
Figure 2. CMTs of mPEG-b-p(HPMAm-Bz/Nt-co-HPMAm-Lac) as a
function of mol % of HPMAm-Bz/Nt (n = 3).
approximately 28.4% have CMTs between 25 and 0 °C, and are
therefore of interest for pharmaceutical application. Higher
contents of aromatic monomers gave polymers that were
insoluble in water at 0 °C.
3.4. Critical Micelle Concentration (CMC). Figure 3
shows that the CMC of mPEG-b-p(HPMAm-Bz-co-HPMAm-
Figure 4. Z-average hydrodynamic diameter (Zave, filled bars) and
polydispersity index (PDI, blocky bars) of empty polymeric micelles. A
total of 0% corresponds to mPEG-b-p(HPMAm-dilactate), and others
are mPEG-b-p(HPMAm-Bz/Nt-co-HPMAm- Lac), with the corre-
sponding amounts of HPMAm-Bz/Nt in the polymers measured by
1H NMR spectroscopy, respectively. All measurements were
performed at 25 °C (n = 3).
micelles as compared to micelles prepared from mPEG5000-b-
p(HPMAm-dilactate) (70 nm, Figure 4) is due to π−π stacking
and the high hydrophobicity of the aromatic groups leading to a
more condensed core of mPEG-b-p(HPMAm-Bz/Nt-co-
HPMAm-Lac) micelles.
Dispersions of DTX-loaded micelles of mPEG-b-p(HPMAm-
Bz25-co-HPMAm-Lac75) and mPEG-b-p(HPMAm-Nt14-co-
HPMAm-Lac86 prepared at a polymer/drug weight ratio of
1.8/1 were opalescent and homogeneous, while those of
mPEG-b-p(HPMAm-dilactate) were cloudy (Figure 5). After
subsequent removing precipitated/aggregated DTX by filtra-
tion through a 0.45 μm membrane, the encapsulation
efficiencies (EE) of DTX in the micellar dispersions were
Figure 3. CMC as a function of mol % of HPMAm-Bz/Nt in the
copolymers at 37 °C (n = 3).
Lac) using pyrene as a fluorescence probe decreased from 0.067
to 0.018 mg/mL with increasing content of HPMAm-Bz from 8
to 27%. The CMC of mPEG-b-p(HPMAm-Bz24-co-HPMAm-
Lac76) was lower than that of mPEG-b-p(HPMAm-dilactate)
(0.023 and 0.080 mg/mL, respectively), while they have similar
CMTs of around 4 °C,46 and therefore it is suggested, π−π
stacking and hydrophobicity of the aromatic groups contribute
to the lower CMC.
When 8,1-ANS was used as fluorescent probe, the CMCs
were approximately two times higher for mPEG-b-p(HPMAm-
Bz-co-HPMAm-Lac) than those obtained using pyrene (Figure
3), likely because the interactions between the hydrophobic
block of the polymers and the more hydrophilic 8,1-ANS is
weaker than that of the very hydrophobic pyrene.37
Corresponding CMC values decreased from 0.12 to 0.060
mg/mL with increasing HPMAm-Bz content (from 7.7 to
26.9%), while those of mPEG-b-p(HPMAm-Nt-co-HPMAm-
Lac) were slightly lower and decreased from 0.087 to 0.054
mg/mL with increasing HPMAm-Nt content from 5.7 to
53.9
1.3, 64.7
2.1, and 2.9
0.2%, for mPEG-b-
p(HPMAm-Bz24-co-HPMAm-Lac76), mPEG-b-p(HPMAm-
Nt15-co-HPMAm- Lac85, and mPEG-b-p(HPMAm-dilactate)
micelles, respectively. This indicates that the micelles
containing the aromatic groups are better capable to
solubilize/disperse DTX. TEM images of drug-loaded and
empty micelles are shown in Figure 6.
The drug loading capacity of the micelles was evaluated for
the chemotherapeutic drugs PTX and DTX45 that both have
aromatic groups and are characterized by a high hydrophobicity
(log Ps are 4.7 and 4.1, respectively37). Figure 7 shows that at a
feed drug concentration of 4 mg/mL mPEG-b-p(HPMAm-
dilactate) had a loading capacity (LC) of 17.8 0.4 and 8.2
0.6% for PTX and DTX, respectively. This was associated with
a rather low EE of 48.7 2.1 and 20.1 1.2%, respectively.
