New Thermo-Sensitive Graft Copolymers Based on a Poly(N-isopropylacrylamide) Backbone and Functional Polyoxazoline Grafts with Random and Diblock Structure

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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

Macromol. Chem. Phys. 2010, 211, 706–716

� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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



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

www.mcp-journal.de 707

J. C. Rueda, S. Zschoche, H. Komber, F. Krahl, K.-F. Arndt, B. Voit

708

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.



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


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)

ol. Chem. Phys. 2010, 211, 706–716

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



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.



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

DOI: 10.1002/macp.200900437


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.



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



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



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


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.



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



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



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


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



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



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

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