Multicompartment aqueous microgels with degradable hydrophobic domains

Polymer 172 (2019) 283–293

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Multicompartment aqueous microgels with degradable hydrophobic domains

T

Dominic Kehrena,∗,1, Catalina Molano Lopeza, Stefan Theilera, Helmut Keula, Martin Möllera, Andrij Picha,b,∗∗ a DWI Leibniz Institute for Interactive Materials e.V, Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstr. 50, D-52056, Aachen, Germany b Aachen Maastricht Institute for Biobased Materials (AMIBM), Maastricht University, Brightlands Chemelot Campus, Urmonderbaan 22, 6167 RD, Geleen, the Netherlands

H I GH L IG H T S

microgels with hydrophobic pockets for uptake/release of water-insoluble molecules. • Degradable free miniemulsion of molten monomer in aqueous phase. • Solvent biodegrasdability and solubilisation of hydrophobic substances. • Thermo-responsiveness, • Star shaped crosslinker provides degradability and hydrophobic pockets.

A R T I C LE I N FO

A B S T R A C T

Keywords: Microgels Degradable Hydrophobic domains Polyvinylcaprolactam Minieemulsion

In this work we successfully synthesized degradable microgels based on poly(N-vinylcaprolactam) (PVCL) with hydrophobic pockets for the uptake/release of poorly water-soluble molecules. The degradability as well as the presence of hydrophobic pockets in the microgel structure are ensured by incorporation of a star-shaped acrylate-functionalised poly(ε-caprolactone) (starPCL) crosslinkers. Microgels with variable amount of star PCL crosslinker, narrow size distribution and a hydrodynamic radius of 200–400 nm were obtained by miniemulsion polymerisation. The microgel size and size distribution was characterised by means of dynamic light scattering (DLS), field emission scanning electron microscopy (FESEM) and sedimentation analysis. The obtained microgels undergo enzymatic degradation in aqueous medium as evidenced by DLS, nuclear magnetic resonance (NMR) and FESEM. Additionally, the uptake of hydrophobic molecules like the dye Nile-Red (model system) and the drug Ibuprofen into the starPCL-based microgels was proven by ultraviolet–visible spectroscopy (UV/Vis). The experimental data indicate that the solubilisation ability of microgels can be regulated by the amount of crosslinker in the microgel structure.

1. Introduction Smart or functional materials are in the focus of material research not only in the fields of polymer science but also for ceramics and metals as well as for composites and biomaterials. Among the innumerable polymeric materials, microgels have aroused significant

interest due to their stimuli-responsiveness. Microgels are crosslinked polymer particles with a network structure that swells in solvents [1–3]. They can have a size between 50 nm and 5 μm and be synthesized from different monomers and polymers, exhibiting a wide range of interesting properties, like deformability, controlled swelling and surfaceactivity [1,4]. Moreover, these intelligent polymeric networks can be

∗

Corresponding author. Federal Institute for Occupational Safety and Health, Unit Particulate Hazardous Substance, Advanced Materials, Nöldnerstraße 40 – 42, 10317, Berlin, Germany. ∗∗ Corresponding author. DWI Leibniz Institute for Interactive Materials e.V, Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstr, 50, D-52056, Aachen, Germany. E-mail addresses: [email protected] (D. Kehren), [email protected] (A. Pich). 1 Dominic Kehren: Federal Institute for Occupational Safety and Health; Unit Particulate Hazardous Substance, Advanced Materials. Nöldnerstraße 40–42, 10317 Berlin. https://doi.org/10.1016/j.polymer.2019.03.074 Received 7 February 2019; Received in revised form 28 March 2019; Accepted 30 March 2019 Available online 05 April 2019 0032-3861/ © 2019 Elsevier Ltd. All rights reserved.

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formed droplets. The crosslinker starPCL [36] was dissolved in the molten VCL monomer. Due to the biodedradability of the crosslinker and its hydrophobic backbone, the microgels can be used for the encapsulation of hydrophobic molecules. We describe herein the properties of obtained microgels like swelling, particle size, biodegradability and their ability to solubilize hydrophobic compounds. This remarkable feature enables their usage as colloidal carriers for transport and release of substances with poor water solubility like oils, dyes or drugs [37,38]. Moreover, their special properties like chemical functionality, degradability and availability of hydrophobic pockets opens new perspectives for design of new functional microgel-based materials and systems.

