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ARTICLE
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Amphiphilic Gradient Copolymers of 2-Methyl- and 2-Phenyl-2-oxazoline:
Self-Organization in Aqueous Media and Drug Encapsulation
Yanna Milonaki,1,2 Eleni Kaditi,1 Stergios Pispas,1 Costas Demetzos2
1
Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vassileos Constantinou Ave.,
11635 Athens, Greece
2
Department of Pharmaceutical Technology, Faculty of Pharmacy, Panepistimiopolis Zografou 15771, University of Athens,
Athens, Greece
Correspondence to: S. Pispas (E-mail: [email protected])
Received 27 October 2011; accepted 8 December 2011; published online 29 December 2011
DOI: 10.1002/pola.25888
ABSTRACT: Gradient (or pseudo-diblock) copolymers were synthesized from 2-methyl-2-oxazoline and 2-phenyl-2-oxazoline
monomer mixtures via cationic polymerization. The selfassembling properties of these biocompatible gradient
copolymers in aqueous solutions were investigated, in an
effort to use the produced nanostructures as nanocarriers for
hydrophobic pharmaceutical molecules. Dynamic and static
light scattering as well as AFM measurements showed that
the copolymers assemble in different supramolecular nanostructures (spherical micelles, vesicles and aggregates)
depending on copolymer composition. Fluorescence spectroscopy studies revealed a microenvironment of unusually high
polarity inside the nanostructures. This observation is related
partly to the gradient structure of the copolymers. The polymeric nanostructures were stable with time. Their structural
properties in different aqueous media—PBS buffer, RPMI solution—simulating conditions used in pharmacological/medicinal studies, have been also investigated and a composition
dependent behavior was observed. Finally, the hydrophobic
drug indomethacin was successfully encapsulated within the
gradient copolymer nanostructures and the properties of the
mixed aggregates were studied in respect to the initial copolymer assemblies. The produced aggregates encapsulating indomethacin showed a significant increase of their mass and
C 2011 Wiley
size compared to original purely polymeric ones. V
Periodicals, Inc. J Polym Sci Part A: Polym Chem 50: 1226–
1237, 2012
INTRODUCTION During the last few years pharmaceutical
nanotechnology based on polymers has become a promising
field for the improvement of existing drug formulations. The
idea of drug delivery via polymeric nanocarriers resulted
from the possibility to synthesize macromolecules, whose
chemical structure allows them to self-assemble and function
as drug carriers. Polymer science has considerably moved up
by introducing self-assembling polymeric nanosystems with
important applications on the improvement of drug delivery.1–4 Among such polymeric nanosystems amphiphilic
block copolymers, consisting of two blocks of different solubility in water, that is, a hydrophobic and a hydrophilic one,
are a widely studied case.5,6 Asymmetric amphiphilic block
copolymers self-assemble in aqueous media, to form a coreshell micellar structure, with a mesoscopic narrow size range
(in the order of 10–100 nm). The core forms the inside of
the particle and is created by the aggregation of the insoluble (minority) blocks and the corona, which is exposed to
water, is made by the soluble blocks.7 Several different aggregate morphologies have been also observed depending on
copolymer composition and physicochemical parameters of
the solution.8,9 The aforementioned applications of block
copolymers in nanomedicine lead to the pursue for a deeper
understanding of their self-organizing properties, which
define their application potential in a significant way. For
example, the nanoscale property of the self-assembled structure allows easy cellular uptake, while the detailed chemical
structure of the copolymer and the morphology of the nanoassemblies formed in solution defines the drug loading ability and capacity of polymeric nanostructures.
KEYWORDS: cationic polymerization; drug delivery systems;
gradient copolymers; polyoxazolines; self-assembly
Poly(2-oxazoline)s, provide an easy access to well-defined
amphiphilic polymeric structures, mainly through cationic
polymerization,10,11 able to form self-assembled nanostructures.12–14 Thanks to their amide unit based chemical structure, resembling to polypeptides, poly(2-oxazoline)s may
present important advantages compared to poly(ethylene oxide) (PEO) and poly(ethylene-glycol) (PEG) for biomedical
applications.15–17 It should also be noted that PMeOx is
more hydrophilic than either poly(2-ethyl-2-oxazoline)
Additional Supporting Information may be found in the online version of this article.
C 2011 Wiley Periodicals, Inc.
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(PEtOx) or PEO.13 The hydrophilic poly(2-methyl-2-oxazoline) (PMeOx) chains are biocompatible and suppress interactions with proteins and the immune system (stealth behavior).18 In addition they undergo rapid renal clearance,
similarly to PEG used for intra venous drug delivery systems.19,20 As far as poly(2-phenyl-2-oxazoline) (PPhOx) is
concerned, it presents increased hydrophobicity, similar to
other poly(2-oxazoline)s based on 2-oxazoline monomers
with side hydrocarbon groups, longer than propyl, and aryl
groups. Additionally, PMeOx monomeric unit structure
resembles to that of the amino acid alanine and PPhOx
monomeric unit is isomeric to phenylalanine.
point of view. This is, partially, due to the increasing interest
of pharmaceutical industries to recover in market already
well established, effective and low cost drugs such as IND,
by improving their effectiveness and reducing their side
effects to patients. IND could also be used as the lead compound, due to its hydrophobic nature, to study the physicochemical properties of the copolymers which are considered
as crucial for producing effective drug delivery systems for
hydrophobic pharmaceutical molecules. Moreover, this study
can offer substantial knowledge concerning the polymeric
formulation of indonethacin in particular and could be a
new approach for producing new and effective polymeric
formulations of NSAID with less adverse drug reactions.
In the present study we focus on the self-assembly behavior
of gradient copolymers containing 2-methyl-2-oxazoline and
2-phenyl-2-oxazoline (MPOx copolymers). The copolymers
were synthesized by cationic polymerization of mixtures of
the two monomers. As it was previously demonstrated, due
to the copolymerization characteristics of the two monomers
the structure of the resulting copolymers can be regarded as
a gradient or a pseudo-diblock structure.6,21,22 Therefore, the
synthetic protocol presents some advantages for scale-up.
The self-assembling properties of linear copolymers, with
gradient compositions of monomers along the polymeric
chain, in selective solvents have been studied to a lesser
extend. The particular macromolecular architecture may
have significant impact on the self-assembly process, as has
been reported earlier for the case of some gradient copolymers in organic and aqueous media.6,21–26 In turn the encapsulation of hydrophobic drugs in the formed nanostructures
may also be largely affected. We have used a gamut of physicochemical techniques, to elucidate structure and properties
of the self-assembled nanostructures created by MPOx gradient copolymers of differing compositions in different aqueous media. We also investigated the nanostructures formed
by encapsulation of the hydrophobic drug indomethacin
(IND). IND belongs to the class of nonsteroidal anti-inflammatory drugs (NSAID). NSAIDs are highly effective in the
treatment of rheumatoid and osteoarthritis, but their long
term use results in gastrointestinal (GI) toxicity in a large
number of cases, like ulceration and structure formation in
esophagus, stomach and duodenum, leading to severe bleeding, perforation and obstruction.27 In view of the required
decreased adverse drug reactions of IND formulations, the
encapsulation within MPOx nanostructures seems to be an
appealing approach from the pharmaceutical manufacturing
EXPERIMENTAL
Materials
All chemicals were purchased from Aldrich unless indicated
otherwise. Indomethacin was supplied by Fluka and was
used as received.