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Figure 5. Photograph of DTX-loaded micelles dispersions. Feed
concentrations were 5 mg/mL DTX and 9 mg/mL of the different
polymers. From left to right: mPEG-b-p(HPMAm-dilactate), mPEG-b-
p(HPMAm-Nt18-co-HPMAm-Lac82) and mPEG-b- p(HPMAm-Bz24-
co-HPMAm-Lac76).
Figure 7. Encapsulation efficiency (EE) and loading capacity (LC) of
PTX- and DTX-loaded micelles (feed drug and polymer concen-
trations were 4 and 9 mg/mL); 0% corresponds to mPEG-b-
p(HPMAm- dilactate); the other polymers are mPEG-b-p(HPMAm-
Bz/Nt-co-HPMAm-Lac) with different amounts of HPMAm-Bz/Nt,
respectively (n = 3).
This polymer had a higher loading capacity for PTX than DTX,
which can probably be ascribed to a better compatibility of
PTX and the polymer than that with DTX. Interestingly,
mPEG-b-p(HPMAm-Bz/Nt-co-HPMAm-Lac) showed a signifi-
cantly increased LC compared to mPEG-b-p(HPMAm-
dilactate). When the feed concentrations of drug and polymers
were 4 and 9 mg/mL, the highest LCs were 27.8 0.3 and 28.0
0.4% for PTX and DTX, and the EEs were 87.1 3.2 and
88.2 3.6%, respectively.
Figure 7 shows that when feed concentrations of drug and
polymers were 4 and 9 mg/mL, respectively, mPEG-b-
p(HPMAm-Bz24/Nt18-co-HPMAm-Lac76/82) were the polymers
that displayed the highest EE and LC for PTX and DTX.
Therefore, the influence of increasing feed drug concentration
on the EE and LC at a fixed concentration of these polymers
was evaluated, with mPEG-b-p(HPMAm-dilactate) as control
(Table 1, Figures S8 and S9). In Figure S8, it is clear that the
EE of DTX decreased when feed concentration increased from
4 to 10 mg/mL. On the other hand, the LC of DTX peaked at
feed concentration of 7 mg/mL (Figure S9). Afterward, passing
the upper limit of the loading capacity results in precipitation of
the drug, and therefore low EE.59−61 The highest LCs obtained
p(HPMAm-Bz24-co-HPMAm-Lac76) and mPEG-b-p(HPMAm-
Nt18-co-HPMAm-Lac82), respectively (Table 1). To the best of
our knowledge, polymeric micelles with the highest PTX
loading reported in literature are those based on PEG-b-p(2-(4-
vinylbenzyloxy)-N,N-diethylnicotinamide).38 For those mi-
celles, the LC of 37.4% for PTX was calculated by weight of
drug per weight of polymer. For comparison, when our results
are calculated in that way, the LC of the mPEG-b-p(HPMAm-
Nt18-co-HPMAm-Lac82) would be equal to 50.4% for DTX. In
other words, we can state that the loading capacity of our
micelles is unprecedentedly high.
Figure 7 shows that with 4 mg/mL of feed drug
concentration, the LC did not increase with increasing
HPMAm-Bz/Nt content, which can be ascribed to an enhanced
interaction between the hydrophobic blocks of the polymers
with increasing HPMAm-Bz/Nt content, which would reduce
for the DTX were 29.5
0.5 and 33.5
0.9 for mPEG-b-
Figure 6. TEM images of empty and DTX-loaded polymeric micelles: polymer, mPEG-b-p(HPMAm-Bz27-co-HPMAm-Lac73); left, empty micelles;
right, DTX-loaded micelles. Feed concentrations of the drug and polymer were 4 and 9 mg/mL, respectively.
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Table 1. DTX Loading and Molar Ratios of DTX/Polymer/Aromatic Groups of Various Micelles for Different Feed Ratios of
Polymer and Drug
a
b
b
aromatic comonomer content
feed drug concentration (mg/mL)
EE (%)
LC (%)
DTX/polymer
DTX/aromatic groups
24% HPMAm-Bz
18% HPMAm-Nt
0%
10
10
10
7
10.1 0.4
33.1 1.1
2.4 0.1
10.1 0.2
26.9 0.7
2.6 0.3
29.5 0.5
33.3 0.9
2.1 0.3
27.4 0.6
28.2 0.3
10.5 0.2
24.8 0.3
28.1 0.4
8.2 0.3
3.7
0.4
2.2
NA
1.4
3.0
NA
1.8
2.3
NA
1.4
2.3
NA
15.5
1.2
24% HPMAm-Bz
18% HPMAm-Nt
0%
53.9 1.3
64.7 2.1
2.9 0.2
14.0
21.2
1.0
7
7
24% HPMAm-Bz
18% HPMAm-Nt
0%
5
67.8 1.9
70.7 2.6
21.1 1.3
74.2 2.3
88.1 3.6
20.1 1.2
12.6
16.5
5.2
5
5
24% HPMAm-Bz
18% HPMAm-Nt
0%
4
11.0
16.5
4.0
4
4
a
0% corresponds to mPEG-b-p(HPMAm-dilactate); the other polymers are mPEG-b-p(HPMAm-Bz/Nt-co-HPMAm-Lac) with different amounts of
b
HPMAm-Bz/Nt, respectively (n = 3). mol/mol.