highly biocompatibile [5], possess reactive groups [6], carry charges [7] or work as template for in situ nanoparticle preparation [8,9]. In addition, microgel properties can easily be tuned by further functionalization with proteins or metal nanoparticles to get access to adaptation, conductivity, magnetic response or catalytic activity [10–15]. In particular, special focus is set on the sensitivity of the microgels by environmental variations. The swelling and de-swelling behaviour of microgels can be triggered by changes in temperature [3,16], pH value [17] or light [18]. Well-known thermo-sensitive microgels are swollen in water below the Volume Phase Transition Temperature (VPTT) due to the physical interactions between the aqueous media and the polymer chains. However, an increase of the temperature above the VPTT leads to rapid shrinking and the formation of collapsed polymer particles. The microgels considered in this work are based on PVCL. PVCL is thermo-sensitive with a VPTT of 32 °C [19] and shows a good biocompatibility [5,20], contrary to the more intensively studied poly(Nisopropylacrylamide) (PNIPAM) microgels [5,21]. Microgels are mainly synthesized by precipitation polymerisation in water [1], therefore the selection of monomers is partially limited to water soluble building blocks. Nevertheless, microgels can also be prepared by miniemulsion polymerisation to avoid this limitation [22]. This was reported for PVCL in a toluene/water O/W emulsion among further successful examples [23,24]. In terms of degradation, different microgels have already been synthesized by inverse miniemulsion polymerisation for the incorporation of an acidic degradable crosslinker [25]. Furthermore, De Geest and coworkers published interesting works about the synthesis of dextran/poly(hydroxyethyl methacrylate) microgels, which are degradable by hydrolysis [26,27]. Precipitation polymerisation was used to synthesize microgels with degradable azoaromatic crosslinker [28]. Bulmus et al. used divinyl-functionalised acetal-based crosslinkers for copolymerisation with hydroxyethyl methacrylate (HEMA) to synthesize acid-degradable microgels [29]. Moreover in 2015, acid degradable PVCL microgels were successfully produced by incorporation of 2,2dimethacroyloxy-1-ethoxypropane (DMAEP) as a crosslinker, and N-(2hydroxypropyl)methacrylamide (HPMA) as a co-monomer [30]. Further work has been specifically performed on the synthesis of aqueous microgels decorated with hydrophobic domains, in view of drug transport and solubilisation of hydrophobic bioactive compounds. For example, PNIPAM was combined with various hydrophobic comonomers like methyl methacrylate, hexylacrylate, hexafluoroisopropyl methacrylate and hexafluorobutyl-methacrylate to obtain hydrogels with hydrophobic interior [31]. In 2010, PVCL microgels were successfully modified by hydrophobic domains by post-modification approach and integration of micellar structures [32]. In addition, microgels composed of covalently crosslinked poly(acrylic acid)-g-poly (ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic-PAA) networks have shown superior capacity for equilibrium loading of hydrophobic drugs such as taxol, camptothecin and steroid hormones, as well as higher capacity for weakly basic drugs such as doxorubicin, mitomycin C, and mitoxantrone [33–35]. In contrast to the previously discussed findings, the aim of the present work was combining three main features in one functional microgel system: thermo-responsiveness, biodegradability and solubilisation of hydrophobic substances. Especially to mention hereby is that the degradability as well as the presence of hydrophobic pockets are achieved both by the utilization of a novel functional crosslinker. In the present manuscript, we investigate the synthesis of PVCL microgels crosslinked with star-shaped acrylate functionalised poly(ε-caprolactone) by miniemulsion polymerisation in water. Contrary to the work of Crespy et al. [23], we modified the microgel synthesis by miniemulsion to avoid the usage of toxic solvents like toluene. Therefore, we used in our approach only molten VCL monomer dispersed in form of small droplets in the aqueous phase. The polymerisation of the monomer VCL and the crosslinking of the PVCL chains occur within the