Synthesis of Poly(2-methyl-2-oxazoline)-grad-poly(2phenyl-2-oxazoline) Copolymers (MPOx)
2-methyl-2-oxazoline and 2-phenyl-2-oxazoline monomers
were distilled from CaH2 in a vacuum line using a glass
home-made short-path distillation apparatus, just before the
polymerization. The MPOx gradient copolymers were synthesized via cationic polymerization, using methyl tosylate as
the initiator.10,28–31 The monomers were mixed in appropriate amounts in a glass reactor equipped with a constriction.
Then the calculated amount of methyl tosylate was introduced to the reactor as a solution in butyronitrile (ca. 20
mL, distilled from CaH2 under reduced pressure just before
the polymerization). Total monomer mass was in the range
5–10 g. Details on calculated stoichiometric molecular
weights, Ms, and monomer mass ratios in the feed utilized in
each case are summarized in Table 1. The reaction mixture
was degassed by three freeze-thaw-freeze cycles on a high
vacuum line, using liquid nitrogen for freezing the solution.
After the last freezing period the constriction was flamesealed and the mixture was allowed to thaw slowly at room
temperature. Then the reactor was placed in a temperature
stabilized oil bath at 100 C and the polymerization was
allowed to proceed for 48–72 h. The polymerization reaction
was terminated with water. The copolymers were precipitated in excess diethyl ether and dried under vacuum. Nearly
TABLE 1 Molecular Characteristics of Poly(2-methyl-2-oxazoline-grad-2-phenyl-2-oxazoline)
Copolymers
Sample
Ms
Mna
Mwb
Mw/Mnb
% wt PhOx
in Feed
% wt PhOxa
DPc Me/Ph
MPOx 1
5,080
4,900
5,200
1.14
32
28
42/9
MPOx 2
5,360
5,100
3,200
1.15
12
10
54/4
MPOx 3
4,860
4,600
3,300
1.26
43
39
33/12
a
b
c
1
By H NMR in CDCl3
By SEC in CHCl3 using polystyrene standards
Calculated degrees of polymerization of MeOx and PhOx, respectively, from NMR data
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quantitative conversion of monomers was observed in all
cases. Three copolymer samples with different composition
in the hydrophobic segments (PhOx) were synthesized to
access the effect of composition on the self-assembly properties of this family of copolymers. Molecular characterization
of the MPOx copolymers was performed by size exclusion
chromatography in CHCl3, complemented by ATR-FTIR measurements in the solid state and solution 1H NMR in CDCl3.
Solution Preparation for Self-Assembly Studies in
Aqueous Media
Stock solutions of the MPOx copolymers were prepared by
directly dissolving the samples in distilled and filtered water
(0.45 lm hydrophilic Teflon filters from Millipore) and left
overnight for complete dissolution and equilibration. All
copolymers were rather readily soluble in water within the
prescribed period of time. Dilutions were performed the next
day to obtain series of solutions with lower concentrations.
Evidence for the presence of aggregates could be obtained
by the naked eye for the case of sample MPOx 3, which has
the highest content in the hydrophobic monomer—its solutions had significant bluish tint or were opaque at higher
concentrations (ca. 1 102 g/mL).
Similarly, copolymer stock solutions were mixed with filtered
(0.45 lm hydrophilic Teflon filters from Millipore) aqueous
phosphate buffer solution (PBS, Sigma Aldrich, pH ¼ 7, ionic
strength 0.15 M) to obtain MPOx 1, MPOx 2 at a concentration of 0.01 g/mL and MPOx 3 at a concentration of 0.002
g/mL and left overnight for equilibration. Dilutions were performed once again to obtain series of solutions with lower
concentrations that would allow performance of light scattering measurements. Finally solutions of MPOx in RPMI 1640
R No. 31870-017, containing sodium
culture medium (GibcoV
bicarbonate, pH ¼ 8.2 6 0.3, osmolality 274% 6 5%
mOsm/Kg H2O,) were prepared by mixing each copolymer
stock solution in PBS (0.5 mL) with filtered RPMI (1.5 mL).
For steady state fluorescence spectra a stock solution was
prepared by dissolving the copolymer directly in filtered
water. After a series of successive dilutions of the stock solution concentrations in the range 6 1010 to 1 102 g/mL
were obtained. Acetone solutions of the hydrophobic probe
pyrene (2.5 mM) were then added into the vials and acetone
was allowed to evaporate.
For the studies on the encapsulation of indomethacin into
the copolymer nanoassemblies, both indomethacin and the
copolymers were dissolved separately in CHCl3 and then left
overnight for complete dissolution and equilibration. The
two solutions were then mixed in appropriate proportions
and chloroform was evaporated at room temperature overnight. To the solid copolymer/drug mixtures 5 mL of PBS
were added and solutions of different copolymer: indomethacin mass ratios were obtained, that is, 1:0.25, 1:0.50, 1:0.75,
and 1:1 w/w.
Methods
Molecular weights and molecular weight distributions of the
MPOx copolymers were determined by size exclusion chro-
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matography (SEC) using a Waters system, with a Waters
1515 isocratic pump, a set of three l-Styragel mixed bed columns, having a porosity range of 102–106 Å, a Waters 2414
refractive index detector (at 40 C) and controlled through
Breeze software. CHCl3 was the mobile phase used, at a flow
rate of 1.0 mL/min at 25 C. The set-up was calibrated with
polystyrene standards having weight average molecular
weights in the range 1200 to 900,000 g/mol. Average composition of the copolymers was determined by 1H NMR spectroscopy, using a Bruker AC 300 spectrometer in CDCl3 at 30
C. Infra-red spectra of the copolymers were taken in the
solid state at room temperature, in the range 550–5000
cm1, using a Bruker Equinox 55 Fourier transform instrument, equipped with an attenuated total reflectance (ATR)
diamond accessory, from SENS-IR, and a press, by averaging
100 scans at 4 cm1 resolution.
