new thermo-sensitive graft copolymers based on a poly(n-isopropylacrylamide) backbone and functional...
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New Thermo-Sensitive Graft Copolymers Basedon a Poly(N-isopropylacrylamide) Backboneand Functional Polyoxazoline Grafts withRandom and Diblock Structure
Juan Carlos Rueda, Stefan Zschoche, Hartmut Komber, Franziska Krahl,Karl-Friedrich Arndt, Brigitte Voit*
New thermo-sensitive functionalized graft copolymers characterized by a poly(N-isopropyl-acrylamide) backbone and grafts containing 2-ethyl-2-oxazoline and 2-(2-methoxycarbonyl-ethyl)-2-oxazoline units were synthesized. The conformation transition temperatures of thegraft copolymers could be modified by variation of themolar composition in the side chain, by different sidechain structure (random distribution of both oxazo-lines vs. diblock structure) and by hydrolysis of themethylester to the acid form. Graft copolymers withlong functional oxazoline side chains allowed thestabilization of aggregates above the phase transitiontemperature of the backbone until the LCST of the sidechain. The temperature window allowing for the for-mation of stable aggregates was widened with acidfunctions in the corona.
Introduction
Temperature-responsive properties of copolymers can be
connectedwith the reversible change of solution properties
B. Voit, S. Zschoche, H. KomberLeibniz Institute of Polymer Research Dresden, Hohe Straße 6,D-01069 Dresden, GermanyFax: (þ49) 351 4658565; E-mail: [email protected]. C. RuedaLaboratorio de Polımeros, Seccion Fısica, DAI, PontificiaUniversidad Catolica del Peru, Lima, PeruF. Krahl, K.-F. ArndtPhysical Chemistry of Polymers, Dresden University ofTechnology, D-01062 Dresden, Germany
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from hydrophilic to amphiphilic depending on tempera-
ture. An interesting property of such amphiphilic water-
soluble copolymers is their potential to form micelles,
lamellar aggregates, vesicles, and hydrogels by self-
assembly as reviewed recently by Dimitrov et al.[1] It was
outlined that a controlled synthesis of well-defined
copolymer structures is one possibility to have control
over the phase behavior of their aqueous solutions.
Thermo-responsive polymeric micelles based on seg-
mented structures arewell-documented.[1–5] Inmany cases
the hydrophobic part of such micelles is formed by poly(N-
isopropylacrylamide) (polyNIPAAm) sequences above their
DOI: 10.1002/macp.200900437
New Thermo-Sensitive Graft Copolymers Based on a Poly(N-isopropylacrylamide) . . .
phase transition temperature.[4,6,7] The property profile of
such micelles can be extended when a second responsive
structure is introduced, i.e., when double-responsive
copolymers form the micelles.[1] Thus, the combination
of temperature-responsive polyNIPAAm and pH-respon-
sive poly(acrylic acid) (PAA)[8] or poly(N-acryloylpyrroli-
dine)[1,9] was reported. In dependence on the applied
temperature and pH value the polyNIPAAm-b-PAA copo-
lymers change their hydrophobic andhydrophilic parts and
form different types of micelles.[8] Temperature-induced
phase transitionover awide range of pHvalue could also be
demonstrated for randompolyNIPAAm-co-PAAand PAA-g-
polyNIPAAm structures by Chen and Hoffman.[10]
Thermosensitivity is also known for several poly(2-alkyl-
2-oxazoline)s and their copolymers.[5,11] Thus, a lowering of
the LCST range of poly(2-isopropyl-2-oxazoline) to 8–46 8Ccould be achieved by copolymerization with more hydro-
phobic 2-alkyl-2-oxazolines.[11] By contrast, Park and
Kataoka[5] controlled precisely the LCST behavior of
poly(2-isopropyl-2-oxazoline) via gradient living cationic
copolymerization with 2-ethyl-2-oxazoline (EtOxa) as a
hydrophilic comonomer in the range of 38–68 8C. The
hydrophilic character of polyEtOxa is utilized in amphi-
philic polymeric micelles where the hydrophilic part of the
copolymer is poly(e-caprolactone)[12] or poly(L-lactide).[13]
Such micelles were used for application in drug delivery.
Recently[14,15] we reported on the synthesis and char-
acterization of new graft copolymers based on a poly-
NIPAAmmain chain and poly(2-methyl-2-oxazoline) (poly-
MeOxa) or polyEtOxa grafts with varying number and
length of the graft arms synthesized through the ‘‘grafting
from’’method. For this, the ‘‘living’’ cationicpolymerization
of the MeOxa or EtOxa was initiated through a statistic
copolymer of NIPAAm and chloromethylstyrene
(CMS).[14,16] The involved temperature dependent studies
on these polymers were focused on the effect of the
different structureson theTtr of thepolyNIPAAmbackbone.
Nevertheless, a double-temperature-sensitive behavior is
observed for copolymers with polyEtOxa grafts. Micelles
were formedwhereas the amphiphilic character is realized
by thehydrophobic polyNIPAAmbackbone aboveTtr on the
one side and hydrophilic poly(2-oxazoline) grafts on the
other. At a balanced relation of both components the graft
copolymers forms stable aggregates over a wide tempera-
ture range.[14,15]
For extending this approach, now additional COOH
groups had been incorporated in the polyEtOxa graft arms.
Basedonthe livingcharacterofoxazolinepolymerization in
a first step side chains based on random and diblock
copolymers of EtOxa and 2-(2-methoxycarbonylethyl)-2-
oxazoline (MEtOxa) were synthesized with varying como-
nomer content. The latter comonomer is to introduce
carboxylic acid functionalities after mild hydrolysis of
themethylester groups. The aimof this study is to elucidate
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the influence of thesemore hydrophilic groups on the LCST
behavior with focus on the formation of stable aggregates
in the physiological range and to achieve a wide
temperature window in which stable micelles are formed.
