On the structure of biocompatible, thermoresponsive poly(ethylene glycol) microgels

Polymer 55 (2014) 6717e6724

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On the structure of biocompatible, thermoresponsive poly(ethylene glycol) microgels Kornelia Gawlitza a, *, Aurel Radulescu b, Regine von Klitzing a, Stefan Wellert a a b

€t Berlin, Stranski-Laboratory for Physical and Theoretical Chemistry, Straße des 17. Juni 124, 10623 Berlin, Germany Technische Universita Forschungszentrum Jülich GmbH, Outstation at MLZ, Lichtenbergstraße 1, Garching 85747, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 September 2014 Received in revised form 29 October 2014 Accepted 30 October 2014 Available online 5 November 2014

The aim of the present study is the preparation and characterization of microgel particles which are, contrary to other microgels, thermoresponsive as well as biocompatible. Hence, monodisperse pMeO2MA-co-OEGMA microgel particles were synthesized by precipitation polymerization. Swelling/ deswelling behavior and the structure of poly(ethylene glycol) (PEG) based microgel particles were investigated. A combination of dynamic light scattering (DLS) and small angle neutron scattering (SANS) was used. Particle size and the volume phase transition temperature (VPTT) are adjustable by changing the amount of comonomer. SANS measurements indicate an inhomogeneous structure of the PEG microgels in the swollen state. At temperatures above the VPTT a compact structure was observed. An increase of the comonomer content leads to a densely packed core and a fuzzy shell in the swollen state. Additionally, nanodomains inside the polymer network were observed in the temperature range around the volume phase transition (VPT). Due to this heterogeneous structure in the swollen state two correlation lengths of the network fluctuations were observed. © 2014 Elsevier Ltd. All rights reserved.

Keywords: PEG microgels Structure Small angle neutron scattering

1. Introduction Microgel particles have gained increasing scientific interest which is mostly related to their swelling/deswelling behavior. Depending on the chemical composition this volume phase transition (VPT) can be achieved by different external stimuli, e.g. temperature [1e3], pH [4], photons [5,6] or ionic strength [7]. Many applications are envisioned, such as drug delivery [8], enzyme supports [9,10], catalysis [11e14], tissue engineering [15,16], optical materials [17] and smart surface coatings [18,19]. Due to the smaller size compared to macrogels these particles respond faster to external stimuli and have a higher surface-to-volume ratio [20]. In the field of temperature responsive materials, e.g. microgels made of poly-N-isopropylacrylamide (p-NIPAM) are intensively studied [21e24]. Due to the lower critical solution temperature (LCST) of linear polymers consisting of the monomer N-isopropylacrylamide (NIPAM) the polymer particles show a reversible volume phase transition (VPT). The volume phase transition temperature (VPTT) occurs at around 32
C. At this temperature the polymerepolymer interactions start to over-compensate the hydration of the polymer [25]. The VPTT can be shifted by integrating * Corresponding author. E-mail address: [email protected] (K. Gawlitza). http://dx.doi.org/10.1016/j.polymer.2014.10.069 0032-3861/© 2014 Elsevier Ltd. All rights reserved.

a comonomer into the p-NIPAM network, e.g. acrylic acid (AAc) [26], allylacetic acid (AAA) [27] or N-isopropylmethacrylamide (NIPMAM) [28]. P-NIPAM microgels serve as a model system for the investigation of the basic principles of microgel supported transport of active substances, e.g. pulsatile drug delivery. However, the NIPAM monomer is carcinogenic or teratogenic. This limits its applicability as a delivery system inside the human body [29,30]. This drawback can be overcome by the replacement of NIPAM and the design of biocompatible microgels with the same properties as p-NIPAM particles. In previous studies it was shown that poly(ethylene glycol) (PEG) polymers only have a weak influence on cells and their viability is at z83% even after long incubation times of 48 h [31]. PEG is widely used as polymeric antifouling coating [32]. Hence, a combination of its intrinsically anti-adhesive properties with the stimuli-sensitivity and fast response of microgel particles benefits from the peculiar properties of both materials. Additionally, low cytotoxicity and immunogenicity make PEG promising for the synthesis of biocompatible microgels. For example, such particles can then serve as building blocks for coatings on implants. PEG microgel particles composed of the monomer 2-(2-methoxyethoxy)ethyl methacrylate and the comonomer poly(ethylene glycol)methyl ether methacrylate (p-MeO2MA-co-OEGMA) can be synthesized by precipitation polymerization [33,34]. The LCST of