the space for encapsulation of drug molecules. A similar effect
that an increasing hydrophobicity of polymers decreased their
LC was reported for micelles based on cholesterol-modified
polymers.62
The ratios of DTX to the number of aromatic groups in the
micelles and that of DTX to the polymers (mol/mol) were
calculated (Table 1). For the polymers with aromatic groups,
the ratio of DTX to polymer with a drug feed concentration of
7 mg/mL and a polymer concentration of 9 mg/mL was up to
20 times higher as compared to mPEG-b-p(HPMAm-dilactate).
The HPMAm-Nt containing polymer encapsulated around two
times more DTX molecules than the HPMAm-Bz polymer.
The ratio of drug/aromatic groups also showed that more DTX
molecules were associated with the HPMAm-Nt than
HPMAm-Bz groups, likely due to the more hydrophobic
character of HPMAm-Nt and its better π−π stacking with the
drug. The difference of DTX molecules associated with
HPMAm-Bz and HPMAm-Nt groups was most pronounced
when the drug feed concentration was increased from 5 to 10
mg/mL.
Figure 8 shows that the size of drug-loaded micelles at feed
concentrations of drug and polymer of 4 and 9 mg/mL,
respectively, increased compared with that of empty micelles
(30−50 nm, Figure 4). Both PTX- and DTX-loaded mPEG-b-
p(HPMAm-dilactate) micelles had a size of around 110 nm,
however, the size of drug-loaded mPEG-b-p(HPMAm-Bz/Nt-
co-HPMAm-Lac) size was between 60 and 80 nm (poly-
dispersity between 0.04 and 0.23), despite their larger drug
contents. The smaller size of the micelles carrying aromatic
comonomers indicates that their cores are more condensed,
which is due to the π−π stacking and hydrophobic effect of the
aromatic groups.
3.6. In Vitro Stability and Drug Retention Studies. The
size and light scattering intensity (LSI) of empty and drug-
loaded micelles of mPEG-b-p(HPMAm-Nt24-co-HPMAm-
Lac76) in pH 7.4 buffer were stable for at least 48 h at 37 °C
(Figures 9 and 10), which indicates a good colloidal stability. It
has been proven that the hydrolysis of the side groups in the
pH range 7 to 10 is first order in OH− concentration,63 which
allows calculation of the stability under physiological conditions
using accelerated degradation conditions. Therefore, a hydro-
lytic stability study was also conducted at pH 10.0. When the
micelles were incubated in pH 10.0 buffer, they began to swell
after 2 h, accompanied by a strong increase of the LSI, and
Figure 8. Z-average hydrodynamic diameter (Zave) of drug-loaded
polymeric micelles at 25 °C. A total of 0% corresponds to mPEG-b-
p(HPMAm-dilactate); the others polymers are mPEG-b-p(HPMAm-
Bz/Nt-co-HPMAm-Lac) with different amounts of HPMAm-Bz/Nt (n
= 3).
dissociated after 7 h as reflected by the sudden decrease in the
LSI. This behavior is similar to what has been reported for
PEG-b-p(HPMAm-dilactate) based micelles47 and is caused by
the hydrolysis of the lactate and naphthoate side groups of
HPMAm, followed by hydration of the core of the micelles due
to its increased hydrophilicity. Destabilization of the micelles
occurs when the lower critical solution temperature (LCST) of
the polymer increases such that it passes the incubation
temperature of 37 °C.47 The t1/2 of the hydrolysis of HPMAm-
Lac at 37 °C and pH 10.0 is calculated to be 0.28 h based on a
previous study,63 and the hydrolysis study of HPMAm-Nt
(Supporting Information, section 4) showed that the t1/2 was
6.8 h under the same condition. Therefore, at the early stage of
the micelles’ destabilization, the hydrolysis of HPMAm-Lac
contributed mainly to the hydration of the micelles’ core,
whereas the full dissociation of the micelles is probably related
to the hydrolysis of the aromatic units. According to the
degradation study of the monomers HPMAm-Bz/Nt (Support-
ing Information, section 4) and HPMAm-Lac,63 the hydrolysis
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Figure 9. Stability of empty micelles of mPEG-b-p(HPMAm-Nt25-co-HPMAm-Lac75) at pH 7.4 (left) and 10.0 (right) measured by DLS at 37 °C.