In addition, the free carboxylic groups might allow
chemical modifications, i.e., covalent or ionic bonds to
biomaterials and other active substances, or complex
formation. Thus, the switching between dissolved state
and aggregated state will not only be related to release or
inclusion of chemically non-bonded substances but also to
accessibility or screening of chemically bonded agents.
Certainly, micelles or soft nanoparticles based on such
scaffold should be useful for various practical applications
such as supports for catalysts, sensors, separation systems,
enzymatic bioconjugates, and drug carriers.[2–5] Similar
objectives were intended, i.e., by Nuyken et al.[17] who
modified carboxylic groups in 2-alkyl-2-oxazoline based
diblock copolymers with catalytical active sides for
homogeneous catalysis in water or by Kim and Healy[18]
who prepared hydrogels composed of NIPAAm and acrylic
acid by redox polymerization with peptide cross-linkers.
They intended to create an artificial extracellular matrix
with the COO� groups supposed to stabilize the gel in cell-
culture media.
This paper presents the synthesis and structural
characterization of graft copolymers containing in the
main chain long segments of polyNIPAAm and in the side
chains polyoxazolines functionalized with carboxylic acid
groups. These graft copolymers were characterized with
respect to their temperature-responsive properties by UV-
vis spectra, turbidity measurements and 1H NMR spectro-
scopy and the structural influences were discussed.
Experimental Part
Materials
NIPAAm (Aldrich) was purified by recrystallization from ethanol
and dried in vacuum. CMS is a mixture of isomers (30% meta and
70%para) andwasdistilled twicebeforeuse. 2,20-Azoisobutyronitrile
(AIBN, Aldrich) was recrystallized twice from methanol. EtOxa
(Aldrich)wasdistilled twice fromcalciumhydrideandstoredunder
dry nitrogen atmosphere. Potassium iodide (Aldrich) was used as
received.All theother substanceswerepurchased fromAldrichand
purified according to standard procedures described in the
literature.
Synthesis of 2-(2-Methoxycarbonylethyl)-2-oxazoline
(MEtOxa)
MEtOxa was synthesized according to the procedure reported by
Nuyken and coworkers[17] which is a modification of the method
proposed by Levy and Litt.[19] 2-Chloroethylamine hydrochloride
was treated with methyl succinate chloride and triethylamine in
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J. C. Rueda, S. Zschoche, H. Komber, F. Krahl, K.-F. Arndt, B. Voit
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dichloromethaneto formtheamideas intermediate.After isolation
it was cyclizised in vacuum with anhydrous sodium carbonate to
produce finally the 2-oxazoline derivative MEtOxa. MEtOxa was
purifiedbyvacuumdistillation. ThepurityandstructureofMEtOxa
was confirmed by NMR.1H NMR (DMSO-d6): d¼2.44 (t, 2H, CH2C(N¼)O), 2.59 (t, 2H,
CH2C(O)O), 3.59 (s, 3H,OCH3), 3.67 (t, 2H, CH2N¼), 4.17 (t, 2H, CH2O).13C NMR (DMSO-d6): d¼ 22.51 (CH2C(N¼)O), 29.58 (CH2C(O)O),
51.36 (OCH3), 53.89 (CH2N¼), 67.04 (CH2O), 165.95 (C(N¼)O), 172.34
(C(O)O).
Synthesis of the Random Copolymer of N-Isopropylacrylamide and Chloromethylstyrene (MI)
The copolymer used asmacroinitiator (MI)was synthesized by free
radical polymerization of NIPAAm and CMS initiated by AIBN in
dioxane at 68 8C as described in a previous publication.[14] The CMS
content was 2.4mol-% as determined by 1H NMR.1H NMR (CDCl3): d¼ 1.1 (CH3, NIPAAm), 1.2–2.5 (CH, CH2,
backbone), 3.7 - 4.1 (CH, NIPAAm), 4.5 (CH2Cl, CMS), 5.5–6.6 (NH,
NIPAAm), 6.6–7.5 (HAr, CMS).13C NMR (CDCl3): d¼22.5 (CH3, NIPAAm), 30–45 (CH, CH2,
backbone), 41.1 (CH, NIPAAm), 45.9 (CH2Cl, p-CMS), 46.2 (CH2Cl,m-
CMS),125–130(CHAr,CMS),135.4 (p-C,p-CMS),137.5 (m-C,m-CMS),
144.7 (ipso-C, CMS), 173.9 (C¼O, NIPAAm).
Synthesis of the Graft Copolymers with Random
(GCR) and Diblock Side Chains (GCB)
The graft copolymers were synthesized by polymerization of the
2-oxazoline monomers EtOxa and MEtOxa initiated by the benzyl
chloride functional groups of the MI. The formation of polyoxazo-
line side chainswith randomdistribution of bothmonomers (GCR)
is expected when both monomers were reacted simultaneously
(monomer mixture) whereas a diblock structure (GCB) is expected
by sequential addition of both monomers.
A typical procedure was the following: In a 100mL reaction
vessel 1.0 g ofMI and 0.140g of potassium iodidewere dissolved in
24mL of benzonitrile under dry nitrogen atmosphere. Then a
mixture of 3.34 g of EtOxa and 0.278 g of MEtOxa was added. The
reactionvesselwasheatedwithagitationat120 8Cfor8 hunderdrynitrogen atmosphere. After this time, the reaction mixture was
cooled to 25 8C and 2mL of a solution of 0.5 g of KOH in 10mL of
methanolwas added to the reactionmixture. After 6 h the polymer
was precipitated in diethyl ether, redissolved in chloroform,
decanted, filtrated, and then precipitated again in diethyl ether.
The final polymer was dried until reaching constant weight. The
graft polymer was characterized by NMR spectroscopy.