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oligomers of the monomer MeO2MA (z20
C) is below body temperature while the LCST of oligomers of the comonomer OEGMA (z90
C) is above it. Due to this, the volume phase transition temperature (VPTT) of the PEG microgels can be adjusted close to body temperature (z37
C) by different amounts of OEGMA [35,36]. To our knowledge, no research work on the changes of the PEG microgel structure during swelling/deswelling is published. Knowledge about this is highly desirable since it will allow adjusting the composition and the thermosensitivity with respect to their functionality. In literature, several experimental techniques such as static and dynamic light scattering (SLS, DLS), elastic and quasielastic small angle neutron scattering (SANS, NSE) [37e40], NMR [41] and imaging techniques were used to characterize the bulk phase morphology, the inner network structure, the dynamics and the swelling/de-swelling behavior of p-NIPAM microgels. From the knowledge about p-NIPAM microgels no direct conclusions on the properties of the PEG microgels can be drawn. Additional experimental work with respect to the peculiarities of the PEG system is necessary. Moreover, concepts developed for the pNIPAM based microgels can be tested. We investigated p-MeO2MA-co-OEGMA particles with different amounts of comonomer (5 mol-%, 17 mol-%, 26 mol-%). The influence of the microgel composition on particle size, VPTT and inner structure was studied. SANS measurements at different temperatures were used to explore the particle structure below, close to and above the VPTT. This study contributes to the investigations on the use of such particles for controlled uptake and release of biologically active molecules. 2. Experimental section 2.1. Materials 2-(2-Methoxyethoxy)ethyl methacrylate (95%) (MeO2MA), poly(ethylene glycol) methyl ether methacrylate (average Mn ¼ 500 g/mol) (OEGMA), ethylene glycol dimethacrylate (99%) (EGDMA) and potassium peroxodisulfate (99%) (KPS) were purchased from SigmaeAldrich (Munich, Germany). The sugar surfactant Glucopon 220 was provided by Henkel. In all SANS experiments the microgel particles were dispersed in deuterium oxide (99.9% D) (D2O) purchased from Eurisotop (Saarbrücken, Germany). All chemicals were used as received. A three-stage Millipore Milli-Q Plus 185 purification system was used for water purification. 2.2. Microgel synthesis Microgel particles based on the monomer 2-(2-methoxyethoxy) ethyl methacrylate (MeO2MA), the comonomer poly(ethylene glycol) methyl ether methacrylate (OEGMA) and the cross-linker ethylene glycol dimethacrylate (EGDMA) were synthesized by precipitation polymerization [36]. In the present paper, the microgel systems are labeled as p-ME3Oy according to their crosslinker and comonomer content where y is the mol-% of comonomer with respect to the amount of monomer. Comonomer concentrations of 5 mol-%, 17 mol-% and, 26 mol-% were used. The amount of cross-linker was kept constant at 3 mol-%. The sugar surfactant Glucopon 220 was used to obtain lower polydispersity. The amount was kept constant at 0.9 mol-%. Briefly, 1.205 g of MeO2MA (6.00 mmol), 0.038 g EGDMA (0.19 mmol, 3 mol-%), 0.020 g Glucopon 220 (0.05 mmol, 0.9 mol-%) and the desired amount of OEGMA were dissolved in 100 mL of water in a three-neck flask. The temperature was increased to 70
C and the solution was degassed for 30 min. Afterwards, 1 mL of an