For dynamic (DLS) and static (SLS) light scattering measurements an ALV/CGS-3 Compact Goniometer System
(ALVGmbH, Germany) was used, equipped with a cylindrical
JDS Uniphase 22 mW HeANe laser, operating at 632.8 nm,
and an Avalanche photodiode detector. The system was interfaced with an ALV/LSE-5003 electronics unit, for stepper
motor drive and limit switch control, and an ALV-5000/EPP
multitau digital correlator. Measurements were made at the
angular range of 30 to 150 . For evaluating the temperature
stability of the systems the cell temperature was varied from
25 to 55 C, in 5 C steps, using a temperature controlled circulating bath (model 9102 from Polyscience). Heating and
cooling cycles were performed, with equilibration of the systems at intermediate temperatures. The autocorrelation functions from DLS were analyzed by the constrained regularized
CONTIN method to obtain distributions of relaxation rates.
The decay rates provided distributions of the apparent diffusion coefficient (D ¼ C/q2), where q is the magnitude of the
scattering vector. The apparent hydrodynamic radii were calculated using the Stokes Einstein equation:
Rh ¼ kT=6pgD
(1)
where k is the Boltzmann constant, g is the viscosity of
water at temperature T, and D is the diffusion coefficient at
a fixed concentration. The polydispersity of the particle sizes
was given as the l2/C2 from the cumulants method, where
C is the average relaxation rate, and l2 is its second
moment. The values of the radii of gyration, Rg, were
obtained from the Zimm plots, which can be described by
the following equation:
KC
Rvv ðqÞ
ﬃ
c!0
1
1
1 þ R2g q2
Mw
3
(2)
where Rvv(q) is known as the Rayleigh ratio, K ¼ 4p2n2(dn/
dC)2/(NAk40 ) and q ¼ (4pn/k0)sin(y/2), with NA, dn/dC, n
and k0 being the Avogadro number, the specific refractive
index increment, the solvent refractive index, and the wavelength of the light in vacuum, respectively. Apparent molecular weight, Mw, virial coefficient, A2, and the number of
molecules participating in the aggregate formation, defined
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as the aggregation number Nw were also calculated from the
Zimm plots. Toluene was used as the calibration standard
for obtaining absolute values for the scattered intensity.32–34
Steady-state fluorescence spectra of pyrene probe in the solutions were recorded with a double-grating excitation and a
single-grating emission spectrofluorometer (Fluorolog-3,
model FL3-21, Jobin Yvon-Spex) at room temperature (25
C). Excitation wavelength was k ¼ 335 nm (for pyrene) and
emission spectra were recorded in the region 350–600 nm,
with an increment of 1 nm, using an integration time of 0.5
s. Slit openings of 1 nm were used for both the excitation
and the emitted beam. The average of three measurements
for the first and the third peak (I1 corresponding to the 372
nm and I3 corresponding to the 383 nm peak of pyrene fluorescence spectra) were used to determine the ratio I1/I3,
which gives an estimate of the polarity of the environment
around the pyrene probe.35–38 No excimer band formation
was observed.
Zeta potential measurements were performed at 25 C using
Zetasizer 3000HS, Malvern Instruments, Malvern, UK. Samples were illuminated at k ¼ 633 nm and measurements
where performed at an angle of 90 . z-potential values were
determined using the Smolukowski equation relating the
ionic mobility with surface charge, and are the average of 10
repeated measurements. The data were analyzed by the Malvern software.
AFM measurements were performed on a Quesant Q-Scope
250 atomic force microscope (Quesant Instrument) in the
tapping mode, under ambient conditions. Samples for imaging were prepared by dipping fresh, dried silicon wafers,
precleaned with isopropanol, in aqueous solutions of the
copolymers, for typically 5–10 min. After withdrawing the
wafer from the solution, excess water was bolted carefully
by filter paper and samples were left to dry in air. In this
way supramolecular structures were absorbed on the wafer
surface.
RESULTS AND DISCUSSION
Synthesis and Molecular Characterization of MPOx
Gradient Copolymers
Previous kinetic studies on the copolymerization of mixtures
of 2-methyl-2-oxazoline and 2-phenyl-2-oxazoline have demonstrated the great differences in the reactivity ratios of the
two monomers, which could be attributed to the lower
nucleophilicity of 2-phenyl-2-oxazoline.10,22 In such cases
2-methyl-2-oxazoline is incorporated in the polymer chain at
the early stages of polymerization and the amount of the
particular monomer is decreasing. Gradually, incorporation
of 2-phenyl-2-oxazoline takes place leading to the formation
of copolymers with gradient composition along the chain. If
sufficient amounts of pure monomer sequences are present
at the two different chain extremities pseudo-diblock copolymers are formed, whereas the pure block sequences do not
have a well defined limit along the copolymer chain.
Although it is not easy to differentiate unambiguously the
two latter cases by subsequent physicochemical characteriza-
SCHEME 1 Reaction scheme for the synthesis of 2-methyl-2oxazoline/2-phenyl-2-oxazoline
gradient
(pseudo-diblock)
copolymers synthesized in the present study.
tion of the resulting copolymers, it can be concluded that the
copolymers synthesized in this work also belong to the general class of gradient copolymers. The favorable kinetic characteristics of the system also allow the synthesis of amphiphilic gradient (pseudo-diblock) copolymers from monomer
mixtures, without the use of a sequential monomer addition
technique, as it is usually done for normal diblock copolymers, which in turn may lead to a schematic representation
of the synthetic procedure shown in Scheme 1.
Methyl tosylate was used as the organic initiator, since it has
been demonstrated that it gives well-controlled polymerizations of 2-oxazoline monomers.10,22 Additionally, cationic polymerization initiating systems incorporating inorganic compounds may be a rather poor choice when the final
copolymers are to be used in biomedical applications,
because contamination of polymers with unwanted metal
trace impurities should be generally avoided. Polymerizations were performed under vacuum conditions (instead of
an inert gas set up) due to the long reaction times usually
needed for 2-oxazoline polymerizations under normal conditions (although Schubert and coworkers have shown that the
use of microwave irradiation reduces substantially polymerization times22). Utilization of glass sealed reaction vessels is
advantageous, since their use excludes the incorporation of
deleterious impurities into the reaction mixture for long
periods of time. Termination with water leads to the incorporation of a terminal hydroxyl group in the copolymer
chain that can be further utilized for additional postpolymerization functionalization reactions, if needed (the kinetic
amino ester end-group can be also formed due to water
attack on the 2-position as has been reported in the literature,39,40 but this does not seem to be significant in the present case due to the low signal at ca. 4.2 ppm). The amount
of copolymer isolated by precipitation in diethyl ether, after
termination of the polymerization reaction, was nearly quantitative indicating complete consumption of the monomers.
The molecular characteristics of the gradient MPOx copolymers are shown in Table 1. The obtained MPOx copolymers
had relatively narrow molecular weight distributions. The
use of polystyrene standards for SEC calibration does not
allow for determination of the true molecular weights of the
MPOx gradient copolymers synthesized, due to the different
conformational and eluting characteristics of polystyrene
standards versus the MPOx copolymers. Therefore, the Mw
values presented in Table 1 should be regarded as apparent
ones. However, calculated stoichiometric molecular weights
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to elucidate the dilute solution behavior of the gradient
MPOx copolymers in different aqueous media.