Example: GCR-31H NMR (CD3OD): d¼ 1.1 (H3), 1.16 (CH3, NIPAAm), 1.5–1.9 (CH2,
NIPAAm backbone), 1.9–2.3 (CH, NIPAAm backbone), 2.3–2.5 (H2),
2.64 (H6), 2.70 (H5), 3.4–3.7 (NCH2), 3.66 (H8), 4.0 (CH, NIPAAm).13C NMR (CD3OD): d¼9.9 (C3), 22.8 (CH3, NIPAAm), 26.85 (C2),
28.7 (C5), 29.8 (C6), 34–40 (CH2, NIPAAm backbone), 42.4 (CH,
NIPAAm), 43.2 (CH, NIPAAm backbone), 44–48 (NCH2), 52.2 (C8),
174.1 (C4), 174.9 (C7), 175.9 (C¼O, NIPAAm), 176–177.5 (C1). Signals
of the reacted CMS moiety could not be observed due to their low
concentration.
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The synthesis of the graft copolymers with block side chains
(GCB-1; -2) followed the same procedure but the EtOxa monomer
wasfirst polymerizedat 120 8C for8 h. Thena solutionofMEtOxa in
10mL benzonitrile was added and the polymerization was
continued at 120 8C for 4 h.
Hydrolysis of the Methoxy Groups of the Graft
Copolymers Resulting in the Acid Forms GCR-x-A andGCB-x-A
To introduce carboxylic acid functional groups themethoxygroups
of theMEtOxa units were removed by basic hydrolysis under mild
conditions and the resulting salt was neutralized with HCl to
produce finally the acid group containing graft copolymers GCR-x-
A and GCB-x-A.
A typicalprocedure is the following: 1.0 gofGCR-3wasdissolved
in 15mLof 0.1 N aqueous sodiumhydroxide and25mL ofmethanol
which was added to keep the polymer in solution. The mixture
was heated at 60 8C for 6 h under agitation. Then the mixture
was cooled to 25 8C and 15mL of 0.1 N HCl and 25mL of methanol
were added until the pH-value was approximately 5. Methanol
and water were removed with a rotary evaporator as well as by
freeze drying. The final product was dissolved in chloroform and
after 24h the precipitated sodium chloride was filtrated off. The
filtrate was evaporated and the resulting polymer was dried
until reaching constant weight. The polymer was characterized
by NMR.
Example: GCR-3-A1H NMR (CD3OD): d¼ 1.1 (H3), 1.16 (CH3, NIPAAm), 1.5–1.9 (CH2,
NIPAAm backbone), 1.9–2.3 (CH, NIPAAm backbone), 2.3–2.5 (H2),
2.55–2.75 (H50; H60), 3.4–3.7 (NCH2), 3.66 (H8), 4.0 (CH, NIPAAm).13C NMR (CD3OD): d¼9.9 (C3), 22.8 (CH3, NIPAAm), 26.85 (C2),
28.8 (C50), 30.1 (C60), 34–40 (CH2, NIPAAm backbone), 42.4 (CH,
NIPAAm), 43.2 (CH, NIPAAm backbone), 44–48 (NCH2), 174.5 (C70),
175.5–177.5 (C1; C70; C¼O, NIPAAm). Signals of the reacted CMS
moiety could not be observed due to their low concentration.
Analytical Measurements
500.13MHz 1H NMR and 125.74MHz 13C NMR spectra were
recorded on a DRX 500NMR spectrometer (Bruker) at 303K.
Deuteratedmethanol (CD3OD, d(1H)¼3.31ppm; d(13C)¼49.0 ppm)
or D2Owas used as a solvent. The spectra recorded inD2O solutions
werereferencedontheinternal standardsodium3-(trimethylsilyl)-
propionate-d4 (d(1H)¼0ppm; d(13C)¼ 0ppm). For temperature-
dependent 1H NMR measurements, the temperature was con-
trolled by theBruker variable temperature accessory BVT-3000and
was calibrated using the standardWilmad ethylene glycol sample.
Size exclusion chromatography (SEC) measurements were
carried out on an Agilent system equipped with a 510pump,
detector UV-486, detector RI-410, and PL gel 10mm mixed-B LS
column. Chloroformwas used as elution solventwith a flow rate of
1mL �min�1. Poly(vinylpyridine) standards (PSS Mainz, Germany)
were used for calibration.
UV-vis spectra turbidity measurements were carried out on a
Varian Cary 100 as it was described in the literature.[14,20] The
polymers were measured as 1wt.-% solution in bi-distilled water.
The solutions were filtered before placing them in the measuring
DOI: 10.1002/macp.200900437
New Thermo-Sensitive Graft Copolymers Based on a Poly(N-isopropylacrylamide) . . .
cell. Each single measurement was detected after 3–5min
equilibrium of temperature. The transmittance at 650nm was
evaluated. The phase transition temperature Ttr of the polymer
was determined as the inflection point of the transmittance versus
temperature curve.
To determine the Ttr behavior by temperature-dependent1H NMR measurements in D2O the signal intensities of the
NIPAAm and oxazoline units, respectively, were followed accord-
ing to our previous report.[14]
Dynamic Light Scattering measurements were carried out on
commercial laser light scattering spectrometer (ALV/DLS/SLS-
5000) equipped with an ALV-5000/EPP multiple digital time
correlator and laser goniometer system ALV/CGS-8F S/N 025. A
helium–neon laser (Uniphase 1145P, output power of 22mW and
wavelength of 632.8nm) was used as the light source.
Samples were prepared by dissolving the graft copolymers
in Millipore water. The concentration
was adjusted to 0.3 g � L�1. Prior to the
measurements the solutions were fil-
tered using 0.22mm CME membrane
filters (Rotilabo, Carl Roth, Germany).
Typically, the sample was immersed in
a test tube (diameter 10mm, 3mL
sample solution) and thermostated
within an error of �0.1 8C in a toluene
bath.