aqueous solution of KPS (0.74 mM) was added. The mixture was stirred continuously. After 4 h of reaction time the temperature was decreased to room temperature. The mixture was stirred overnight under N2-atmosphere. The crude microgel particles were purified by filtering over glass wool. Afterwards, they were dialyzed for 2 weeks with daily water exchange. Finally, the particles were freeze dried at 85
C and 1  103 bar for 48 h. 2.3. Experimental techniques 2.3.1. Dynamic light scattering The swelling/deswelling behavior of the PEG microgel particles was investigated by Dynamic Light Scattering (DLS). The intensity auto-correlation functions were recorded at a constant scattering angle of 60
using an ALV/CGS-3 compact goniometer system equipped with an ALV/LSE-5004 correlator and a HeeNe laser (l ¼ 632.8 nm, 35 mW). Data analysis was done using the inverse Laplace transformation algorithm (CONTIN [42]). The measurements were carried out in a temperature range between 15
C and 60
C using a Huber compatible control thermostat. 2.3.2. Small angle neutron scattering (SANS) SANS measurements were carried out at the KWS-2 instrument of the JCNS at the FRM-II (Garching, Germany). Three sample-todetector distances (2.05 m, 8 m and 20 m) were combined to cover a q-range from q ¼ 0.003 Å1e0.3 Å1. The neutron beam (l ¼ 5 Å) collimation was adapted the sample-detector distances. Samples of the microgel systems p-ME3O5, p-ME3O17 and, pME3O26 were dispersed in D2O and adjusted to a mass concentration of 2 wt%. The samples were measured in quartz cells with 2 mm neutron path way. All samples were measured at seven temperatures in the range from 15
C to 60
C. The desired temperature was kept constant using a thermocycler with a stability of ±0.1 K. Prior to each measurement the samples were equilibrated for 15 min. Raw scattering data were initially treated, radially averaged and brought to absolute scale by the procedures provided by the JCNS using the QtiKWS10 software package. The treated scattering data were further analyzed using the SasView 2.2.1 software. The fit model is discussed in Section 3.2. 3. Theoretical background e analysis of SANS data Samples of the PEG microgel systems were investigated in SANS measurements in a wide temperature range to explore the structural evolution during the deswelling process. The resolution of the full spherical particle form factor of the microgel particles depends on the particle size, especially in the swollen state and the chosen SANS instrument configuration. In case of large particles, the data are usually modeled using a superposition of the OrnsteineZernike scattering function and the Porod law [40,43]. This model has been used to fit SANS data of neutral microgels and corresponds to a scattering contribution from the particle surface and the scattering from solvated fluctuating polymer chains. In macrogels no Porod scattering occurs. Instead one observes a Guinier-like decay related to the distance between the chemical cross-links [44]. In microgels this length scale related to the chemically fixed network cannot be resolved. Only the dynamic solution-like correlation length is observed. Previous studies mostly dealt with the modeling of SANS data of p-NIPAM microgels. Considering this work, we assume a combination of several effects to contribute to the scattering intensity distributions of PEG microgels [45,46]. During the synthesis of p-NIPAM microgels the cross-linker consumption is faster than that of the monomer. This unavoidably results in an inhomogeneous polymer network with a radial

K. Gawlitza et al. / Polymer 55 (2014) 6717e6724

polymer density gradient or, in other words, a coreeshell like structure within the microgel particles [47]. These inhomogeneities are detectable by SANS measurements. Their contribution to the scattering intensity can be modeled by adding a Gaussian distribution of the scattering length density to the radial box profile of a homogeneous sphere [46]. In copolymerized microgels the monomer and the comonomer exhibit different LCSTs. For such systems an additional bump in the scattering profiles I(q) was observed during the VPT [45]. This was explained by phase separation of comonomer rich and spherical comonomer poor nanodomains. During the collapse of one component the other component remains swollen. Furthermore, the fluctuations of the polymer network always have an influence on the scattering intensity profile. In case of p-NIPAM microgels high cross-linker contents (10 mol-%) lead to coreeshell like structures. Hence, network fluctuations at different length scales might be observable. For example, it was reported that two correlation lengths can be found in the swollen state, a short fluctuation length is present in the core and a second one in the shell [48]. In general, the scattering intensity contains the coherent and incoherent scattering contributions

IðqÞ ¼ DrNSðqÞPðqÞ þ Iinc

(1)

The coherent scattering intensity is given by the product of structure factor S(q) and particle form factor P(q) and proportional to the number N of scatterers and the scattering length density difference between scatterers and solvent Dr. In highly diluted microgel suspensions no influence on the scattering signal due to particle interaction occurs and the structure factor can be assumed as S(q) ¼ 1. Hence, the intensity distribution is determined by the form factor P(q). Depending on sample temperature and q-range the scattering data can be best described by a sum of at least five contributions to the scattering intensity profile