Results from DLS measurements on MPOx copolymer solutions in water showed that the solutions of samples MPOx 1
and MPOx 2 contained three populations differing in size as
shown in Figure 2.
FIGURE 1 1H NMR spectrum of sample MPOx 1 in CDCl3.
are in the same range with apparent Mw determined by SEC
(SEC traces are included in Supporting Information Figs. S1
and S2). ATR-FTIR analysis of MPOx copolymers reveals several characteristic peaks that can be correlated with the
expected molecular structure of the materials. The spectrum
of sample MPOx1 is shown in Supporting Information Figure
S3 as a typical example. The peak at 1626 cm1 is attributed
to the carbonyl group vibrations in the amide functionality
of both monomeric units. The peak centered at about 1415
cm1 is coming from the asymmetric stretching vibrations of
the ANACH2A groups on the polymer chain and also from
the C-N stretching bond vibrations in the amide moieties.
Several peaks are resolved in the region 800–600 cm1 associated with the aromatic CAH out-of-plane bending vibrations of the phenyl groups of the monomeric units, resulting
from the incorporation of 2-phenyl-2-oxazoline monomer
within the copolymer chain. The 1H NMR spectra of MPOx
copolymers in CDCl3 (Fig. 1) are dominated by the peak at
about 3.3 ppm attributed to the NACH2 protons of the main
chain, present in all monomeric units. The peaks at 7–7.8
ppm are originating from the protons of the phenyl ring of
the hydrophobic units in the copolymer, resulting from the
incorporation of 2-phenyl-2-oxazoline monomers. From the
areas of these peaks the composition in hydrophobic units
has been calculated for each copolymer, as it is reported in
Table 1. The amphiphilic gradient MPOx copolymers
obtained have different compositions in hydrophilic and
hydrophobic units, a parameter, that is, expected to influence
their self-assembly in aqueous media.
Self-Assembly of Gradient Copolymers in Water
Due to the gradient molecular structure of the MPOx copolymers and the expected amphiphilic character it is interesting
to examine their self-assembly behavior in aqueous media.
So far no systematic studies on the self-assembly behavior of
such macromolecules has been presented. Only Schubert and
coworkers have examined the aggregation characteristics of
a gradient MPOx copolymer in water by transmission electron microscopy.6 Here we employ a larger number of gradient copolymers and a gamut of physicochemical techniques
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Judging from the relative sizes they can be tentatively
assigned to free copolymer chains, micelles and aggregates,
starting from the lower to the highest Rh values. The most
intense peak for the case of sample MPOx 1 was found at
about Rh ¼ 9 nm, which presumably corresponds to copolymer micelles. This peak is narrow. The small peak at about
Rh ¼ 1.8 nm should correspond to unimers and it is also
narrow. The one at about Rh ¼ 120 nm may be attributed to
aggregates of larger size. One possibility regards the formation of micellar clusters, that is, promoted by the gradient
structure of the copolymer chains, however, clusters of
micelles have been observed also in the case of oxazoline
based triblock copolymers.40 The gradient polymer chain
structure allows for a greater exposure of hydrophobic segments to the aqueous environment and this in turn can facilitate hydrophobic interactions between micelles.
For the case of sample MPOx 2 the peak assigned to micelles
is shifted to higher Rh values (ca. Rh ¼ 15 nm) and it is considerably broader. The low Rh peak assigned to unimers is
relatively more intense. Formation of larger aggregates is
also evident in this case and more pronounced (ca. Rh ¼ 140
nm). The differences should be correlated to the hydrophobic composition of each sample.41 MPOx1 has a higher
hydrophobic content, therefore it forms more well defined
micelles of smaller size (the higher hydrophilicity of MPOx 2
may result in greater chain dimensions within the micelles)
and the micelle-unimer equilibrium is shifted more towards
micelles. The presence of unimers should be a result of the
low molecular weight of the samples and partially to their
FIGURE 2 Hydrodynamic radii distributions of samples MPOx
1 (dashed line) and MPOx 2 (solid line) in water solutions,
measured at a scattering angle y ¼ 90 , at 25 C, and at concentrations C ¼ 2 103 g/mL and C ¼ 6 103 g/mL,
respectively.
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1 and MPOx 2, further conclusions can be definitely drawn.
The results are summarized in Table 2. A typical KC/DRy versus concentration plot is given in Figure 4.
FIGURE 3 Hydrodynamic radius distribution of sample MPOx 3
in water solutions measured at scattering angle y ¼ 90 , at 25
C and at concentration C ¼ 7 104 g/mL.
gradient molecular structure. It has been indicated earlier
that copolymer chains with gradient structure, or more generally copolymer chains where some mixing of monomers is
taking place, show lower tendency for aggregation.6,21–26,42
We also assume that the presence of aggregates is partially
due to the gradient structure of the copolymers, as well as
to their composition (note that sample MPOx 2 with the
lower hydrophobic content tents to form larger aggregates,
probably of ill defined structure, because its hydrophobic
content is not high enough to organize in small more welldefined micelles).
In the case of sample MPOx 3 only one population was
observed, having a relatively narrow size distribution (Fig. 3).
The hydrodynamic radius of the assemblies was found at
about 39 nm. Formation of more well defined structures in
this case should be related to the higher hydrophobic content
of sample MPOx 3. Obviously the size of the assemblies is
very large to be characterized as spherical core-shell micelles
(the calculated length of fully extended MPOx 3 chains, taking
into account only bond lengths and not the bond angles and
restrictions to rotation, is ca. 11 nm, considerably smaller
than the radius of the assemblies). So assemblies of MPOx 3
should have a different morphology.
SLS was used to extract additional information on the structure and properties of the MPOx gradient copolymer assemblies. Although SLS results cannot be interpreted in a
straightforward manner in cases where several different species are present in solutions, as in the case of samples MPOx
The first feature to be observed are the low A2 values (negative in some cases) indicating aggregation of the copolymer
chains in water. Apparent Mw values are larger that those
expected for single chains and together with the values of
the aggregation number, Nw, follow the trend in copolymer
composition. The results reveal that assemblies with higher
mass are formed as the hydrophobic content of MPOx
copolymers increases. This is typical also for regular block
copolymer micelles, including those from oxazoline based
amphiphilic block copolymers.21,43,44 The relatively low Nw
determined for samples MPOx 1 and MPOx 2 should be
attributed to the low hydrophobic content and low molecular
weights of the samples, as well as to the gradient structure.