2. Experimental part 2.1. Materials Acetone (99.9%, VWR), 2,2′-azobis[2-methylpropionamidine] dihydrochloride (AMPA, 97%, Aldrich), cetyl trimethylammonium bromide (CTAB, 99.9%, Aldrich), 9-diethylamino-5-benzo[α]phenoxazinone (Nile-Red, 98%, Aldrich), N,N′-methylen-bis-(acrylamide) (BIS, 99%, Sigma-Aldrich), phosphate buffered saline (PBS, Aldrich), (RS)-2(4-(2-methylpropyl)phenyl)propanoic acid (Ibuprofen, > 98%, Aldrich), tetrahydrofuran (THF, 99.9% VWR), distilled water. All these materials are used as delivered without having undergone any other cleaning procedures. N-Vinylcaprolactam (VCL, 98%, Aldrich) has been was purified by vacuum distillation under nitrogen before use. 2.2. Synthesis of starPCL crosslinkers The synthesis of the star-shaped acrylate-funtionalized poly(ε-caprolactone) crosslinker (starPCL) was carried out following the procedure presented in previous reports [36,39,40]. Scheme 1 shows the synthetic pathway to achieve various starPCL crosslinkers. In the present study, we used starPCL with 4 or 6 arms and the number of caprolactone repeating units per arm was adjusted to 5 and 15 (Table 1). Di(trimethylolpropane) or di(pentaerythritol) were dissolved in 3caprolactone and Zn(oct )2 was added. The mixture was stirred for 24 h at 130 °C; the polymer was recovered by precipitation in cold hexane. The precipitate (PCL) was separated by decantation and was dried in vacuum at 40 °C up to constant weight. Star-shaped PCL was dissolved in dichloromethane and acryloylchloride was slowly added to stirred solution. Stirring for 5 h at 60 °C was followed by precipitation in cold hexane. The precipitate was separated by decantation and dried in vacuum at room temperature until constant weight was reached. Characterisation data for the most frequently used crosslinker in this study (starPCL having 4 arms and 5 repeating units per arm): yield: > 97%. Mn, SEC = 4900 g/mol with repeating units n = 5.1H NMR (CDCl3): d (ppm) ¼ 0.80–0.92 (m, H4); 1.32–1.48 (m, H3, H9); 1.58–1.72 (m, H8, H10); 2.31 (tr, H7); 3.22–3.36 (m, H1); 3.96–4.10 (m, H5, H11); 4.10–4.20 (tr, H11E) 5.82 (d, H14); 6.11 (dd, H13); 6.40 (d, H14). 2.3. Synthesis of microgels 2.3.1. Miniemulsion polymerisation The synthesis of microgels, illustrated in Scheme 2, is divided in three steps. To synthesize sample 1 (Table S1) at the first stage aqueous solution of surfactant (CTAB (0.005 g; 0.014 mmol) in 100 g water) is prepared and heated to 70 °C. VCL (1 g; 7.180 mmol) was melted at 70 °C and starPCL crosslinker (0.07 g; 0.025 mmol) was dissolved in the liquid monomer melt under nitrogen atmosphere. Both solutions were mixed by keeping the temperature at 70 °C to form a pre-emulsion. This pre-emulsion was further homogenized with MRT-CR5 microfluidizer (Microfluidics corp., USA) (1800 bar, 8 cycles) to form a stable miniemulsion. The obtained miniemulsion was then directly transferred into a double-wall glass reactor preheated to 70 °C and equipped with a 284

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Scheme 1. Two-step synthesis route to various starPCL crosslinkers: (i) polymerisation of ε-caprolactone with multifunctional alcohols as initiators; (ii) incorporation of acrylate end-groups by modification of terminal hydroxyl groups with acryloyl chloride.

N,N-methylenebis(acrylamide) BIS (0.07 g; 0.025 mmol) were dissolved in 100 g water at 70 °C under nitrogen atmosphere in a double-wall glass reactor equipped with a stirrer and reflux condenser. The watersoluble initiator AMPA (0.04 g; 0.147 mmol) was added and the polymerisation continued for 6 h under continuous stirring at 70 °C. The microgel dispersion was purified by dialysis (72 h) (Millipore Labscale TFF System) using a regenerated cellulose membrane with MWCO 10.000 g/mol.

Table 1 Main characteristics of starPCL crosslinkers used in present study. starPCL

Yield, [%]

DF, [%]

CL r.u.

Mn NMR [g/ mol]

Mn SEC [g/ mol]

Mw/Mn

star4PCL5* star4PCL10 star4PCL15 star6PCL5 star6PCL10 star6PCL15

98 98 99 97 98 99

93 95 98 99 98 99

5 10 15 5 10 15

2800 5000 7300 4000 7400 10700

4300 9000 14800 7400 14600 19500

1.34 1.40 1.33 1.23 1.24 1.12

2.4. Degradation of the microgel particles

DF: degree of functionalization; CL r.u.: ε-caprolactone repeating units per arm.

To investigate the degradation behaviour of the microgel particles the microgel solutions have been diluted down to a concentration of 8 mg/mL. For the degradation 15 mg of Candida rugosa (Sigma, EC 3.1.1.3, 2 U/mg) was added to the diluted microgel solution. The solution was kept at pH 7 and at 37 °C, which are the optimal conditions for the enzyme. Samples were taken out in several time intervals and investigated by DLS and FESEM.

stirrer, reflux condenser and was again purged with nitrogen. The water-soluble initiator 2,2′-Azobis-(2-methylpropionamidine) dihydrochloride (AMPA) (0.04 g; 0.147 mmol) was added and the polymerisation continued for 6 h under continuous stirring at 70 °C. The provided recipe was used for all syntheses, while varying two parameters like the crosslinker type and concentration (see Table 2). A detailed list of the used ingredients for all microgel samples can be found in the Supporting Information (Table S1). The microgel dispersions were purified by dialysis (72 h) (Millipore Labscale TFF System) using a regenerated cellulose membrane with MWCO 10.000 g/mol.