Figure 10. Stability of PTX-loaded micelles of mPEG-b-p(HPMAm-Nt24-co-HPMAm-Lac76) at pH 7.4 (left) and 10.0 (right) measured by DLS at
37 °C.
is a first-order reaction in hydroxyl ion concentration. The
degradation rate of the monomers at pH 10.0 is therefore about
400 times faster than that at pH 7.4. Consequently, based on
the swelling and dissociation time of the micelles at pH 10.0, it
is calculated that the micelles start to swell after ∼800 h and
fully dissociate after ∼3000 h of incubation at pH 7.4.
The micellar dispersion turned clear after hydrolysis at 37 °C
and pH 10.0. This means that after hydrolysis, the polymers are
fully water-soluble and can be eliminated by renal filtration44 as
the molecular weights are below 31 kDa (Supporting
Information, Table S1). Additionally, the ester bond between
mPEG5000 and the thermosensitive block is hydrolyzable (t1/2
=
34 h at pH 8.5 and 37 °C),63 and the molecular weight of the
polymers therefore further decreases after hydrolysis of the
ester bond connecting the two blocks.
Figure 11. PTX release from micelles in PBS 7.4 at 37 °C (n = 3).
A release study was carried out under nonsink conditions.
The water solubility of PTX is rather low (0.3 μg/mL), and it is
consequently practically difficult to maintain sink conditions
during release. To explain, to keep the PTX concentration in
the medium below the saturation concentration requires a large
volume or a frequent refresh of the release medium, or addition
of surfactants to solubilize the released PTX. However, the first
two options might result in a too low drug concentration for
detection and in destabilization of the micelles due to passing
the CMC of the polymers. The addition of surfactants also
might result in destabilization of the micelle and/or the
formation of mixed micelles. Nevertheless, despite the nonsink
conditions, the payload of mPEG-b-p(HPMAm-dilactate)
micelles was almost fully released and precipitated after 240 h
of incubation at pH 7.4. On the contrary, there was still around
50% of loaded PTX solubilized in micelles of mPEG-b-
p(HPMAm-Bz/Nt-co-HPMAm-Lac) at the same time (Figure
11). Therefore, a better retention of PTX in the micelles with
the aromatic groups (benzoyl and naphthoyl) is shown as
compared to micelles lacking aromatic units in their polymer
chains, which can be explained by π−π stacking and
hydrophobic interaction between aromatic groups, the micelles’
core, and those of the drug.
3.7. In Vitro Cytotoxicity Study. The in vitro cytotoxicity
of both PTX-loaded and empty micelles based on mPEG-b-
p(HPMAm-Bz/Nt-co-HPMAm-Lac) was studied using B16F10
cells. For comparison, mPEG-b-p(HPMAm-dilactate) micelles,
which have a good cytocompatibility and Cremophor EL/
ethanol with and without PTX, were included as well (Figure
12). Cremophor/ethanol showed a strong cytotoxicity at high
concentrations (0.1 and 1.0 mg/mL), while the polymeric
micelles hardly affected the cells’ viability up to 1 mg/mL. At
high concentrations (>0.01 mg/mL), micelles of mPEG-b-
p(HPMAm-Bz/Nt-co-HPMAm-Lac) showed a slightly higher
cytotoxicity than the mPEG-b-p(HPMAm-dilactate) ones.
Figure 12 also shows that the cytotoxicity of mPEG-b-
p(HPMAm-Nt28-co-HPMAm-Lac72) micelles was slightly high-
er than that of mPEG-b-p(HPMAm-Bz27-co-HPMAm-Lac73)
micelles. Figure 13 shows that PTX-loaded polymeric micelles
at low PTX concentration (up to 0.1 μg/mL) showed
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1
lead to a shift of aromatic H NMR signals to lower ppm
values.65,66 In addition, cross peak intensities observed in two-
dimensional correlation experiments such as NOESY67 spectra
report on internuclear geometry.65,68,69 Intermolecular inter-
actions such as π−π stacking should, hence, increase the
magnitude and number of cross peak correlations among
aromatic protons. Finally, previous solid-state NMR work in
semisolid systems such as hydrogels70,71 has shown that a
decrease in molecular tumbling leads to an increase in solid-
1
state NMR line width. Figure 14 compares two 1D H spectra
Figure 12. In vitro cytotoxicity of empty micelles on B16F10 after 72
h of incubation (n = 3).