Another conclusion that can be drawn regards the large
aggregates observed by DLS. From their contribution to the
scattering intensity, it turns out that their number should be
low and/or their structure not so dense. They can be also
considered as loosely bound aggregates (clusters) of
micelles, since their large size does not coincide with the
determined low values of Nw. If their mass was high and
their structure compact the determined Mw and Nw should
have been higher. Nw for sample MPOx 3 is considerably
larger. For this particular sample it was possible to determine unambiguously the apparent Rg values of the single
population (Rg ¼ 41 nm). The ratio Rg/Rh, which indicates
the overall morphology/shape of the copolymer’s superstructures, was calculated as 1.05, very close to the value characterizing vesicular structures. This observation in conjunction
with the fact that the value of the Nw number for MPOx 3 is
large (Table 2) leads to the conclusion that probably vesicles
are formed in the MPOx 3 solutions. It must also be pointed
out that the MPOx 3 assemblies have a relatively small size
polydispersity index at all concentrations studied (l2/C2 ca.
0.1, see also Fig. 3).
To get a more direct picture of the nature and morphology
of MPOx 3 nanoassemblies AFM was used. AFM images (Fig.
5) show spherical shaped particles with heights in the range
6–30 nm and lateral dimensions in the range 40–200 nm.
The dimensions determined by AFM show differences
TABLE 2 Apparent Weight Average Molecular Weight, Mw,
Virial Coefficients, A2, and Aggregation Numbers, Nw,
Determined by Static Light Scattering for MPOx Gradient
Copolymers in Water, at 25 8C
Sample
MPOx 1
MPOx 2
MPOx 3
Mw (g/mol)
69,000
25,300
4.4 106
A2 (mol mL/g2)
9.2 10
5
4
7.6 10
4.2 105
Nw
13 6 3
862
1,333 6 230
FIGURE 4 KC/DRy versus C plot for sample MPOx 1 in water.
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FIGURE 5 AFM image showing MPOx 3 vesicular nanoassemblies at different collapsed states at C ¼ 1.7 104 g/mL.
compared to those determined by light scattering. Also the
size polydispersity observed in AFM images contradicts with
the low size polydispersity observed in water solutions by
DLS. These observations must be explained as a consequence
of particle deformation during the adsorption on the surface
and after solvent evaporation in the case of AFM images,
while DLS is an in situ technique giving the hydrodynamic
size of the particles in solution. The large difference between
the particles’ height and lateral dimensions indicates a large
deformability of the particles on the z-direction perpendicular to the surface. This is consistent with a vesicular morphology of the MPOx 3 nanoparticles, where the solvent containing space within the nanoassembly enhances
deformation of the particle on the solid surface after solvent
removal. Overall, AFM observations together with light scattering measurements seem to point towards the formation of
vesicles for sample MPOx 3 in water. It is rather interesting
that gradient copolymers have also the ability to form
vesicles in analogy to regular block copolymers.7–9
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FIGURE 6 Scattered intensity (solid line) and Rh (from cumulants analysis, dashed line) versus temperature for MPOx 2
aqueous solutions measured at a scattering angle y ¼ 90 and
at C ¼ 8 103 g/mL (the lines are guides to the eye).
carry no net charge. Stability of Rh values was also verified
after 5 months. Therefore, it is safe to conclude that MPOx
nanoassemblies remain stable even for a considerably long
period of time.
Fluorescence spectroscopy has been utilized in an attempt to
extract some information on the internal nanostructure and
microenvironment of MPOx nanostructures in water solutions. The ratio I1/I3 in the fluorescence emission spectrum
of pyrene is used as a measure of the polarity of the environment of the pyrene probe. In aqueous or similarly polar
environment this ratio is found between 1.6 and 1.9. For regular amphiphilic block copolymer micelles, the less polar
environment of the core results in a characteristic decrease
of the I1/I3 ratio by increasing concentration above the critical micelle concentration (cmc). Quite surprisingly for the
nanoassemblies formed by the amphiphilic gradient copolymers, the I1/I3 ratio remained stable at 1.7–1.9 regardless of
the copolymer concentration (Fig. 7 and Supporting Information Figs. S6 and S7). It was therefore impossible to
Samples MPOx 2 and MPOx 3 were selected as the most and
the least hydrophilic, respectively, to study the effect of temperature on the structural stability of the nanoassemblies. As
it is evident in Figure 6 the mass and size remain virtually
unchanged for sample MPOx 2 in the temperature range 25
to 55 C, because the scattered intensity from the solution
(analogous to the mass of the nanoassemblies), as well as
the Rh values remain almost constant within experimental
error. A similar temperature behavior was observed for sample MPOx 3 (Supporting Information Fig. S5). The observations prove that the assemblies should be stable both at
body temperature and at more extreme environmental temperature conditions.
The z-potential values were measured for stock and diluted
solutions of all three copolymers directly after their preparation and after 5 months. The z-potential was found in all
cases close to zero, which is expected since the copolymers
1232
FIGURE 7 I1/I3 ratio determined from pyrene fluorescence
spectra in solutions of sample MPOx 3 in water at different copolymer concentrations.
JOURNAL OF
POLYMER SCIENCE
FIGURE 8 Distributions of hydrodynamic radius for MPOx 1
(dashed line) and MPOx 2 (solid line) in PBS solutions, measured at 25 C, at a scattering angle y ¼ 90 and at concentrations C ¼ 3 103g/mL and C ¼ 5 103 g/mL, respectively.
determine a CMC for these copolymers. At first we assumed
that this is an indication that pyrene failed to enter the
nanoassemblies. That assumption was shortly after rejected,
not only because pyrene is particularly friendly with hydrophobic phenyloxazolines found within the micellar core, but
also because indomethacin, which is extremely hydrophobic,
was successfully entrapped within MPOx nanoassemblies, as
it will be described later on. Kabanov and coworkers
reported on pyrene fluorescence measurements on regular
diblock copolymers composed of 2-n-butyl- and 2-ethyl-2oxazoline or 2-methyl-2-oxazoline monomers.44 The authors
observed unusually high values of the I1/I3 ratio which interestingly increased further as the block copolymer concentration increased. They concluded that the behavior should be
related to the significantly hydrophilic nature of the oxazoline monomers, even for the ones that carry a hydrophobic
side group. We believe that the pyrene behavior is probably
due to the unusually polar microenvironment of the micellar
cores, attributed at least partially to the gradient structure of
the copolymers in the present case (which allows partial
mixing of hydrophilic and hydrophobic monomeric units
within the assemblies). In other words, hydrophobic domains
may not be large enough, compared to the size of the probe,
in order for the pyrene to ‘‘feel’’ a hydrophobic environment.
In any case we believe a micellar like structure, with the
more hydrophobic PhOx segments residing in the inner part
of the assembly and the hydrophilic MeOx segments distributed mostly in the periphery, can describe the structure of
the middle population in the solutions of samples MPOx 1
and MPOx 2. This particular characteristic of the assemblies
may be advantageous for the encapsulation of less hydrophobic drug molecules.