2.5. Loading of hydrophobic dye Nile Red in microgels To determine the efficiency of the microgels in the uptake of hydrophobic substances we used the water-insoluble fluorescent dye Nile Red as model substance. The dye was dissolved in a mixture of 2:1 acetone/THF (vol/vol) with a concentration of 0.3 mg/mL and 10 μL were successively added to the microgel dispersion (20 mg/mL). The dye uptake by microgels was investigated with UV/Vis spectroscopy using an aqueous microgel solution without dye as reference sample.

2.3.2. Precipitation polymerisation The synthesis of the reference microgels was performed via precipitation polymerisation according to the previously published procedure [16,41]. Sample 20 (Table S1, Supporting Information) was synthesized in following way. VCL (1 g; 7.180 mmol) and crosslinker

Scheme 2. Microgels with degradable hydrophobic domains. 285

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Table 2 Comparison of the droplet- and microgel size, the monomer and free polymer concentration in the reaction medium before and after polymerisation. Nr

crosslinker

21 12 14 6 20c a b c

BIS BIS/xPCL (50/50) BIS/xPCL (50/50) xPCL BIS

crosslinker amount

0.07 g; 6 mol% 0.07 g; 3 mol% 0.14 g; 6 mol% 0.3 g; 1.5 mol% 0.07 g; 6 mol%

before polymerisation

after polymerisation

d (PDI) emulsion at 70 °C

VCL in water [%]

193 nm 197 nm 203 nm 204 nm –

17.3 10.5 11.2 12.9 100

(0.098) (0.09) (0.11) (0.12)

a

d (PDI) microgel at 20 °C

sol fraction [%]b

345 nm 400 nm 650 nm 800 nm 720 nm

14.1 9.3 10.4 11.2 8

(0.08) (0.09) (0.09) (0.3) (0.05)

-% of total monomer used. -% of total polymer formed; d – microgel diameter. Precipitation polymerisation.

chemical design. The structure of the microgel particles is shown in Scheme 2. In our concept, the degradable hydrophobic star-shaped polymer crosslinkers integrated into the microgel network act as crosslink sites holding together the water-soluble polymer chains and simultaneously forming hydrophobic binding domains to load hydrophobic molecules (or potentially poor water-soluble drugs). During the degradation process the polymer network is decomposed to water-soluble polymer chains, crosslinker fragments and the payload is released into the aqueous phase. The biggest challenge in the chemical design of such microgels is an efficient and controlled incorporation of the hydrophobic polymeric crosslinker and its homogeneous distribution within the amphiphilic microgel. In this case it is not possible to use conventional precipitation polymerisation because of the poor water-solubility of the crosslinker.

2.6. Loading and release of (RS)-2-(4-(2-methylpropyl)phenyl)propanoic acid (ibuprofen) The procedure described above was used to study the loading of Ibuprofen, as model drug, which has a poor solubility in water. After the drug loading the microgels have been freeze-dried, to prevent any release. The release of the Ibuprofen from microgels was then studied by re-dispersing them in water followed by UV–Vis measurements with 10 min intervals. 2.7. Characterisation methods The size of the microgel particles was determined by dynamic light scattering (DLS) using ALV/LSE-5004 Light Scattering Multiple Tau Digital Correlator with the scattering angle set at 90°. The samples were also measured at different temperatures (from 10 °C to 60 °C) and the temperature fluctuations were below 0.1 °C. Prior to the measurement microgel samples were diluted with doubly distilled water and filtered through 2.5 μm filter. The sedimentation velocity of the microgel dispersions was measured with the separation analyser LUMiFuge (LUM GmbH, Germany). The samples have been measured in device specific Polycarbonate (PC) cuvettes at an acceleration velocity of 2000 rpm. The slope of the sedimentation curves was used to calculate the sedimentation velocity, which then is used to compare the colloidal stability of the different microgel dispersions. The samples were analysed by Field Emission Scanning Electron Microscopy (FESEM) with a Hitachi FE-SEM S4800. Two droplets of diluted microgel dispersion were placed and spread on a piece of aluminium foil and dried at room temperature. No metal sputtering was used during sample preparation. The FTIR-spectra of the samples were measured with a Thermo Nicolet Nexus 470 Fourier transform infrared spectrometer. The dried samples have been embedded in a potassium bromide matrix before measuring. The UV/Vis spectroscopy investigations have been done with a Jasco V-630 photometer. The different samples were measured against a reference sample and the data was analysed with the device software. 1 H NMR spectra were recorded on a Bruker AV 400 FT-NMR spectrometer at 400 MHz. Deuterated chloroform (CDCl3) was used as solvent, and tetramethylsilane (TMS) as internal standard.