1
Figure 14. Comparison of 1D solid-state NMR H spectra obtained
under MAS at 37 °C (red) and 1 °C (black). Numbering of the
aromatic proton positions is given in the inset.
Figure 13. In vitro cytotoxicity of PTX formulations on B16F10 after
72 h of incubation (n = 3).
obtained at 37 °C (red) and 1 °C (black) using an MAS rate of
1 kHz. Upon increasing the temperature, aromatic signals
broadened significantly in line with an overall increase in
effective molecular size.70,71 Furthermore, aromatic signals
shifted toward lower ppm values, as expected for π−π
interactions. To investigate such interactions by 2D NMR, we
acquired 2D NOESY67 spectra obtained at both temperatures
(Figure 15). In general, cross peaks were more intense at 37 °C
(red) and correlations shifted upfield. Although a detailed
structural analysis is precluded because cross-peak intensities
not only depend on distance but also molecular correlation
time and MAS rate69 the most likely explanation for the
observed correlations (indicated by dashed boxes) are proximal
intra- or intermolecular nuclear interactions within aromatic
rings.71 Taken together, the NMR results (line width, chemical
shifts and 2D cross peak intensities) are consistent with an
overall reduction in molecular mobility due to micelle
formation that involves π−π stacking66,65 among aromatic
moieties at higher temperature.
comparable cytotoxicity on B16F10 as Taxol. At both 1 and 10
μg/mL of PTX, Taxol showed a higher cytotoxicity than PTX-
loaded polymeric micelles formulations which can be ascribed
to the high toxicity of the Cremophor EL/ethanol vehicle. The
concentrations of the polymers were below the CMC at PTX
concentration <2 μg/mL and thus the drug was present in its
free form in the cell culture medium. At the PTX concentration
of 10 μg/mL, the polymer concentration was higher than the
CMC. However, as seen in Figure 11, around 30% of PTX was
released in 72 h (the incubation time of the cell with the
formulations). This means that the cytotoxicity of the
formulation is therefore likely due to released PTX either in
the incubation medium and/or after endocytosis of the micelles
in the cytosol. The released PTX concentration in the medium
for the micellar formulation of 10 μg/mL of PTX is probably at
least 3 μg/mL, which explains that the cell viability of 10 μg/
mL of PTX was similar to that of 1 μg/mL of PTX.
3.8. Detection of the π−π Stacking in Micelles. We
conducted one- and two-dimensional 1H solid-state NMR
experiments on the micellar suspension prepared in D2O with a
polymer concentration of 10 mg/mL at 37 and 1 °C. Magic
Angle Spinning (MAS,64) was employed to increase spectral
resolution.
In general, such solid-state NMR experiments report on
molecular structure via the measurement of isotropic chemical
shifts and internuclear interactions, that is, dipolar spin−spin
interactions. The chemical shift of hydrogen atoms is strongly
influenced by external electrons providing a means to study
packing of π-electron systems. In particular, π−π interactions
CONCLUSION
■
Thermosensitive amphiphilic polymers of mPEG-b-p-
(HPMAm-Bz/Nt-co-HPMAm-Lac) were synthesized and they
self-assembled into polymeric micelles above their critical
micelle temperature and critical micelle concentration. Solid-
state NMR data are consistent with the formation of π−π
stacking in the micellar core among the aromatic groups. The
poorly water-soluble anticancer drugs paclitaxel and docetaxel
were encapsulated into the polymeric micelles with an
unprecedented high loading efficiency. The mPEG-b-p-
(HPMAm-Bz/Nt-co-HPMAm-Lac) micelles had a better drug
loading and retention compared with mPEG-b-p(HPMAm-
J
dx.doi.org/10.1021/bm400234c | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
1
Figure 15. Spectral cutouts of 2D H−1H NOESY spectra obtained at 37 °C (red) and 1 °C (black) using a mixing time of 30 ms. Dotted lines
connect diagonal and cross-peak correlations.
dilactate) micelles that do not contain aromatic units in the
polymer chains due to π−π stacking and hydrophobic
interaction by aromatic groups. The empty polymeric micelles
did not affect the viability of B16F10 cells up to 1 mg/mL and
PTX-loaded polymeric micelles showed comparable cytotox-
icity on B16F10 as Taxol, which means that the therapeutic
effect of PTX was not affected by loading it into the polymeric
micelles. The high loading capacity, strong drug retention in the
micelles and low cytotoxicity of the synthetic polymers show
the potential for in vivo application. The paclitaxel-loaded
micelles will be further evaluated in vivo in a suitable animal
model.