Self-Assembly of Gradient Copolymers in Phosphate
Buffer Solution (PBS)
Light scattering measurements were also performed for
MPOx gradient copolymer solutions in PBS, since the pH and
the ionic strength of PBS resembles the conditions met
within the human body. DLS measurements showed that free
polymeric chains are present in PBS solutions of MPOx 2 (Rh
ARTICLE
ca. 2–3 nm depending on the sample), while both for MPOx
1 and MPOx 2 micelles, having hydrodynamic radius equal
to 10 and 18 nm, are formed similar to the case of water
solutions. However, larger aggregates were not observed
(Fig. 8), in contrast to the water solutions. Taking into consideration the greater ionic strength of PBS one can assume
that the absence of aggregates may be due to differences in
the solvation state of polymeric chains when ions are present in solution.45–48 The last observation indirectly indicates
that large MPOx aggregates are formed in salt free aqueous
solutions due to secondary interactions, which are decreasing in the presence of salt. Furthermore, based on the previous discussion it is more plausible to assume that larger
aggregates are formed by secondary interactions between
micelles. Another explanation can be based on the changes
in the hydration power of water, due to the presence of ions
in the PBS solutions, as well as the, at least partial, coordination of these ions to the amide moieties of the gradient
copolymers, something that would make the chains more
hydrophilic.49,50 As far as sample MPOx 3 is concerned some
significant differences in Rh values of the single population
of nanoassemblies have been observed. The obtained Rh was
found to be 100 nm. The increase in Rh is followed with an
increase in polydispersity of the aggregates (l2/C2 values ca.
0.2 or larger where obtained). The calculated Rg/Rh ratio
was equal to 0.75. AFM imaging shows spherical nanoparticles with a relatively large size polydispersity on the dry
SiO2 surface (Supporting Information Fig. S9).
Some results from SLS are shown in Table 3. Values for the
parameters Mw, A2, and Nw follow the same trends observed
also in salt free aqueous solutions. It is important to note
that sample MPOx 3 most probably forms compound
micelles in PBS,9 since the Nw value determined is very large
to be correlated with a simple core-cell micellar structure
and the Rg/Rh value is close to 0.775, the value for hard
spheres. Thus, the presence of salt induces some changes in
the solution behavior of MPOx gradient copolymers and the
structural properties of their assemblies. Measurements performed after a 9 days period on MPOx solutions in PBS
revealed that the characteristics of the MPOx 1 nanoassemblies were unaltered. For sample MPOx 2 two populations
were resolved with Rh values at 20 and 450 nm. One population with Rh ¼ 140 nm was observed in aged solutions of
MPOx 3 in PBS. Their polydispersity values were in the
range 0.2–0.3. The results suggest that secondary aggregation takes place after this period in the case of samples
TABLE 3 Apparent Weight Average Molecular Weights, Mw,
Virial Coefficients, A2, and Aggregation Numbers, Nw,
Determined by Static Light Scattering for MPOx Gradient
Copolymers in PBS, at 25 8C
Sample
MPOx 1
Mw (g/mol)
87,000
A2 (mol mL/g2)
6 10
5
Nw
17 6 3
4
MPOx 2
26,000
4.2 10
862
MPOx 3
3.8 106
1.3 105
1,152 6 180
1233
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can be concluded that the nanoassemblies formed by sample
MPOx 3 are the more prone to morphological changes, as a
result of alterations in the physicochemical parameters of
the surrounding solutions. Such a behavior seems to be
closely correlated to the hydrophobic/hydrophilic balance of
the copolymer and the rather fragile vesicular structure of
MPOx 3 assemblies in water.
The copolymer solutions mixed in RPMI were also tested for
their stability during time. As it is shown in Figure 10 both
size and mass remained relatively unchanged after initial
mixing of PBS solutions with RPMI.
FIGURE 9 Distribution of Rh for sample MPOx 3 in PBS/RPMI
mixed solutions, measured at 25 C and at a scattering angle y
¼ 90 . Copolymer concentration is C ¼ 2 103 g/mL.
MPOx 2 and MPOx 3. This may be correlated to the hydrophobic ratios of the copolymers. MPOX 2 is very hydrophilic
but existing hydrophobic interactions may result in the formation of ill-defined aggregates in aged solutions. The gradient chain structure may play some role. MPOx 3 is the most
hydrophobic and this results in the formation of larger structures as time passes (probably with a change in the aggregates morphology). Colloidal temporal stability of the initially
formed structures is greatly affected by the hydrophilic/
hydrophobic ratio in different ways. It seems that as far as
the temporal stability is concerned sample MPOx 1 presents
the optimum case.
Self-Assembly of Gradient MPOx Copolymers in RPMI
The behavior of the nanoassemblies formed by MPOx gradient copolymers were investigated after mixing PBS solutions
of the copolymers with RPMI 1640, which is a culture medium for human cells. The results of such studies are useful
for determining stability of drug nanocarriers during application in drug delivery. The measurements were made at 37 C
after examining the properties of RPMI by light scattering
techniques. RPMI contains several different molecular species—dextrose, aminoacids, inorganic salts, vitamins. However, the scattering intensity was small compared to that of
copolymer solutions and therefore it was possible to differentiate peaks originating from the MPOx solutions (Fig. 9
and Supporting Information Figs. S10 and S11). Light scattering measurements performed after mixing MPOx 1 and
MPOx 2 PBS solutions with RPMI presented similar results
to those in aqueous solutions. Therefore, MPOx 1 and MPOx
2 nanoassemblies are not disturbed by the presence of RPMI
components. On the other hand, the size of MPOx 3 assemblies was found to increase by more than 100 nm within the
first 5 minutes after adding RPMI. Their size distribution
was also increased. Apparently, the MPOx 3 compound
micelles interact fast with the RPMI components forming
aggregates of large size. Most probably the interaction is
facilitated by the structure of the nanoassemblies, since no
significant differences were observed for samples MPOx 1
and MPOx 2, which are forming mainly spherical micelles. It
1234
Indomethacin Encapsulation into MPOx Gradient
Copolymer Assemblies
Having established a detailed picture regarding the self-assembly behavior of the different MPOx copolymers in aqueous solutions it was decided to investigate the possibilities
for encapsulation of indomethacin within the copolymer
assemblies. Indomethacin consists of aromatic rings and contains an amide group similar to phenyloxazolines based segments, while its carboxylic group allows formation of hydrogen bonds. The chemical structure, the amphiphilic nature
and the biocompatibility of MPOx gradient copolymers make
them good candidates for encapsulating indomethacin.51–54
As it was discussed in the experimental section indomethacin was encapsulated within MPOx in different proportions
(copolymer: indomethacin ¼ 1:0.25, 1:0.50, 1:0.75, 1:1 w/
w). It can be observed in Figure 11 that turbidity of the solutions increases with increasing concentration of indomethacin. Precipitation was only observed in the mixed copolymer/drug solutions 1:1, while 1:0.25, 1:0.5, 1.0.75 mixed
solutions remained stable after addition of water and for a
period of several days. The color of the solutions is attributed to the yellow color of indomethacin when found in solution. Presumably the whole quantity of drug was encapsulated within the copolymer nanostructures, as the absence of
precipitate for the particular weight ratios indicates.53
DLS measurements indicate the presence of copolymer-drug
mixed aggregates in the solutions. Two populations were
FIGURE 10 Scattered intensity (solid line) and Rh (dashed line)
as a function of time for MPOx 1 solutions in PBS mixed with
RPMI at 37 C.