At every temperature the intensity–
intensity time correlation functions
g(2)(t,q) were measured angular depen-
dent from 30 to 1008 in steps of 108.g(2)(t,q) is related to thenormalizedfirst-
order electric field time correlation
function g(1)(t,q) as[21]
Macrom
� 2010
gð2Þðt;qÞ ¼ Ið0;qÞIðt;qÞh i
¼ A½1þ bjgð1Þðt;qÞj2� (1)
where A is the measured base line, b is
a parameter depending on the coher-
ence of the detection, t is the delay
time, and q is the scattering vector
(q¼ (4pn/l)sin(u/2), with n, l and u
being the refractive index of the
medium, the wavelength of the inci-
dent beam in vacuum, and the scatter-
ing angle, respectively). For a
polydisperse sample, g(1)(t,q) is related
to the line-width distribution G(G) by
gð1Þðt;qÞ ¼ Eðt;qÞE�ð0;qÞh i
¼Z1
0
GðGÞe�GtdG (2)
Scheme 1. Synthesis routes to the macroinitiator (MI) and the graft copolymers (GC) withpoly(2-oxazolines) grafts containing MEtOxa in a random copolymer with EtOxa (GCR-x) or in adiblock copolymer (GCB-x). The methyl ester of the incorporated MEtOxa units is reacted to theacid form (-A) by alkaline hydrolysis under mild conditions followed by acidification.
Using the Laplace inversion program
CONTIN G(G) was calculated from
g(2)(t,q) on the basis of Equation (1)
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WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and(2). IndilutesolutionsG is relatedtoG¼D/q2 forapurediffusive
relaxation where D is the translational diffusion coefficient. D can
be converted into the hydrodynamic radius Rh using the Stokes–
Einstein equation: D¼ kB T/6phRh, where kB, T, and h are the
Boltzmann constant, the absolute temperature and the solvent
viscosity respectively.
Results and Discussion
Polymer Synthesis
The MI is a random copolymer of CMS and NIPAAm
(Scheme 1) synthesized as described in our previous
publication.[14] The CMS content of 2.4mol-% in the
copolymer was determined by 1H NMR which is in good
agreementwith themolar composition of the reaction feed
www.mcp-journal.de 709
J. C. Rueda, S. Zschoche, H. Komber, F. Krahl, K.-F. Arndt, B. Voit
Table 1. Synthesis of graft copolymers based on poly(NIPAAm-co-CMS) as MI and 2-alkyl-2-oxazolines (EtOxa and MEtOxa) randomlydistributed in the side chains: experimental details and results (solvent: benzonitrile; T¼ 120 8C; nitrogen atmosphere; [KI]/[CMC]¼4).
Graft copolymer [PP
Oxa]/[CMS]a) Content of
MEtOxa
Yieldb) Monomer units in the side chainc) Content of
MEtOxac)
mol �mol�1 mol-% % mol-%
GCR-1 164 2.7 78 150 2.5
GCR-2 168 5 87 150 4.7
GCR-3 175 10 90 157 9.3
a)Molar ratio of oxazoline monomers and initiating CMS groups in the copolymerization feed; b)Yield of the graft copolymerization;c)Averaged number of monomer units was determined by 1H NMR from the intensity of the CH(CH3)2 proton signal of the MI taking into
account the CMS content of 2.4mol-% in the MI and the signal intensities of C(O)CH2 protons of EtOxa and C(O)CH2CH2C(O) protons of
MEtOxa units in the side chain. The latter were used to calculate the MEtOxa content. Estimated relative error for both values:�10%.
710
(2.5mol-% CMS). SEC verified a monomodal molecular
weight distribution with Mn ¼ 43 500 g �mol�1,
Mw ¼ 126000 g �mol�1, and a polydispersity index of 2.9.
ThegraftcopolymerspolyNIPAAm-co-CMS-graft-2-oxazo-
line were synthesized by the ‘‘grafting from’’ method
through ring-opening cationic polymerization of the EtOxa
and theMEtOxamonomers initiated by the aforementioned
MI (Scheme1)andwithpotassiumiodideasanactivator.The
potassium iodide induces an interchange between chlorine
and iodine resulting in the in situ formation of benzyl iodide
groups which were more efficient initiators than benzyl
chloride groups.[14,16] Using the ‘‘living’’ character of this
2-oxazoline polymerization two types of graft copolymers
were synthesized with the aforementioned monomers. The
first typecontainsa randommixtureof themonomersEtOxa
andMEtOxa in the side chains (GCR) and thesecondcontains
in the side chains a first block of polyEtOxa and a second
blockofpolyMEtOxa (GCB) (Scheme1). Thegrafts arebonded
to a backbone with properties determined by long poly-
NIPAAm sequences.
The ‘‘living’’ character of the polymerization of 2-
oxazolines allows further adjusting the total polymeriza-
Table 2. Synthesis of graft copolymers based on poly(NIPAAm-co-CMCMEtOxa in the second block: experimental details and results (solve
Graft
copolymer
[EtOxa]/[CMS]a) [MEtOxa]/[CMS]a) Yie
mol �mol�1 mol �mol�1 %
GCB-1 150.5 8 8
GCB-2 144 18 8
a)Molar ratio of EtOxa andMEtOxamonomer, respectively, and initiati
of the first block and second block, respectively; b)Yield of the graft co
Table 1.
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tiondegreeof thesidechainsandthepolymerizationdegree
of each block in the case of the sequential polymerization of
themonomersEtOxaandMEtOxa. Table 1and2 summarize
the experimental details and the obtained results. The yield
of the graft copolymers varied from 78 to 90%.