IðqÞ ¼ Ipart þ Inano þ Ifluct þ IPorod þ Iinc :

(2)

Here, Ipart is the contribution of the inhomogeneous spherical microgel particles, Inano the contribution of nanodomains formed during the VPT and Ifluct the contribution of the network fluctuations to the scattering intensity distribution. The Porod scattering (IPorod ¼ Cq4) reflects the scattering from the whole particle which shows a huge influence on the scattering profile above the VPTT of the microgel systems. Iinc represents the incoherent scattering background.

radius where half of the core SLD is reached and sF is the width of the diffuse particle surface. The overall size of the microgel particles which can be obtained by SANS measurements is given by RSANS ¼ R þ 2sF [46]. The radius of the dense core is described by Rbox ¼ R  2sF. A continuous gradient in cross-linking density ends up in a fuzzy sphere with R ¼ 2sF which represents a microgel without a dense core. In this case Rbox ¼ 0. 3.2. Nanophase separation during the VPT The PEG microgels contain two monomers with strongly different LCSTs at z20
C (MeO2MA) and z90
C (OEGMA). While one of the monomers induces the VPT of the microgel particles at 20
C the other one tends to stay in the swollen state. Hence, an increase in temperature can generate strains inside the polymer network. This leads to the formation of nanodomains. These nanodomains show differences in the scattering length density and can therefore contribute to the scattering intensity distributions. Similar behavior was found for p-NIPAM microgels copolymerized with N-isopropylmethacrylamide (NIPMAM) [45]. Assuming spherical shape of the nanodomains, the form factor of these phases is given by the box function

Inano ðqÞzPnano ðqÞ " #2 scale 3VðDrÞ½sinðqrnano Þ  qrnano cosðqrnano Þ ¼ ; V ðqrnano Þ3 (4) where rnano denotes the radius of the nanodomains. 3.3. Network fluctuations inside the microgel network [40,43] The liquid-like correlations in the polymer network decay with the dynamic correlation length x. x is smaller compared to the mean distance between the chemical cross-links. Their characteristic signature in scattering curves is a broad shoulder where the density fluctuations are enhanced at the length scale of the liquid-like polymer fluctuations [49]. To model this contribution in the scattering curve the OrnsteineZernike equation is applied [40,43]

Ifluct ðqÞ ¼ 3.1. Fuzziness of the microgels For p-NIPAM microgels synthesized under the same conditions like the PEG microgels a higher degree in cross-linking density in the core region of the microgel network than in the shell was reported. SANS measurements confirmed the assumption of a fuzzy particle morphology [46]. This fuzziness has to be included into the model. In this case, the form factor of the spherical microgel particles can be described by convoluting the radial box profile of a homogeneous sphere with a Gaussian:

! "

scale 3ðDrÞ½sinðqRÞ  qR cosðqRÞ Ipart ðqÞzPpart ðqÞ ¼ e V ðqRÞ3

ðsF qÞ 2

2

#2

(3) where scale is equivalent to the volume fraction of the microgel, V is the specific volume of the microgel, Dr is the difference in scattering length density between the microgel and the solvent, R is the

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A

(5)

1 þ ðqxÞ2

A is the scattered intensity at q ¼ 0 and x denotes the dynamic correlation length. Theoretically, x can only be detected until the VPTT is reached. At this point x diverges and is not observable at higher temperatures [50]. Above the VPTT fluctuations are still present if the network is deswollen but not fully collapsed [51]. In the present work we observed a second correlation length in the system. As described in more detail in Section 4.2 for the PEG microgels we observed two clearly distinguishable correlation lengths in the swollen state. Hence, the contribution to the scattering profile is given by

Ifluct ðqÞ ¼

A 2

1 þ ðqx1 Þ

þ

B 1 þ ðqx2 Þ2

;

(6)

where, again, A and B denote the intensity at q ¼ 0 and x1, x2 account for the correlation lengths of the fluctuations. From analyzing x1(T) and x2(T) we identified x1 as the contribution of the outer microgel region with its fuzzy morphology and x2(T) as the correlation length associated with the fluctuations in the core region (see Section 4.2).