JOURNAL OF
POLYMER SCIENCE
ARTICLE
This behavior was also observed in other works.51–54 Giacomelli et al. noticed a similar increase in Rh, when indomethacin was encapsulated in poly(ethylene oxide)-b-poly[2-(diisopropylamino)-ethyl
methacrylate]
(PEO-b-PDPA)
and
poly(glycerol monomethacrylate)-b-PDPA (PG2MA-b-PDPA)
block copolymer nanoassemblies. Furthermore, their results
indicated the creation of junctions between micelles at
higher concentration of indomethacin.53 Our copolymers can
therefore be named ‘‘active’’ according to the definition of
Zhang et al.54 It should also be mentioned that the micellar
size presented a slight increase, while augmenting the indomethacin loading, contrary to Zhang et al. observations on
another class of block copolymers.54 Chemical structure,
architecture and molecular weight of the copolymer, as well
as specific copolymer-drug interactions should determine the
behavior of different mixed copolymer/drug systems.
FIGURE 11 Solutions of samples (a) MPOx 1 and (b) MPOx 2,
at a copolymer concentration 5 103g/mL containing indomethacin at increasing mass proportions (from right to left: copolymer: indomethacin ¼ 1:0.25, 1:0.50, 1:0.75, 1:1 w/w).
observed, suggesting the existence of micelles, as well as
larger aggregates. Those aggregates should be regarded as
hyper-micellar nanostructures resulting from the fact that indomethacin can act as a strong hydrophobic aggregation
center.
The measured scattered intensity increases significantly with
indomethacin content, which must reflect an increase of the
mass of the copolymer aggregates encapsulating the hydrophobic drug (Fig. 12). This change should be attributed to
the formation of new micellar like aggregates that include
the insoluble indomethacin within their core. As far as micellar size is concerned, Rh increased by almost 50% (from 9 to
16 nm) compared to indomethacin-free particles (Fig. 12).
FIGURE 12 Scattered intensity (solid line) and Rh values
(dashed line) determined in solutions of MPOx1 in PBS as a
function of indomethacin content, at temperature 25 C and a
scattering angle of y ¼ 90 . Copolymer concentration is 5 103 g/mL.
The formation of aggregates of larger dimensions occurred
for all copolymers. Apparently, the presence of hydrophobic
drug modulates the aggregation behavior of the gradient
copolymers/drug mixed systems in an effort to minimize
drug-water interactions and the effects depend on the composition of the copolymer. This conclusion is corroborated by
the fact that for copolymer: indomethacin solutions 1:0.25
and 1:0.5, in the case of MPOx 1 the largest population was
the micellar one, while for MPOx 2 was the one consisting of
larger aggregates (Fig. 13 and Supporting Information Fig.
S12). The MPOx/indomethacin mixed aggregates still have
sizes in the nanometer scale range making the particular
systems promising for drug delivery applications through
utilization of copolymer nanocarriers.
CONCLUSIONS
A series of gradient (pseudo-diblock) copolymers were synthesized via living cationic polymerization, from hydrophilic
2-methyl-2-oxazoline and hydrophobic 2-phenyl-2-oxazoline
mixtures. Copolymers had low molecular weights and varying composition. Studies on the self-assembling behavior, in
different aqueous environments, showed the formation of
organized supramolecular nanostructures of different
FIGURE 13 Distribution of Rh, for MPOx 1 copolymer:indomethacin solutions 1:0.75 w/w, measured at 25 C and at scattering angle y ¼ 90 . Copolymer concentration is 5 103 g/mL.
1235
ARTICLE
morphologies and structural characteristics that were primarily dependent on copolymer composition. In particular,
the more hydrophobic gradient copolymer sample formed
well defined vesicles in analogy to regular block copolymers.
The characteristics of copolymer nanoassemblies were found
to be relatively unaltered for long periods of time and unaffected by temperature changes. The internal microenvironment of the nanoassemblies was shown to be unusually polar, partly because of the gradient/pseudo-diblock molecular
structure of the copolymers. Some secondary aggregation
effects were observed in RPMI solutions. Finally, encapsulation of the hydrophobic drug indomethacin was achieved
within copolymer nanostructures. The mixed copolymer/
drug aggregates produced in these cases, presented higher
mass and sizes compared to the original MPOx copolymers.
These results can be used as a road map concerning the
self-assembly processes in pure gradient copolymers systems, as well as for producing new drug delivery systems
composed of such copolymers. Additionally, the results of
the encapsulation of the hydrophobic drug IND, showed that
the drug can regulate the aggregation behavior of the system, probably by minimizing the drug-water interactions,
and thus can offer advantages as a nanocarrier system for
hydrophobic NSAID. The relatively high drug encapsulation
capacity and the nanometer size of the mixed gradient copolymer/indomethacin assemblies are good assets of the present systems. Further optimization of the formulation protocol
and pharmacological studies are needed to prove the effectiveness of the studied drug delivery system, taking into
account that the reduction of the serious drug adverse reactions of IND, and of NSAID in general, should be minimized.
JOURNAL OF
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15 Adams, N.; Schubert, U. S. Adv. Drug Delivery Rev. 2007,
59, 1504–1520
16 Barz, M.; Luxenhofer, R.; Zentelb, R.; Vicent, M. J. Polym.
Chem. 2011, 2, 1900–1918.
17 Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S.
Angew. Chem. Int. Ed. 2010, 49, 6288–6308.
18 Konradi, R.; Pidhatika, B.; Mühlebach, A.; Textor, M. Langmuir 2008, 24, 613–616.
19 Woodle, M. C.; Engbers, C. M.; Zalipsky, S. Bioconjugate
Chem. 1994, 5, 494–496.
20 Gaertner, F. C.; Luxenhofer, R.; Blechert, B.; Jordan, R.; Essler, M. J. Controlled Release 2007, 119, 291–300.
21 Hoogenboom, R.; Thijs, H. M. L.; Wouters, D.; Hoeppener,
S.; Schubert, U. S. Macromolecules 2008, 41, 1581–1583.
22 Lambermont-Thijs, H. M. L.; Heuts, J. P. A.; Hoeppener, S.;
Hoogenboom, R.; Schubert, U. S. Polym. Chem. 2011, 2,
313–322.