TheNMRspectraconfirmtheoverall structureof thegraft
copolymers. Besides the characteristic signals of NIPAAm
units of the MI backbone, signals of both monomers
incorporated in the polyoxazoline grafts were observed
proving the copolymerization of the monomers EtOxa and
MEtOxa by the MI (Figure 1). The content of incorporated
MEtOxa units as well as the averaged number of oxazoline
units in the side chains were determined from 1H NMR
signal intensities (comp. Table 1). The MEtOxa content
covers 2.5–9.3mol-%whereas the polymerization degree of
the side chains was approximately 150 monomeric units
(EtOxaþMEtOxa) for all the synthesized copolymers.
As stated for similar graft copolymers[14] the SEC
characterization of these polymers is problematic because
of their amphiphilic characterwhich can result in enthalpic
interactions with the column material and also the
formation of aggregates cannot be ruled out. As alternative,
) as MI with diblock side chains containing EtOxa in the first block andnt: benzonitrile; T¼ 120 8C; nitrogen atmosphere; [KI]/[CMC]¼4).
ldb) Monomer units
in the side chainc)
Content
of MEtOxac)
Total
(nRm)
EtOxa
block (n)MEtOxa
block (m)
mol-%
3 149 145 4 2.7
8 148 135 13 8.8
ng CMS groups in the graft copolymerization feed for the synthesis
polymerization; c)Determined by 1H NMR, compare footnote c in
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Figure 1. 13C NMR spectra of a graft copolymer with randomdistribution of 8.7 mol-% of MEtOxa units in the polyoxazolinegraft arm in the ester form (GCR-3, bottom) and in the acid formafter hydrolysis of the methylester groups (GCR-3-A, top). Solvent:CD3OD. The numbering corresponds with Scheme 1, for assign-ments of the MI signals see Experimental Part.
number averaged molecular weights were estimated
(Table 3) based on Mn of the MI (43 500 g �mol�1) as
obtained from SEC, full conversion of the 2.4mol-% CMS
units as initiator, the number averaged length of the grafts
and the graft composition as determined by 1H NMR and
given in Table 1 and 2. The polymers are quite uniform in
their calculated molecular weights covering the region of
about 180 000–192000 g �mol�1.
Both types of graft copolymers containing side chains of
the randomtypeandwithdiblock sidechainswereobjected
to basic hydrolysis to remove the methylester groups
contained in the monomer MEtOxa. The sodium salt form
was finally converted in the acid (CEtOxa) form adjusting
weak acidic conditions with HCl. In this way, from the
copolymers GCR-x (x¼ 1–3) and GCB-y (y¼ 1, 2) the
corresponding copolymers GCR-x-A and GCB-y-A bearing
carboxylic acid groups were obtained. The completeness of
Table 3. Molecular characteristics of the graft copolymers.
Graft
copolymer
Composite
side chain
(EtOxa/CEtOxa)
Mna) single
side chain
g �mol�1
GC-0 150/– 14 850
GCR-1 146/4 15 026
GCR-2 143/7 15 158
GCR-3 142/15 16 203
GCB-1 145-b-4 14 927
GCB-2 135-b-13 15 224
a)Calculated by (number of EtOxa units�MEtOxa)þ (number of CEtO
grafts�Mn single side chain); GC-0: Mn (MI)¼31000 g �mol�1 with �grafts.
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hydrolysis under reaction conditions could be proved by
disappearance of the methylester signal at 3.66 pm in the1H NMR spectra and at 52.2 ppm in the 13C NMR spectra
(Figure 1). Hydrolysis of amide groups resulting in
�CH2�NH�CH2� moieties would result in methylene
signals at about 2.8 ppm in CD3OD. However, there is no
hint that such a hydrolysis occurred as side reaction.
Phase Transition Temperatures
For the comparative study of the temperature-dependent
conformational behavior the transmittance at 650nmwas
evaluated for 1wt.-% polymer solutions to determine the
conformational transition temperature Ttr. The MI used in
this study showed a Ttr of 29 8C (Figure 2) which is lower
than that of the polyNIPAAm homopolymer at 32 8C. Thevalue is inaccordancewithdatadetermined inourprevious
study for polyNIPAAm-co-CMSs with different CMS con-
tent.[14] It couldbe shownthatwith increasingCMScontent
Ttr decreases probably due to the incorporation of the
hydrophobic comonomer. By contrast, an increase of Ttrwith respect to the MI was expected for the graft
copolymers bearing more hydrophilic segments in the
polyoxazoline grafts and, after hydrolysis of the MEtOxa
units, additionally carboxylic acid groups.
Our previous study[14] showed that Ttr of the polyNI-
PAAm main chain changed from 29 to 40 8C depending on
number and length of polyEtOxa side chains. The behavior
at higher temperatures was not investigated at that time,
but it was assumed that above Ttr of the backbone the
macromolecules are amphiphilic andmight be able to form
stable micelles.[14,15] Actually, only when the hydrophilic
polyEtOxa side chains are long enough they sufficiently
stabilize the aggregates. At even higher temperatures one
NIPAAm/PP
Oxa Mnb) graft
copolymer
Content
CEtOxa
mol �mol�1 g �mol�1 wt.-%
0.227 146000 –
0.271 181000 2.9
0.271 182000 5.0
0.259 192000 10.2
0.273 180000 2.9
0.275 183000 9.3
xa units�MCEtOxa);b)Calculated by Mn (MI)þ (number of POxa
7.75 grafts; GCR and GCB: Mn (MI)¼43 500g �mol�1 with �9.15
www.mcp-journal.de 711
J. C. Rueda, S. Zschoche, H. Komber, F. Krahl, K.-F. Arndt, B. Voit
Figure 2. Transmittance versus temperature curves for 1 wt.-%solutions in water of the MI, a random graft copolymer with4.7 mol-% MEtOxa before (GCR-2) and after hydrolysis (GCR-2-A)and a polyNIPAAm-g-polyEtOxa copolymer with comparabledegree of grafting and side chain length (GC-0).
Table 4. Phase transition temperatures determined by turbiditymeasurements and hydrodynamic radius of the aggregates ofselected samples at 42 8C (as determined by DLS).