3.1. Synthesis of microgels The polymerisation method used for the synthesis of microgels in present study is very similar to miniemulsion polymerisation (Scheme 3). Firstly, we prepared a miniemulsion consisting of molten VCL and starPCL crosslinker droplets stabilized by a surfactant in aqueous phase at 70 °C. After addition of the water-soluble initiator, the polymerisation in droplets occurs as well as the formation of the colloidal networks crosslinked by starPCL. Since VCL shows no tendency to self-crosslinking unlike NIPAm [42], we assume that microgels are crosslinked only by star polymers. After consumption of the monomer in the droplets, the formed microgels are in a collapsed state because the reaction temperature is far above the VPTT of PVCL microgels in water (32 °C [19]). When the temperature is reduced below VPTT, the microgels swell and remain stabilized in aqueous phase by the initiator charges incorporated into the polymer chains as well as the surfactant molecules that are still present in the reaction mixture. In a classical miniemulsion process, the monomer is not soluble or has very low solubility in water. In the present system we used VCL as a monomer for the synthesis of microgels due to the good biocompatibility of the PVCL [5,21]. VCL is soluble in water exhibiting a solubility value of 40 g/L at 20 °C. The amount of VCL used for the polymerisation process in our system can be completely dissolved in the water used as continuous medium. However, the dissolution of VCL in water is quite slow and this allows preparation of a colloidally stable miniemulsion at 70 °C, which consists of molten VCL confined in droplets stabilized by surfactant molecules. In addition, VCL in liquid form is an excellent solvent for the hydrophobic starPCL crosslinkers what allows their homogeneous distribution within miniemulsion droplets. We performed a series of experiments to characterize the VCL/water emulsions to answer the following questions: a) how stable is the VCL/water emulsion? b) what is the concentration of VCL in the continuous phase (water) after the emulsification process before the initiator was added? and c) what is the amount of non-crosslinked polymer (sol fraction) in water after polymerisation? The experiments performed include

3. Results and discussion In the present study, we aimed at the synthesis of a new class of multicompartment microgels for drug delivery applications. Our goal was the synthesis of microgels that can be loaded with hydrophobic molecules and degraded by hydrolytic or enzymatic pathways thus inducing the release of the payload. The novelty of our concept is that both functions, namely degradability as well as the ability to solubilize hydrophobic substances are achieved by the crosslinker with a special 286

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Scheme 3. Illustration of the microgel synthesis process: a) N-vinylcaprolactam droplets containing dissolved starPCL crosslinker are stabilized in aqueous phase at 70 °C by surfactant molecules (not shown); b) after addition of water-soluble initiator polymerisa tion/crosslinking in monomer droplets takes place at 70 °C (above VPTT); c) when polymerisation is completed and temperature is reduced to 20 °C (below VPTT) microgels swell due to the intensive water uptake.

in water. It should be noted that no organic solvents are used during this polymerisation process. After the monomer consumption, the microgel particles are well dispersed in water. This modified emulsion method for the synthesis of microgels allows the incorporation of hydrophobic monomers and crosslinkers in hydrophilic microgels and provides an interesting option to obtain polymer colloids with extraordinary properties.

synthesis of microgels by classical precipitation polymerisation in presence of the crosslinker BIS (sample 20, Table S1 Supporting Information) and series of microgels prepared by a miniemulsion route using starPCL (405) or a mixture of starPCL (405) and bisacrylamide (BIS) (1:1) (samples 12–19, Table S1 Supporting Information). For all samples, we measured the concentration of VCL in water before addition of the initiator, as well as the concentration of the linear polymer chains in water after polymerisation, and the size of the monomer droplets and microgels. The experimental data are presented in Table 2. The time-dependent behaviour of the VCL/water emulsion was investigated by DLS measurements at 70 °C without adding the initiator. The droplet size was measured every 10 min for 4 h. The experimental data indicates that the droplet size remains constant for all investigated samples (Fig. S5, Supporting Information). Furthermore the droplet size is not influenced by the crosslinker amount. Nevertheless, the microgel diameter after the polymerisation is two to three times larger than the diameter of the emulsion droplets. This is because of the fact that the microgel formation takes place in the molten VCL droplet, excluding water at a temperature above the VPTT. After the polymerisation is completed, the microgel particles are formed and the dispersion cools down to a temperature below the VPTT, the microgels are swollen and therefore bigger than the emulsion droplets. Thus, the diameter of the droplets correlates with the diameter of the collapsed microgels at 50 °C. Additionally, we analysed the amount of monomer that can be found in the water phase before the polymerisation. This was done by separation of the VCL phase by centrifugation of the emulsion followed by freeze-drying of the supernatant to determine the amount of VCL in water. As shown in Table 2, the amount of the VCL that was found in water before polymerisation is between 10% and 13% from the total monomer amount for the samples in which starPCL crosslinker was used. Interestingly, for the sample in which BIS was used in the miniemulsion process (sample 21) the amount of the VCL in water was considerably higher (17%). The microgel samples were dialysed after the polymerisation process to separate non-reacted monomers and noncrosslinked polymer chains from microgels. Table 2 indicates a good correlation between the VCL amount in water before and the amount of polymer after the polymerisation. Interestingly, the microgel sample prepared by conventional precipitation polymerisation (sample 20) exhibited almost the same amount of non-crosslinked polymer. These experimental data support strongly the hypothesis that VCL remains in droplet form when polymerisation starts and the formation of polymer particles occurs in monomer droplets similar as in the standard miniemulsion polymerisation process. We assume that the polymerisation rate of VCL in droplets is much higher than dissolution of the monomer