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4. Results

Table 1 Summary of the results obtained by DLS and viscometry on samples of p-ME3O5, pME3O17 and p-ME3O26. The hydrodynamic radii RH are given at 15
C and 60
C as well as the deswelling ratio a and the VPTT.

4.1. Characterization of PEG microgels by DLS Three PEG microgels p-ME3O5, p-ME3O17 and, p-ME3O26 (fixed cross-linker content of 3 mol-% and comonomer contents of 5 mol%, 17 mol-% and 26 mol-%) were investigated. The swelling behavior was characterized by DLS measurements. Fig. 1a shows the temperature dependent hydrodynamic radii RH(T). Data of measurements at different scattering angles at 15
C are shown in Fig. S1a in the supporting material. The linear progression of the relaxation rates G as a function of the square of the scattering vector q is observed for all samples. This confirms that only translational diffusion is detected in the sample. Consequently, the StokeseEinstein equation was used to determine the hydrodynamic radii from the mean values of G. Fig. S1b exemplarily shows the size distributions of the three samples at 15
C. The PEG microgel particles show a small polydispersity. To compare their sizes the hydrodynamic radii (RH) in the deswollen state (60
C) are important due to the polymerization temperature above the VPTT. The results are summarized in Table 1. Larger particles are obtained by increasing the comonomer content during the synthesis. This might be related to a larger amount of material accumulated in the polymer network. The efficiency of the swelling behavior is characterized by the deswelling ratio a defined as

a¼

R3 VH ¼ 3H VH;0 RH;0

(7)

Here RH and RH,0 are the hydrodynamic radii in the deswollen and swollen state, respectively. From the hydrodynamic radii at 60
C and 15
C the deswelling ratios were calculated. As can be seen in Fig. 1b, the deswelling ratio increases when the content of OEGMA rises. The VPTTs were determined at the inflection point of the swelling curves and are summarized in Table 1. In addition, Fig. 1a shows a broadening of the VPT with an increase in amount of OEGMA. Both effects can be explained by the large difference in the LCSTs of the monomer and comonomer.

4.2. Investigation of the microgel structure by SANS SANS measurements on samples of the three PEG microgel systems were done over a broad q-range (q ¼ 0.003 Å1e0.3 Å1). All samples were measured at seven temperatures below, close to and above the VPTT. As an example, the scattering curves of pME3O5 at different temperatures are shown in Fig. 2a. Solid lines are fits to the data according to equation (2). Fits and experimental

Sample

RH at 15
C [nm]

RH at 60
C [nm]

a

VPTT [
C]

p-ME3O5 p-ME3O17 p-ME3O26

144.6 ± 5 171.3 ± 5 218.0 ± 4

90.1 ± 2 107.3 ± 2 141.3 ± 1

0.24 0.25 0.27

28 33 36

data are in good agreement. For the scattering curves of p-ME3O17 and p-ME3O26 we refer to Fig. S2 in the supporting material. The scattering curves significantly change with increasing comonomer content and increasing sample temperature. These changes mainly occur in the low q-range where the scattering intensity is determined by the overall structure of the PEG microgels and at larger q-values where the fluid like network fluctuations strongly contribute to the scattering signal. Fig. 2b compares the fits to the scattering intensity profile of p-ME3O5 at 15
C using one correlation length (blue line) and two correlation lengths (red line). The assumption of two distinguishable correlation lengths result in a better matching of the fit to the experimental data and lower residual values. The fit parameters are summarized in Table 2 and will be discussed in detail in Section 5. 5. Discussion 5.1. Characterization of the PEG microgels by DLS measurements The results summarized in Table 1 confirm that particles size, swelling behavior and the VPTT of the PEG microgels can be tuned by changing the amount of integrated comonomer OEGMA. Most likely, these modifications are related to changes in the microgel network properties. In case of an increase in cross-linker content the size and the stiffness of microgels increases [3,52]. Speculatively, an increase in the amount of the used uncharged comonomer can be compared with an increase in cross-linker content. This increase in polymer volume fraction leads to denser microgel particles. This explains the decrease in water uptake during the VPT due to a less flexible polymer network. Moreover, the VPTT can be tuned in a wide temperature range between 28
C and 36
C by changing the amount of comonomer between 5 mol-% and 26 mol-%. Hence, a VPTT close to body temperature (37
C) can be achieved. This is an important feature and beneficial for future biomedical applications, like coating of implants. Beside, a further increase in the comonomer content could lead to much higher VPTTs.