23 Hoogenboom, R.; Wiesbrock, F.; Leenen, M. A. M.; Meier,
M. A. R.; Schubert, U. S. J. Comb. Chem. 2005, 7, 10–13.
24 Hondrokoukes, P.; Pispas, S.; Hadjichristidis, N. Macromolecules 2002, 35, 834–840.
25 Hamada, T.; Kudo, K. Chem Lett. 2010, 39, 1285–1287.
26 Schmitz, C.; Mourran, A.; Keul, H.; Moller, M. Macromol.
Chem. Phys. 2008, 209, 1859–1871.
27 Garuso, I.; Bianchi Porro, G. Brit. Med. J. 1980, 12, 75–78.
28 Hoogenboom, R.; Thijs, H. M. L.; Fijten, W. M.; Van Lankvelt, B. M.; Schubert, U. S. J. Polym. Sci. Part A: Polym. Chem.
2007, 45, 416–422.
29 Hoogenboom, R. Macromol. Chem. Phys. 2007, 208, 18–25.
30 Reiss, G. Prog. Polym. Sci. 2003, 28, 1107–1170.
31 Hoogenboom, R.; Fijten, M. W. M.; Schubert, U. S. J. Polym.
Sci.: Part A: Polym. Chem. 2004, 42, 1830–1840.
32 Pecora, R. Dynamic Light Scattering applications of Photon
Correlation Spectroscopy, Springer, 1985.
REFERENCES AND NOTES
33 Huglin, M. B. Light Scattering for Polymer Solutions, Academic Press, New York, 1972.
1 Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai,
Y. J. Controlled Release 1993, 24, 119–132.
34 Schartl, W. Light scattering from polymer solutions and
nanoparticle dispersions, Springer Laboratory, Springer-Verlag,
Berlin-Heidelberg, 2007.
2 Kwon, G. S.; Okano, T. Adv. Drug Delivery Rev. 1996, 21,
107–116.
3 Haag, R. Angew. Chem. Int. Ed. 2004, 43, 278–282.
4 Duncan, R. Nat. Rev. 2003, 2, 347–360.
5 Tuzar, Z.; Kratochvil, P. Adv. Colloid Interface Sci. 1976, 6,
201–232.
6 Hoogenboom, R.; Thijs, H. M. L.; Wouters, D.; Hoeppener, S.;
Schubert, U. S. Soft Matter 2008, 4, 103–107.
7 Gohy, J.-F. Adv. Polym. Sci. 2005, 190, 65–136.
8 Blanazs, A.; Armes, S. P.; Ryan, A. J. Macromol. Rapid Commun. 2009, 30, 267–277.
9 Zhang, L. F.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118,
3168–3181
10 Kobayashi, S.; Uyama, H. J. Polym. Sci. Part A: Polym.
Chem. 2002, 40, 192–209
11 Schlaad, H.; Diehl, C.; Gress, A.; Meyer, M.; Demirel, A. L.;
Nur, Y.; Bertin, A. Macromol. Rapid Commun. 2010, 31,
511–525.
12 Aoi, K.; Okada, M. Prog. Polym. Sci. 1996, 21, 151–208.
fevre, N.; Hoogenboom, R.; Schubert, U. S.;
13 Fustin, C.-A.; Le
Gohy, J.-F. Macromol. Chem. Phys. 2007, 208, 2026–2031.
14 Hoogenboom, R. Angew. Chem. Int. Ed. 2009, 48, 7978–7994
1236
35 Wilhelm, M.; Zhao, C.-L.; Wang, Y.; Xu, R.; Winnik, M. A.;
Mura, J.-L.; Riess, G.; Croucher, M. D. Macromolecules 1991,
24, 1033–1040.
36 Zhao, C.-L.; Winnik, M. A.; Riess, G.; Croucher M. D. Langmuir 1990, 6, 514–516.
37 Nivaggioli, T.; Alexandridis, P.; Hatton, T. A.; Yekta, A.; Winnik M. A. Langmuir 1995, 11, 730–737.
38 Miguel, M. G. Adv. Colloid Interface Sci. 2001, 89–90, 1–23.
39 Hoogenboom, R.; Fijten, M. W. M.; Thjis, H. M. L.; Van Lankvelt, B. M.; Schubert, U. S. Des. Monom. Polym. 2005, 8,
659–671.
40 Hoogenboom, R.; Wiesbrock, F.; Huang, H.; Leenen, M. A.
M.; Thijs, H. M. L.; Van Nispen, S. F. G.; Van der Loop, M.; Fustin, C.-A.; Jonas, A. M.; Gohy, J.-F.; Schubert, U. S. Macromolecules 2006, 39, 4719–4725.
41 Bonne, T. B.; Ludtke, K.; Jordan, R.; Papadakis, C. M. Macromol. Chem. Phys. 2007, 208, 1402–1408.
42 Hadjichristidis, N.; Pispas, S.; Floudas, S. Block copolymers:
Synthetic strategies, physical properties and applications, J
Wiley & Sons, Hoboken, 2003.
43 Trzebicka, B.; Koseva, N.; Mitova, V.; Dworak, A. Polymer
51, 2010, 2486–2493.
JOURNAL OF
POLYMER SCIENCE
44 Luxenhofer, R.; Schulz, A.; Roques, C.; Li, S.; Bronich, T. K.;
Batrakova, E. V.; Jordan, R.; Kabanov, A. V. Biomaterials 2010,
31, 4972–4979.
45 Deyerle, B. A.; Zhang, Y. Langmuir 2011, 27, 9203–9210.
46 Patel, K.; Bahadur, P.; Guo, C.; Ma, J. H.; Liu, H. Z.; Yamashita, Y.; Khanal, A.; Nakashima, K. Eur. Polym. J. 2007, 43,
1699–1708.
ARTICLE
49 Kempe, H.; Kempe M.; Anal. Bioanal. Chem. 2010, 396,
1599–1606.
50 Wielema, T. A.; Engberts, J. B. F. N. Fur. Polym. J. 1990, 26,
639–642.
51 Shin, I. G.; Kim, S. Y.; Lee, Y. M.; Cho, C. S.; Sung, Y. K. J.
Controlled Release 1998, 51, 1–11.
52 Kim, S. Y.; Shin, I. G.; Lee, Y. M.; Cho, C. S.; Sung, Y. K. J.
Controlled Release 1998, 51, 13–22.
47 Patel, K.; Bharatiya, B.; Kadam, Y.; Bahadur, P. J. Surfact
Deterg. 2010, 13, 89–95.
53 Giacomelli, C.; Schmidt, V.; Borsali, R. Langmuir 2007, 23,
6947–6955.
48 Soo, P. L.; Luo, L.; Maysinger, D.; Eisenberg, A. Langmuir
2002, 18, 9996–10004.
54 Zhang, J.; Li, S.; Li, X.; Li, X.; Zhu, K. Polymer 2009, 50,
1778–1789.
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