3.2. Incorporation of starPCL crosslinkers into microgels After the synthesis of the microgels, the first important step was to prove that the starPCL crosslinker was incorporated into the microgels. We found that NMR is a useful analytical tool to demonstrate the integration of starPCL crosslinkers into microgel network. Fig. 1 shows two 1H NMR spectra; one spectrum of the starPCL (405) crosslinker with 4 arms each consisting of 5 caprolactone (CL) units and one spectrum of the microgel particles prepared in the presence of starPCL (405). The broad peaks in the microgel spectrum are due to a reduced mobility of the chains in the crosslinked molecules. In general, the PVCL peaks overlap with the peaks of the crosslinker since they have similar positions. Only the methyl-group (number 4 in structure B) at δ = 0.81 ppm is separated and still visible in the microgel spectrum. The signals of the double bonds are not visible in the microgel spectrum, which indicates that all are consumed during the polymerisation process. Moreover, the NMR investigation shows clear evidence that the starPCL crosslinker was successfully incorporated into PVCL microgels and no residual vinyl group can be found in polymer structure. StarPCL crosslinkers with variable architectures (arm number) and molecular weights were synthesized to investigate the influence of the crosslinker structure on the incorporation efficiency and polymerisation yield. Table 3 summarizes the crosslinkers used in the present study. The number of caprolactone repeating units per arm was 5 and 15, and the arm number was adjusted to 4 and 6. The polymerisation yields for the microgel synthesis at crosslinker concentration 0.025 mmol decrease with the increase of the molecular weight and arm number of xPCL (Table 3). A reason for this might be a restricted mobility of the crosslinker macromolecules in the monomer mixture that leads to increase of the crosslink inhomogeneity and higher fraction of the noncrosslinked PVCL chains within the microgels, which were removed during dialysis. Fig. 2 shows the 1H NMR spectra of the microgels synthesized with various types of xPCL crosslinkers. In the samples 405/0.5–405/3, the amount of starPCL (4 arms, 5 CL repeat units per arm) is varied from 0.05 g to 0.3 g (from 0.25 mol.-% to 1.5 mol%). In the microgel samples 605/1.0 and 415/1.0, starPCL crosslinkers (0.1 g (0.5 mol%)) with 6 287

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Fig. 1. Chemical structures of starPCL(405) crosslinker (B) and PVCL microgel crosslinked by starPCL(405) (A) on the left with the corresponding 1H NMR spectra on the right.

to a lower incorporation efficiency of crosslinker into microgel network. Therefore, we selected a starPCL crosslinker 405 with 5 caprolactone repeating units per arm and 4 arms for further studies. The microgels synthesized in presence of starPCL crosslinkers were also analysed by FTIR spectroscopy. Fig. 3A shows the spectrum of a sample with a starPCL content of 0.1 g (0.5 mol%). The spectrum has been normalised to the amide band. The v (OH ) absorption band at 3450 cm−1 results from residual water after freeze-drying and uptake of air moisture into the microgel network. Beside the v (CH2 , CH3) absorption bands at 2927 and 2856cm−1 and v (CN ) at 1479cm−1, two typical bands are labelled. At 1637cm−1 the amide band appears, indicating the presence of PVCL. Since PVCL is the major component, this absorption band is the strongest. At 1735 cm−1the carbonyl signal of the ester can be observed, confirming the presence of the crosslinker in the microgel. With higher crosslinker concentration, the intensity of this peak increases (Fig. 3B). Fig. 1S (Supporting Information) shows complete FTIR spectra of the microgels synthesized with various starPCL amounts. The theoretical and experimental ester:amide ratios are in a good agreement indicating controlled incorporation of crosslinker into microgels. The FTIR studies support the NMR results and this correlation confirms that the microgel synthesis with starPCL as crosslinker was successful. Furthermore, based on the experimental data presented above, we can conclude an increasing amount of starPCL in the miniemulsion droplets leads to an increase of the amount of incorporated crosslinker within microgels.