Fig. 1. Swelling curves of p-ME3O5, p-ME3O17 and p-ME3O26 (a.) and the deswelling ratio a in dependence of the comonomer content (b.) measured by DLS.

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Fig. 2. SANS scattering profiles of p-ME3O5 at different temperatures (a.) where the lines represent fits according to equation (1). SANS scattering profile of p-ME3O5 at 15
C (b.) fitted with one correlation length (blue line) and two correlation lengths (red line) and corresponding colored residuals. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

5.2. Investigations on the internal microgel structure by SANS 5.2.1. Fuzziness of the microgels The SANS curves for p-ME3O5, p-ME3O17 and p-ME3O26 (Fig. 2 and Fig. S1) show significant changes as a function of temperature in the low q-range (0.005 Å1e0.1 Å1). The form factor minima are shifted to higher q values and become more pronounced. This is related to the shrinkage of the microgel particles and the polymer network properties. The radii and the widths of the fuzzy shell determined from the fitting procedure are shown in Table 2. In case of swollen p-ME3O5 at T < 28
C the radius is equal to 2sF. The total size of the microgel particles can be calculated by R þ 2sF as described in Section 3.2. Fig. 3a shows the profile of the polymer volume fraction as well as the total size of the microgel particle with 5 mol-% comonomer at 15
C. Here, the total size of the microgel particle is given by the width of the fuzzy shell. At T > 28
C the contribution of the fuzzy shell disappears and the polymer volume fraction profile can be described by the box profile of a homogeneous sphere. Fig. 3b shows the decrease of the total size of the microgel particles. At 60
C the total size of the microgel particles is given by the size of the dense core (Rbox).

Table 2 Summary of the fit parameters R, sF, rnano, x1 and x2 of p-ME3O5, p-ME3O17 and pME3O26 at different temperatures. Sample

T [
C]

x1 [nm]

x2 [nm]

p-ME3O5

15 20 25 28 40 50 60

67.4 73.7 77.0 76.1 77.7 78.4 81.6

35.0 30.8 29.2 21.5 13.9 0 0

0 0 4.9 6.5 5.6 6.7 6.9

11.2 10.4 0 0 0 0 0

1.6 2.2 8.9 8.9 10.1 6.6 3.6

p-ME3O17

15 20 29 33 40 50 60

81.9 83.0 88.5 90.2 90.2 96.2 91.6

36.0 37.3 37.0 36.0 18.9 14.1 0

0 0 7.6 7.4 7.7 9.3 8.5

11.1 10.8 0 0 0 0 0

1.3 1.8 4.9 5.3 10.2 2.6 1.6

15 20 29 36 40 50 60

100.4 97.1 100.0 100.0 109.0 108.0 109.0

35.5 36.2 34.4 35.0 30.9 20.0 0

0 0 22.3 19.7 11.9 11.5 13.8

9.9 9.9 9.9 0 0 0 0

1.1 1.3 1.4 6.2 7.8 3.2 1.9

p-ME3O26

R [nm]

sF [nm]

rnano [nm]