Sample Ttr1a) Ttr2a) Rh
-C -C nm
MI 27.5 –
GC-0 34 58
GCR-2 34.5 54.5 55
GCR-1-A 38 69.5
GCR-2-A 38.5 67.5 90
GCR-3-A 37 57.5
GCB-1 33 48 >200
GCB-1-A 36.5 67 55
GCB-2-A 36 –
a)Approximated as the middle of the corresponding transition
regions in the turbidity curves.
712
has to take into account that also polyEtOxa shows LCST
behavior.[5,11,14,22] The Ttr of PEtOxa is strongly depending
on the molecular weight and the weight fraction of water
solution and was observed in the range of 62–78 8C.[22]
Therefore, a double thermo-responsive behavior of the
polyNIPAAm-g-polyEtOxa polymers and their derivatives
studied in this work is expected. In fact, light scattering
studies indicate thatmicelles are no longer stable and form
larger aggregates or precipitate above a temperature of
about 60 8C which is attributed to the LCST behavior of the
polyoxazoline side chains.[15]
This double thermo-responsive behaviorwas considered
when the number and length of the polyoxazoline side
chains used in this study were selected. In the previous
work it was found that two well separated and sharp
conformational transitions at 33 and 58 8C can be observed
for a polyNIPAAm-g-polyEtOxa (GC-0) with 2.9mol-% CMS
in the polyNIPAAm backbone as initiator and about
150 EtOxa units in the side chain (Figure 2; Table 4).[15]
Here, copolymerswith a slightly lower content of initiating
units (2.4mol-% CMS) but also 150units in the side chains
were synthesized. Within these side chains the content of
MEtOxa and CEtOxa units, respectively, as well as the
architecture of the chains (random vs. block) was varied
with the aim to study the influence of these changes on the
Ttrs.
As a first result one can assert that all samples show two
transitions in the turbidity measurements (Figure 2–4).
Temperature-dependent 1H NMR measurements were
carried out on sample GCB-1-A to correlate the two
temperature regions with significant changes of transmit-
tance with the thermal behavior of substructures of the
graft copolymers. Figure 5 depicts the 1H NMR spectra
recordedbetween25and80 8C in5Ksteps. It is obvious that
the first decrease in transmittance is caused by the LCST
Macromol. Chem. Phys. 2010, 211, 706–716
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
behavior of the polyNIPAAm backbone. Those signals
diminish and disappear finally between 35 and 40 8Cdue to
the solid-like structure of the collapsed backbone. The
polyoxazoline signals remain more or less unchanged in
shape and intensity up to about 60 8C. A slight broadening
of the signals is observed up to this temperature also
resulting in the seemingly significant changes for the
methyl signals. However, between 60 and 65 8C a slight but
significant low-field shift of all signals was observed
followed by line broadening with increasing temperature.
Similar effects were observed in the 1H NMR spectra of a
polyEtOxa homopolymer sample with a molecular weight
comparable with that of the polyoxazoline grafts which
was studied for comparison. A visual inspection of the
sample tubeat75 8Cshoweddropletsat thewall.Obviously,
the decrease in transmittance between 60 and 70 8C for
GCB-1-A (Figure 3) is caused by the LCST behavior of the
polyoxazoline grafts resulting in phase separation. Increas-
ing polyoxazoline concentration in the droplets and
different interactions compared with the initial solution
can explain the abrupt rise of the chemical shifts and the
increasing line width by changes in mobility. With respect
to solutionNMR, there is not a complete immobilization for
the polyoxazoline graft arms as observed for the poly-
NIPAAm backbone but still sufficient mobility.
Figure 2 compares the turbidity measurements for the
parent MI and three graft polymers with polyalkyloxazo-
line side chains of about 150 units length; whereas GC-0
contains only EtOxa units, about 4.7mol-% (7 units) are
randomly replaced byMEtOxa and CEtOxa, respectively, in
GCR-2 and GCR-2-A. The measurements on the graft
DOI: 10.1002/macp.200900437
New Thermo-Sensitive Graft Copolymers Based on a Poly(N-isopropylacrylamide) . . .
Figure 3. Transmittance versus temperature curves for 1 wt.-%solutions in water of the MI and different copolymers withpoly(EtOxa-random-CEtOxa) grafts with different CEtOxa con-tent: 2.5 mol-% (GCR-1-A), 4.7 mol-% (GCR-2-A), and 9.3 mol-%(GCR-3-A).
Figure 4. Transmittance versus temperature curves for 1 wt.-%solutions in water of the MI and two copolymers with polyEtOxa-b-polyMEtOxa grafts before (GCB-1/-2) and after hydrolysis (GCB-1-A/-2-A) with different length of the two blocks 145-b-4 (-1) and135-b-13 (-2).
Figure 5. 1H NMR spectra of GCB-1-A (solvent: D2O) obtained atdifferent temperatures. The filled signals are due to polyNIPAAm.The signals of the polyEtOxa-b-polyCEtOxa side chain areassigned.
copolymers clearly show a two-step process, i.e., a double
temperature sensitive systemhas to be discussed.Whereas
theonsetof thefirstprocess isquitesharp forall samples the
following curve shapes are characterized by less sharp
curvatures. Therefore, the determination of transition
temperatures from the middle of transition regions was
favored over the first derivative. The values given in Table 4
should allow to reveal trends caused by the structural
changes.
The first transition temperature is polyNIPAAm-based
and less influencedby incorporationofMEtOxaunitsbutan
increase is obvious which is more pronounced after
hydrolyzing the methylester to the acid (GCR-2-A). This is
in accordancewith the known effect of hydrophilic units in
the side chain on the LCST behavior of the polyNIPAAm
backbone. It is accompanied with changing from a sharp
transition temperature to a broader temperature region.