Netherlands Organization for Scientific Research (Grants
700.26.121 and 700.10.443 to M.B.).
REFERENCES
■
(1) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev.
2001, 47, 113−131.
(2) Torchilin, V. Pharm. Res. 2007, 24, 1−16.
(3) Talelli, M.; Rijcken, C.; van Nostrum, C.; Storm, G.; Hennink, W.
E. Adv. Drug Delivery Rev. 2010, 62, 231−239.
(4) Miller, T.; Hill, A.; Uezguen, S.; Weigandt, M.; Gopferich, A.
̈
Biomacromolecules 2012, 13, 1707−1718.
(5) Du, J. Z.; Tang, L. Y.; Song, W. J.; Shi, Y.; Wang, J.
Biomacromolecules 2009, 10, 2169−2174.
(6) Gaucher, G.; Marchessault, R. H.; Leroux, J. C. J. Controlled
Release 2010, 143, 2−12.
ASSOCIATED CONTENT
■
(7) Sethuraman, V. A.; Bae, Y. H. J. Controlled Release 2007, 118,
216−224.
S
* Supporting Information
The detailed GPC and 1H NMR characterizations of polymers,
excitation spectra of the polymers and pyrene, and HPMAm-
Bz/Nt hydrolytic degradation are described. This material is
available free of charge via the Internet at http://pubs.acs.org.
(8) Bae, Y.; Jang, W. D.; Nishiyama, N.; Fukushima, S.; Kataoka, K.
Mol. BioSyst. 2005, 1, 242−250.
(9) Oerlemans, C.; Bult, W.; Bos, M.; Storm, G.; Nijsen, J. F. W.;
Hennink, W. E. Pharm. Res. 2010, 27, 2569−2589.
(10) Lammers, T.; Aime, S.; Hennink, W. E.; Storm, G.; Kiessling, F.
Acc. Chem. Res. 2011, 10, 1029−1038.
AUTHOR INFORMATION
■
(11) Yang, R.; Meng, F.; Ma, S.; Huang, F.; Liu, H.; Zhong, Z.
Biomacromolecules 2011, 8, 3047−3055.
Corresponding Author
(12) Wei, H.; Zhang, X.; Cheng, C.; Cheng, S. X.; Zhuo, R. X.
Biomaterials 2007, 28, 99−107.
*Tel.: + 31 30 253 6964. Fax: + 31 30 251 7839. E-mail: w.e.
hennink@uu.nl.
(13) Soga, O.; van Nostrum, C. F.; Hennink, W. E. J. Controlled
Release 2005, 101, 383−385.
Notes
The authors declare no competing financial interest.
(14) Censi, R.; Fieten, P. J.; di Martino, P.; Hennink, W. E.;
Vermonden, T. Macromolecules 2010, 43, 5771−5778.
(15) Hsiue, G. H.; Hsu, S.; Yang, C. C.; Lee, S. H.; Yang, I.
Biomaterials 2002, 23, 457−462.
ACKNOWLEDGMENTS
■
The research was partially supported by China Scholarship
Council. The solid-state NMR work was supported by The
(16) Paasonen, L.; Romberg, B.; Storm, G.; Yliperttula, M.; Urtti, A.;
Hennink, W. E. Bioconjugate Chem. 2007, 18, 2131−2136.
K
dx.doi.org/10.1021/bm400234c | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
(17) Neradovic, D.; Soga, O.; van Nostrum, C. F.; Hennink, W. E.
Biomaterials 2004, 25, 7491−7498.
(18) Rijcken, C. J.; Snel, C. J.; Schiffelers, R. M.; van Nostrum, C. F.;
Hennink, W. E. Biomaterials 2007, 28, 5581−5593.
(19) Liu, J.; Zeng, F.; Allen, C. J. Controlled Release 2005, 103, 481−
497.
(20) Lo, C. L.; Huang, C. K.; Lin, K. M.; Hsiue, G. H. Biomaterials
2007, 28, 1225−1235.
(21) Opanasopit, P.; Yokoyama, M.; Watanabe, M.; Kawano, K.;
Maitani, Y.; Okano, T. J. Controlled Release 2005, 104, 313−321.
(22) Joralemon, M. J.; Rachel, K. O. R.; Hawker, C. J.; Wooley, K. L.