The same analysis was done for all measurements to investigate the trend during the VPT (Fig. 4). For all comonomer contents the size of the fuzzy shell decreases with increasing temperature. Probably, for all compositions the deswelling of the microgel particles is accompanied by the stiffening of the polymer network. A comparison of the results for p-ME3O5, p-ME3O17 and p-ME3O26 at 15
C shows that an increase of the comonomer content leads to the formation of a dense core even in the swollen state. This gives proof of tuning the internal structure by changing the composition of the microgel particles. From literature, it is known that the cross-linker consumption is faster than the consumption of the monomer [46]. This leads to a denser core with increasing amount of cross-linker. Due to the fact that we kept the amount of cross-linker constant it can be assumed that the size of the dense core is additionally dependent on the comonomer content. Probably, in the beginning of the polymerization reaction the cross-linker as well as the comonomer consumption is faster than the monomer consumption. Additionally, also the total size of the PEG microgels increases. This trend agrees with the results of the DLS measurements (Fig. 1, Table 1). Nevertheless, the deviation in particle sizes determined from DLS and SANS measurements increases with rising comonomer content. In principle, the larger sizes determined by DLS can be explained by dangling polymer chains which are attached to the particle surface. These polymer chains contribute to the hydrodynamics but are not concentrated enough to be detected by SANS [46]. The increase in deviation with increasing comonomer content is caused by the initial composition of monomer, comonomer and cross-linker. Due to the fact that the concentration of the comonomer is changed while the amount of monomer, cross-linker and total volume is fixed more polymer material is present and larger microgel particles are formed. Hence, in case of higher comonomer content the same amount of cross-linker is used for the synthesis of larger particles. This leads to a larger region of dangling polymer chains which are detected by DLS but not by SANS. 5.2.2. Nanophase separation at the VPT Fig. 5 shows the radii of the nanodomains in dependence on the temperature. Obviously, no nanophase separation was observed in the swollen state at 15
C but occurs at higher temperatures. A similar behavior for p-(NIPAM-co-NIPMAM) microgels was found. The additional bump in the scattering profiles was explained by the different VPTTs of the corresponding homopolymers pNIPAM (VPTT ¼ 34
C) and p-NIPMAM (VPTT ¼ 44
C) [45]. In contrast to the study of Richtering and co-workers, we considered the formation of nanodomains as additive contribution to the scattering intensity distributions (Section 3.2). The validity of this assumption is supported by two facts. Firstly, in the DLS

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Fig. 3. Schematic drawing of the polymer volume fraction profile and the overall size of p-ME3O5 consisting of the radius of the dense core (dark gray bar) and the width of the fuzzy shell (light gray bar) at (a.) 15
C (Rbox ¼ 0) and (b.) 60
C redrawn from Ref. [46].

Fig. 4. Overall size of p-ME3O5 (a.), p-ME3O17 (b.) and p-ME3O26 (c.) consisting of the dense core (dark gray bars) and the fuzzy shell (light gray bars) at different temperatures.

measurements only one size distribution was observed (Fig. S1b). Hence, there is no spatial separation of the nanodomains from the whole microgel particles. Secondly, the contrast (Dr) in the fit procedure was calculated assuming MeO2MA as nanodomains (rMeO2 MA z 0.7  106 Å2) and OEGMA as surrounding medium (rOEGMA z 1.1  106 Å2).

Fig. 5. Nanodomain-sizes of p-ME3O5, p-ME3O17 and p-ME3O26 determined by SANS scattering curves.

The microgel systems studied in the present paper consist of compounds which show a huge difference in their LCSTs. As shown in Fig. 5, at 15
C both monomer and comonomer are in good solvent condition and fully swollen. At increasing temperature the polymer chains of the monomer MeO2MA start to collapse while the chains of the comonomer OEGMA remain in the good solvent condition. This leads to two different phases within the microgel particles. A microscopically separated phase has a higher concentration of the monomer MeO2MA in the deswollen state. The second phase has a lower polymer density and mainly consists of the comonomer OEGMA in its swollen state. The nanophase separation is sketched in Fig. 5. At 60
C the nanodomains seem still to be present since this temperature is still below the VPTT of the comonomer. Furthermore, the size of the nanodomains increases with increasing amount of comonomer. Stronger forces counteracting the collapse of the monomer chains occur at a higher amount of comonomer. Hence, the nanodomains are less dense after collapsing than in case of a low monomer content. 5.2.3. Thermal network fluctuations Two results were found for the PEG microgels: 1.) Two correlation lengths x1,2 of distinguishable length were found in the swollen state. 2.) It is x2  x1 and at increasing sample temperature

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Fig. 6. Long and short correlation-lengths of p-ME3O5 (a.), p-ME3O17 (b.) and p-ME3O26 (c.) determined by SANS scattering curves. The lines are guides to the eye.