Macromol. Chem. Phys. 2010, 211, 706–716
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The temperature region increases with increasing content
of randomly distributed CEtOxa units in the side chain
(Figure 3). One can argue that with increasing content of
carboxylic groups in the side chain also the probability
increases that such carboxylic groups are nearby the
polyNIPAAm backbone. They could interfere with the
repelling of water in the hydrophilic–hydrophobic LCST
transition of polyNIPAAm and so result in a broader
temperature region for this process.
When the CEtOxa units are located as block at the end of
the side chains (GCB-1, -2; Figure) the first transition is
steeper, Ttr1 slightly lower and the temperature region
narrower as for GCR samples with the same content of
CEtOxa units. This seems to confirm the ‘‘softening’’ effect
of carboxylic groups nearby the polymer backbone because
the hydrophobic ends of the side chains should have no or
less interaction with the polymer backbone.
The second transition is related to the LCST behavior of
the polyoxazoline side chains and results for nearly all
samples to complete loss of transmittance. The number-
averaged molecular weight of all side chains is about
15 000 g �mol�1. From Figure 2 it can be seen that replacing
EtOxa units randomly by MEtOxa units results in a lower
Ttr2 for this transition. Such a behavior is characteristic for
increasing hydrophobicity of the polyoxazoline back-
bone.[5,11,22–24] If one compares a ethyl moiety with a
methoxycarbonylethyl moiety it is difficult to predict
which is the more hydrophobic one. Definitely, the
hydrophilicity of the polyoxazoline side chains increases
after hydrolysis of the methylester to the carboxylic acid.
The general trend is that CEtOxa units within the
polyoxazoline side chains increase Ttr2. However, a clear
dependence on the content of CEtOxa is not obvious
www.mcp-journal.de 713
J. C. Rueda, S. Zschoche, H. Komber, F. Krahl, K.-F. Arndt, B. Voit
Figure 6. Hydrodynamic radius versus temperature of samplesbefore (GCR-2 and GCB-1) and after hydrolysis (GCR-2-A and GCB-1-A).
714
(Figure 3; Table 4).Whereas a low content (2.5mol-%) shifts
theTtr2valuesignificantlybyabout10KcomparedwithGC-
0, a further increase up to 4.7mol-% seems to result in the
same or a slightly lower Ttr2 as for the 2.5mol-%.
Surprisingly, a further increase of the CEtOxa content to
9.3mol-% (GCR-3-A) results nearly in the same low Ttr2 as
GC-0 without CEtOxa units. Furthermore, there is a low
turbidity at low temperature (transmittance� 90%). This
behavior is not understood. It is known that polyNIPAAm
forms compact hydrogen-bonding inter-polymer com-
plexes with PAA.[10,25] Perhaps, we have a similar interac-
tion between the polyNIPAAm main chain and carboxylic
acid groups nearby the polyNIPAAm backbone for the
sample with the highest CEtOxa content in the side chains.
When the same content of CEtOxa units is not randomly
distributed in thesidechainsbut located inendblocksof the
polyoxazolinegrafts suchabehavior isnotobserved.A clear
increase in Ttr2 is observed with increasing CEtOxa content
(Figure4).Moreover, thesampleswith the largestdifference
in Ttr2 have nearly the samehigh CEtOxa content (GCR-3-A/
9.3mol-%vs.GCB-2-A/8.8mol-5) butdifferentlydistributed
in the side chain.When the carboxylic groupsare located far
away from the polyNIPAAm backbone the expected
behavior for increase in hydrophilicity in the polyoxazoline
graft arms is observed supporting the assumption that
hydrogen bonds between polyNIPAAm backbone and
CEtOxa units of the GCR graft arms could influence the
LCSTbehavior of thepolyoxazoline side chain. Contrary, the
short terminal acid block of GCB-2-A is able to stabilize the
formed aggregates very well because even at 75 8C there is
still transmittance for this sample. However, the non-
hydrolyzed precursors GCB-1 and GCB-2 are not able to
effectively stabilize the aggregates and thus, a continuous
increase in turbitity with temperature increase is observed
which is very different to the very broad temperature
window of stable micelle formation observed for the
corresponding samples GCB1-A and GCB-2-A (Figure 4).
Generally, the turbidity curves clearly indicate that the
polyoxazoline side chains prevent at least partially the
formation of large aggregates after collapse of the poly-
NIPAAm backbone due to an amphiphilic behavior of the
graft copolymers. This stabilization ability is enhanced
significantly by incorporation of even a very small amount
CEtOxo units, especially when they are placed at the end of
thegraft chains.Micelles ormicelle-like structures seemtobe
formed which are stabile over a broad temperature region.
However, theyareno longerstablewhenthephasetransition
of the polyoxazoline side chains occurred.
Dynamic Light Scattering Measurements
In order to support the turbitity measurement results
temperature dependent dynamic light scattering experi-
ments were carried out on diluted solutions (0.3 instead of
Macromol. Chem. Phys. 2010, 211, 706–716
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
10 g � L�1 used for turbititymeasurements) of selected graft
copolymers in water. These confirm in general the above-
discussed trends. Stable aggregates above Ttr1 of the
polyNIPAAm backbone and below the second transition
temperature related to the polyoxazoline graft arms are
formed when CEtOxa groups are present in the samples.
Thus, a clear temperature window for the formation of
stable micelles can be identified for a well-balanced
composition of the graft copolymers. Within this tempera-
ture window, the size distribution of the aggregates is
rather narrow and does not vary much with temperature.