J. Am. Chem. Soc. 2005, 127, 16892−16899.
(23) O’Reilly, R. K.; Hawker, C. J.; Wooley, K. L. Chem. Soc. Rev.
2006, 35, 1068−1083.
(24) Read, E. S.; Armes, S. P. Chem. Commun. 2007, 3021−3035.
(25) van Nostrum, C. F. Soft Matter 2011, 7, 3246−3259.
(26) Kang, Y.; Taton, T. A. Angew. Chem., Int. Ed. 2005, 117, 413−
416.
(27) Xu, P.; Tang, H.; Li, S.; Ren, J.; van Kirk, E.; Murdoch, W. J.;
Radosz, M.; Shen, Y. Biomacromolecules 2004, 5, 1736−1744.
(28) Chen, H.; Kim, S.; He, W.; Wang, H.; Low, P. S.; Park, K.;
Cheng, J. X. Langmuir 2008, 24, 5213−5217.
(29) Chen, H.; Kim, S.; Li, L.; Wang, S.; Park, K.; Cheng, J. X. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 6596−6601.
(47) Soga, O.; van Nostrum, C.F.; Ramzi, A.; Visser, T.; Soulimani,
F.; Frederik, P. M.; Bomans, P. H. H.; Hennink, W. E. Langmuir 2004,
20, 9388−9395.
(48) Reddy, P. M.; Venkatesu, P. J. Phys. Chem. B 2011, 115, 4752−
4757.
(49) Zhang, Y.; Furyk, S.; Sagle, L. B.; Cho, Y.; Bergbreiter, D. E.;
Cremer, P. S. J. Phys. Chem. C 2007, 111, 8916−8924.
(50) Soga, O.; van Nostrum, C. F.; Hennink, W. E. Biomacromolecules
2004, 5, 818−821.
(51) Rijcken, C. J. F.; Veldhuis, T. F. J.; Ramzi, A.; Meeldijk, J. D.;
van Nostrum, C. F.; Hennink, W. E. Biomacromolecules 2005, 6, 2343−
2351.
(52) Huh, K. M.; Min, H. S.; Lee, S. C.; Lee, H. J.; Kim, S.; Park, K. J.
Controlled Release 2008, 126, 122−129.
(53) Cho, Y. W.; Lee, J.; Lee, S. C.; Huh, K. M.; Park, K. J. Controlled
Release 2004, 97, 249−257.
(54) Jette, K. K.; Law, D.; Schmitt, E. A.; Kwon, G. S. Pharm. Res.
2004, 21, 1184−1191.
(55) Verweij, J.; Clavel, M.; Chevalier, B. Ann. Oncol. 1994, 5, 495−
505.
(56) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. J. Chem. Phys.
1979, 71, 4546−4553.
(57) Moad, G.; Rizzardo, E.; Thang, S. H. Acc. Chem. Res. 2008, 41,
1133−1142.
(58) Braunecker, W. A.; Matyjaszewski, K. Prog. Polym. Sci. 2007, 93,
93−146.
(30) Talelli, M.; Iman, M.; Varkouhi, A. K.; Rijcken, C. J. F.;
Schiffelers, R. M.; Etrych, T.; Ulbrich, K.; van Nostrum, C. F.;
Lammers, T.; Storm, G.; Hennink, W. E. Biomaterials 2010, 30, 7797−
7804.
(59) Jones, M. C.; Leroux, J. C. Eur. J. Pharm. Biopharm. 1999, 48,
101−111.
(60) Yokoyama, M.; Satoh, A.; Sakurai, Y.; Okano, T.; Matsumura,
Y.; Kakizoe, T.; Kataoka, K. J. Controlled Release 1998, 13, 219−29.
(61) Kwon, G.; Naito, M.; Yokoyama, M.; Okano, T.; Sakurai, Y.;
Kataoka, K. J. Controlled Release 1997, 48, 195−201.
(62) Lee, A. L. Z.; Venkataraman, S.; Sirat, S.; Gao, S.; Hedrick, J. L.;
Yang, Y. Y. Biomaterials 2011, 33, 1921−1928.
(63) Neradovic, D.; van Steenbergen, M.; Vansteelant, L.; Meijer, Y.;
van Nostrum, C. F.; Hennink, W. E. Macromolecules 2003, 36, 7491−
7498.
(31) Talelli, M.; Rijcken, C. J. F.; Oliveira, S.; der Meel, R.;
Henegouwen, P. M. P.; Lammers, T.; van Nostrum, C. F.; Storm, G.;
Hennink, W. E. J. Controlled Release 2011, 1, 93−102.