Table 3 Influence of the crosslinker structure on the polymer yield during microgel synthesis. Nr

Crosslinker type

arm number

CL repeating units per arm

Mn (NMR), [g/mol]

polymer yield, [%]

1 2 7

405* 415 605*

4 4 6

5 15 5

2749 7315 4003

73.1 17.9 6.7

3.3. Microgel size, size distribution and swelling in water

Fig. 2. 1H NMR spectra of microgels with various starPCL types (frame shows the spectral region where the broadening of the signals is observed).

Further analysis of the microgels was carried out by DLS and FESEM. Fig. 4A and B show the size distribution spectra for the microgels synthesized with different starPCL and starPCL/BIS crosslinker concentrations respectively. Microgels that are crosslinked only by starPCL have a bimodal size distribution for low starPCL contents. The microscopy images in Fig. 4C and E prove that the size distribution can be improved by increase of xPCL content up to 0.3 g(1.5 mol%). The reason for the bimodal size distribution is probably not sufficient crosslinking at low starPCL contents and leaching of the PVCL polymer chains into aqueous phase forming small aggregates. In additional experiments, we tried to combine starPCL with BIS, which is a conventional crosslinker for the microgel synthesis [16,41]. By using starPCL/ BIS mixture, we obtained microgels with a narrow size distribution. Later on, we synthesized microgels with different starPCL:BIS ratios to find an optimal crosslinker composition. As it is shown in Fig. 4B, particles with a narrow size distribution can be obtained. The best

arms and 5 CL repeating units per arm and one with 4 arms and 15 CL repeating units per arm have been incorporated. By analysing the NMR spectra of samples 405/0.5–405/3, we notice that the signal assigned to the protons of the caprolactam ring broadens with an increase of starPCL crosslinker. Peak broadening is usually an indication for reduced chain mobility that is originated from the higher crosslink density. Indeed, the more crosslinker we add, the denser the microgels becomes what is reflected in the lower mobility of polymer chains. A direct comparison of the crosslinker with 4 arms and with 15 caprolactone repeating units or with 6 arms with 5 caprolactone repeating units to the 405 crosslinker shows in the calculated integrals no further obvious broadening of signals in the NMR spectra. Taking into account the low yields of the synthesis (Table 1), we assume that the increase of the molecular weight and increase of the arm number lead 288

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Fig. 3. FTIR spectrum of the synthesized microgels with 0.1 g (0.5 mol%) starPCL 405 (A); close-up of a part of the spectrum, which shows the increase of the ester group signal intensity indicating increase of starPCL content in microgels (B).

polydispersity index, the sedimentation velocity and the VPTT (measured by DSC) for various microgel samples. The size of the microgels in water is determined by the swelling degree. With an increase of the crosslinking density, the particles become stiffer, so that their ability to swell decreases. It has been reported that with a higher amount of crosslinker also the microgel size increases [43]. In our case, the bulky and hydrophobic crosslinker leads to a more hydrophobic microgel interior which induces repelling of water due to the formation of hydrophobic domains [44,45]. The size of the microgels increases with an increasing amount of starPCL, whereas it has also to be taken into account that the particle size distribution, expressed by the PDI, broadens. Looking at the microgel samples crosslinked only with starPCL the

particle quality can be achieved with a starPCL/BIS ratio of 50:50 wt%. This is additionally confirmed by the FESEM picture in Fig. 4D, which shows that the microgels are not agglomerated and are uniform in size. This observation is similar in the case of higher total amounts of crosslinker, were also particles with a narrow size distribution can be found at starPCL/BIS ratio of 50:50 wt%. Additionally, it should be noted that the use of BIS in the synthesis leads to yields up to 90% for a crosslinker ratio of 50:50, which is 20% higher than without BIS (compare with Table 3). The combination of two crosslinkers in the polymerisation process allows the synthesis of microgels with tuneable amounts of permanent and degradable crosslinks. Table 4 shows the values of the hydrodynamic radius, the

Fig. 4. Size distribution curves for microgels synthesized with starPCL (A) and starPCL + BIS (B); FESEM images of microgels with: low amount (0.07 g; 0.35 mol%) of starPCL (C), 50:50 ratio of BIS and starPCL (0.07 g; 3 mol%) (D) and high amount (0.3 g; 1.5 mol%) of starPCL (E). 289

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Table 4 Hydrodynamic radius (Rh), polydispersity index (PDI), sedimentation velocity (ν) and volume phase transition temperature (VPTT) for different microgel samples. Nr

crosslinker

Crosslinker amount

Rh [nm]

PDI

ν [μm/s]

VPTT (DSC) [°C]

21 12 18 14 16 17 3 4 6

BIS xPCL/BIS(50/50) xPCL/BIS(66/33) xPCL/BIS(50/50) xPCL/BIS(66/33) xPCL/BIS(70/30) xPCL xPCL xPCL

0.07 g; 6 mol% 0.07 g; 3 mol% 0.07 g; 3 mol% 0.14 g; 6 mol% 0.14 g; 6 mol% 0.14 g; 6 mol% 0.07 g; 0.035 mol% 0.1 g; 0.5 mol% 0.3 g; 1.5 mol%

175 200 275 350 375 400 325 350 420

0.08 0.09 0.2 0.09 0.3 0.36 0.9 0.75 0.2

0.8 1.1 1.4 1.7 2.1 4.8 5.3 4.8 7.5

32.5 32.8 32.6 33.1 33.3 33.2 32.8 33.1 32

υ-sedimentation velocity at 20 °C.