Figure 6 shows the observed aggregate formation
behavior in dependence of the temperature exemplary
for the randomgraft copolymersGCR-2 andGCR-2-Aaswell
as the block graft copolymers GCB-1 and GCB-1-A. GCB-1
having about 4 units ofMEtOxa at the graft chain end is not
able to from stable micelles above the polyNIPAAm
transitionwhich leads to a rapid increase in hydrodynamic
radius with temperature, whereas the corresponding
hydrolyzed sample GCB-1-A forms stable aggregates of
about55nmupto70 8C.Within thesetof thepolymerswith
the random graft arm composition but in general a higher
content of the functional oxazoline, the difference in the
aggregation behavior is not so pronounces: both samples,
GCR-2 andGCR-2-A, are able to form stable aggregates up to
about 65 8C, but these have ahydrodynamic radius of about
90nm for the hydrolyzed sample and again of about 55nm
for the non-hydrolyzed sample. Thus, GCB-1-A and non-
hydrolyzed GCR-2 show on first glances a similar ability to
stabilize small micelle-like aggregates. Figure 7 shows the
distribution of the hydrodynamic radius of the aggregates
formed by GCR-2-A at different temperatures. At 34 8C still
some unimers are visible and the formed aggregates are
smaller. When the temperature regime of stable-micelle
formation is reached (42 8C) the average hydrodynamic
radius stays constant at about 90nm up to 65 8C and the
particle size distributions becomes even a little more
DOI: 10.1002/macp.200900437
New Thermo-Sensitive Graft Copolymers Based on a Poly(N-isopropylacrylamide) . . .
Figure 7. Distribution of hydrodynamic radii of sample GCR-2-A atdifferent temperatures.
Figure 8. Distribution of hydrodynamic radii of sample GCB-1-A atdifferent temperatures.
narrow with increasing temperature. Figure 8 demon-
strates the excellent ability of GCB-1-A having only about
four carboxylic acid units at the graft chain ends: stable
micelles of only about 50nm and with a very narrow size
distribution are formed already at 38 8C and up to 65 8C.It was also verified that the micelle formation is fully
reversible. The sampleshavebeencooledafterheating to65
or 70 8C and after standing over night at room temperature
no micelles or aggregates could be identified in the sample
solution by DLS.
Further measurements to determine the aggregation
numbers with static light scattering are under investiga-
tion.
Scheme 2. Proposed conformation of the graft copolymer withdiblock side chains poly((2-ethyl-2-oxazoline)-block-(2-carboxy-ethyl-2-oxazoline)) and its temperature induced collapse of thepolyNIPAAm main chain leading to micelles with functionalcorona.
Conclusion
Graft copolymers with a main chain of polyNIPAAm and
side chains of poly(EtOxa-co- or -b-MEtOxa) could be
synthesized by a ring-opening cationic polymerization of
EtOxa and MEtOxa. This polymerization was initiated by
benzyl chloride groups contained in the random copolymer
Macromol. Chem. Phys. 2010, 211, 706–716
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of NIPAAm and CMS used as MI. The length of the side
chains could be controlled by themolar initial ratio of the 2-
oxazolines and benzyl chloride. Side chains both with
random distribution of both 2-oxazolines and with a
polyEtOxa block followed by a polyMEtOxa block were
synthesized. Furthermore, after hydrolysis of the methyl
ester groups of MEtOxa units in the side chains, carboxylic
acid functionalizationwas introduced in the polyoxazoline
side chains. Thus, it was possible to control the content and
the assembly of the functional groups in the side chains of
these thermo-sensitive graft copolymers where both, main
chainandsidechainsareable todemonstrateLCSTbehavior
in water. Hence, side chain block copolymers are available
with predefined block length and block placement of
functional groupswhich allows to control the temperature
dependent aggregation behavior in water.
It was shown that the phase transition of the poly-
NIPAAm main chain of the graft copolymers can be
controlled through the balance between the content and
length of hydrophilic 2-alkyl-2-oxazolines side chains and
the polyNIPAAm segments in the main chain. When
the content of the hydrophilic part is big enough and the
content of graft units is small enough, the phase transition
can be confined to the segments in themain chain and it is
possible thatmacromolecular aggregates are formed in the
aqueous solution stabilized by the hydrophilic side chains
of poly(2-alkyl-2-oxazolines). The temperature range for
these stabilized aggregates is dependent on the character of
the monomer units and their content and arrangement in
the side chain. In general the aggregates are stable until the
transition temperature of the graft arms is reached.
Already the incorporation of a small amount of
carboxylic acid groups, especially when placed at the graft
chain end, significantly enhances the amphilicity of the
graft copolymers after the collapse of the polyNIPAAm
backbone and leads to stable micelle formation in a broad
temperature range (Scheme2). ForexampleGCB-1-Ahaving
only about four carboxylic acid groups at the graft arm
www.mcp-journal.de 715
J. C. Rueda, S. Zschoche, H. Komber, F. Krahl, K.-F. Arndt, B. Voit
716
chain end forms stable micelles of about Rh¼ 55nm with
narrow size distribution between 38 and 70 8C.The double-temperature sensitive graft copolymers
synthesized in this paper having the ability to form
stabilized aggregates with a thermo-sensitive nucleus
and hydrophilic side chains with modifiable functional
groups in controlled architecture, can be useful tools for
various biomedical application like cellular crop systems,
controlled release of bioactive substances, functional
hydrogels or also nanocarriers for catalyst for specific
reactions. The presence of the carboxylic acid groups also
offers the chance for additional pH dependence of the
aggregation and de-aggregation behavior and present the
possibility for chemical binding of further functionalities.
Acknowledgements: Juan Carlos Rueda gratefully acknowledgesthe Deutscher Akademischer Austauschdienst (DAAD) for afellowship for a research stay in the Leibniz Institute of PolymerResearch Dresden, Germany. The help of A. Lederer in SECmeasurements is gratefully acknowledged.
Received: August 25, 2009; Revised: October 30, 2009; Publishedonline: January 7, 2010; DOI: 10.1002/macp.200900437
Keywords: functionalization of polymers; graft copolymer;micelles; self-organization; stimuli-sensitive polymers
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DOI: 10.1002/macp.200900437