(32) Talelli, M.; Oliveira, S.; Rijcken, C. J. F.; Pieters, E. H. E.;
Etrych, T.; Ulbrich, K.; van Nostrum, C. F.; Storm, G.; Hennink, W.
E.; Lammers, T. Biomaterials 2013, 34, 1255−1260.
(33) Crielaard, B. J.; Rijcken, C. J. F.; Quan, L.; van der Wal, S.;
Altintas, I.; van der Pot, M.; Kruijtzer, J. A. W.; Liskamp, R. M. J.;
Schiffelers, R. M.; van Nostrum, C. F.; Hennink, W. E.; Wang, D.;
Lammers, T.; Storm, G. Angew. Chem., Int. Ed. 2012, 51, 7366−7370.
(34) Carstens, M. G.; de Jong, P. H.; van Nostrum, C. F.; Kemmink,
J.; Verrijk, R.; de Leede, L. G. J.; Crommelin, D. J. A.; Hennink, W. E.
Eur. J. Pharm. Biopharm. 2008, 68, 596−606.
(35) Yokoyama, M.; Fukushima, S.; Uehara, R.; Okamoto, K.;
Sakurai, Y.; Okano, T. J. Controlled Release 1998, 50, 79−92.
(36) Huh, K. M.; Min, H. S.; Lee, S. C.; Lee, H. J.; Kim, S.; Park, K. J.
Controlled Release 2008, 126, 122−129.
(37) Engelkamp, H.; Middelbeek, S.; JM, R. Science 1999, 284, 785−
788.
(64) Andrew, E. R.; Bradbury, A.; Eades, R. G. Nature 1958, 182,
1659−1661.
(65) Schnell, I.; Spiess, H. W. J. Magn. Reson. 2001, 151, 153−227.
(66) Mafra, L.; Santos, M. S.; Siegel, R.; Alves, I.; Almeida Paz, F. A.;
Dudenko, D.; Spiess, H. W. J. Am. Chem. Soc. 2011, 134, 71−74.
(67) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. J. Chem. Phys.
1979, 71, 4546−4553.
(68) Baldus, M. Prog. Nucl. Magn. Reson. Spectrosc. 2002, 41, 1−47.
(69) Lange, A.; Seidel, K.; Verdier, L.; Luca, S.; Baldus, M. J. Am.
Chem. Soc. 2003, 125, 12640−12648.
(70) Ader, C.; Frey, S.; Maas, W.; Schmidt, H. B.; Goerlich, D.;
Baldus, M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 6281−6285.
(71) Allix, F.; Curcio, P.; Pham, Q. N.; Pickaert, G.; Jamart-Gregoire,
B. Langmuir 2010, 26, 16818−16827.
(38) Carstens, M. G.; Bevernage, J. J. L.; van Nostrum, C. F.; van
Steenbergen, M. J.; Flesch, F. M.; Verrijk, R.; de Leede, L. G. J.;
Crommelin, D. J. A.; Hennink, W. E. Macromolecules 2007, 40, 116−
122.
(39) Lee, S. C.; Huh, K. M.; Lee, J.; Cho, Y. W.; Galinsky, R. E.; Park,
K. Biomacromolecules 2007, 8, 202−208.
(40) Yan, J.; Ye, Z.; Chen, M.; Liu, Z.; Xiao, Y.; Zhang, Y.; Zhou, Y.;
Tan, W.; Lang, M. Biomacromolecules 2011, 12, 2562−2572.
(41) Lai, Y.; Long, Y.; Lei, Y.; Deng, X.; He, H.; Sheng, M.; Li, M.;
Gu, Z. J. Drug Targeting 2012, 20, 246−254.
(42) Kim, B. S.; Park, S. W.; Hammond, P. T. ACS Nano 2008, 2,
386−392.
̀
(43) Kang, N.; Perron, M. E.; Prud’Homme, R. E.; Zhang, Y.;
Gaucher, G.; Leroux, J. C. Nano Lett. 2005, 5, 315−319.
(44) Zhang, J.; Wang, L. Q.; Wang, H.; Tu, K. Biomacromolecules
2006, 7, 2492−2500.
̌
̌
̌
(45) Etrych, T.; Subr, V.; Strohalm, J.; Sírova, M.; Ríhova, B.;
́
́
Ulbrich, K. J. Controlled Release 2012, 164, 346−354.
(46) Neradovic, D.; van Nostrum, C. F.; Hennink, W. E.
Macromolecules 2001, 34, 7589−7591.
L
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