x1 and x2 converge. Above the VPTT network fluctuations still seem to be observable in the deswollen states. The values determined for x1,2 are summarized in the graphs of Fig. 6. At increasing sample temperature x1 decreases due to the induced collapse of the MeO2MA chains. Concurrently, x2 increases. This behavior was previously observed for the temperature dependence of the correlation length of p-NIPAM based microgels [48]. Both fluctuation lengths converge and finally, only one average correlation length above a temperature of 20
C exists. A further increase in the temperature above the VPTT leads to a continuous decrease of x. In this temperature range the microgel network deswells and the polymer density increases. This restricts the fluctuation of the polymer chains which is related to a decrease in the blob size of the polymer network. From the progression of x1(T) and x2(T) we conclude that x1 is the contribution from the outer microgel region with its fuzzy morphology and x2(T) stems from the fluctuations in the core region. x1(T) decreases since the shell region becomes denser during the deswelling. x2(T) shows the typical increase of the correlation length close to the VPT and decreases at temperatures above the VPT. Above the VPT shell and core can hardly be distinguished and only one mean correlation length was found. This observation can be attributed to the heterogeneous structure of the microgel particles. The fuzzy microgel structure as well as a partially inhomogeneous distribution of the monomer MeO2MA and the comonomer OEGMA could lead to the observations described above. In particular, at higher amounts of OEGMA, it is likely that the microgel particles consist of a dense core and a fuzzy shell. Moreover, such polymerization reactions never lead to a homogeneous distribution of cross-linking points in the network. From Fig. 6 we conclude that for all three microgel systems at temperatures below the VPTT two fluctuation regimes exist. The corresponding correlation lengths x1(T) and x2(T) differ by a factor of 5e6. Comparing the different microgel systems it can be observed that the point at which x1 and x2 converge is shifted to higher temperatures with an increase in the comonomer content. Furthermore, Fig. 6 indicates that network fluctuations are still present at a temperature of 60
C. This supports the assumption made in Section 5.2.2 that PEG microgels are deswollen but not collapsed at this temperature due to the high VPTT of the comonomer (90
C). 6. Conclusion In the present study, biocompatible microgel particles were polymerized by using the monomer 2-(2-methoxyethoxy)ethyl methacrylate (MeO2MA), the comonomer poly(ethylene glycol)

methyl ether methacrylate (OEGMA) and the cross-linker ethylene glycol dimethacrylate (EGDMA). Their bulk phase properties were investigated by several methods, such as light scattering and SANS. DLS measurements showed that the VPTT of the microgel particles can be easily tuned over a broad range by changing the amount of comonomer. Moreover, the VPTT can be adjusted to body temperature which is important for potential applications as drug release system inside the human body. The progression of the particle structure as a function of sample temperature was investigated at different amounts of comonomer (5 mol-%, 17 mol-%, 26 mol-%) in the temperature range between 15
C and 60
C. Below the volume phase transition the PEG microgels consist of a dense core surrounded by a fuzzy shell. This morphology changes during the deswelling around the VPTT. Above the VPTT the fuzzy shell is collapsed. With an increase in the comonomer content the size of the dense core increases while the fuzziness decreases. This inhomogeneous structure becomes manifest in two separated correlation lengths of the liquid-like network fluctuations. In addition, spherical nanodomains are formed during the collapse which is related to the large differences in the LCSTs of the monomer and the comonomer. Due to their biocompatibility and the ease of VPTT tuning PEG derivative microgels are good candidates for smart packaging of drugs like growth factors or other biologically active molecules. Acknowledgments The authors thank the DFG (G8 “Multiscale smart Coatings”, KL 1165/15-1) for financial support. JCNS and ILL are gratefully acknowledged for financial support to perform the neutron scattering measurements. This work benefited from DANSE software developed under NSF award DMR-0520547. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.polymer.2014.10.069. References [1] Senff H, Richtering W. Temperature sensitive microgel suspensions: colloidal phase behavior and rheology of soft spheres. J Chem Phys 1999;111:1705e11. [2] Berndt I, Richtering W. Doubly temperature sensitive core-shell microgels. Macromolecules 2003;36:8780e5. [3] Kratz K, Hellweg T, Eimer W. Structural changes in PNIPAM microgel particles as seen by SANS, DLS and EM techniques. Polymer 2001;42:6631e9. [4] Hoare T, Pelton R. Highly pH and temperature responsive microgels functionalized with vinylacetic acid. Macromolecules 2004;37:2544e50. [5] Suzuki A, Tanaka T. Phase transition in polymer gels induced by visible light. Nature 1990;346:345e7.

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