3.4. Enzymatic degradation of microgels

effect of an increasing particles size with an increasing amount of crosslinker seems to be more obvious, since the PDI decreases at the same time. In this case, the higher amount of crosslinker leads also to larger microgel sizes. It should be noted that the increase of the starPCL concentration increases also the size of miniemulsion droplets as proved by DLS measurements (Table S2, Supporting Information). The reason for this effect is probably the increase of the viscosity of molten VCL due to the starPCL addition. To demonstrate that the synthesized microgels are colloidally stable, we analysed the sedimentation behaviour of the microgels by measuring their sedimentation velocity (Fig. S2, Supporting Information). The sedimentation velocity values measured in the accelerated sedimentation process at 4000 rpm are very low (Table 4). The increase of the starPCL content within the microgels increases their sedimentation velocity due to the increase of the microgels size and density (see Fig. S3, Supporting Information for more details). Another important property of microgels is their temperature-responsive behaviour in water. VPTT of microgels was investigated by DLS and differential scanning calorimetry (DSC). The DSC results are summarized in Table 4. Surprisingly, the presence of starPCL does not shift the VPTT of the microgels to lower temperatures as it may be expected due to the enhanced hydrophobicity of the microgel interior. Fig. 5 shows the swelling ratio of four different samples as a function of the temperature. The swelling ratio is defined by the hydrodynamic radius Rh divided by the hydrodynamic radius at 50 °C (Rh0) of each sample. These data show that the microgels still show the thermosensitive behaviour even crosslinked with 0.3 g (1.5 mol%) starPCL. Furthermore we notice that both samples with starPCL have a higher swelling degree, which can be explained by a lower crosslinking density.

The obtained microgels are supposed to undergo degradation due to the degradability of the PCL backbone of the starPCL crosslinker. To prove this, we added the enzyme Candida rugosa to the buffered microgel solutions and investigated size and morphology of the polymer particles by means of DLS and FESEM over 40 days. Fig. 6 B-E shows the FESEM pictures of microgels prepared with starPCL crosslinker (0.3 g; 1.5 mol%) after 11 d (B) and 30 d (C) of degradation, microgels with a 50:50 wt% ratio of xPCL and BIS (D) and microgels prepared only with BIS (0.07 g) (E, reference sample). The microscopy images show clearly that microgels with starPCL in their structure loose their spherical shape upon degradation and form non-defined polymer aggregates. Microgels synthesized with 50:50 wt% starPCL/BIS degrade only partially and the deformation effects are not very strong (Fig. 6D). Contrary, microgels crosslinked only with BIS show no visual changes even after 40 days of enzymatic treatment. Fig. 6A shows DLS measurements of the microgel particles prepared with 0.3 g (1.5 mol%) of starPCL. The size distribution curves of the samples after different time intervals during the enzymatic degradation are presented. Before the degradation begins, the size distribution is monomodal and narrow. After some days the peak begins to broaden and to form a bimodal shape towards smaller radii. As the degradation takes place, the intensity of the original peak decreases and the intensity for smaller radii increases followed by the appearance of particles of 10 nm radius. The main peak disappears after 33 days of degradation as well as the 10 nm particles and the later appearing peak at even smaller radii some days later. The microgel degradation process is different from particles that consist solely of a degradable polymer. These colloids usually show continuous decrease of the particle size by the stepwise degradation of their surface [46–48]. The microgel particles synthesized in the present study consist of non-degradable polymer chains crosslinked by a degradable crosslinker and therefore the degradation process is more complex. It shows a broadening of the particle size distribution as well as appearing and vanishing peaks, instead of a simple shift to smaller sizes. In addition, we used 1H NMR spectroscopy to study the degradation products. The spectrum of the degradation product mixture as well as a spectrum of the VCL monomer for comparison is shown in Fig. S4 (Supporting Information). Traces of signals of the crosslinker can be still found and other signals are not present or covered by the dominant signals of the PVCL chains.

3.5. Solubilisation of hydrophobic organic molecules in microgels To prove that the incorporation of starPCL in the microgels gives the possibility to load hydrophobic substances, we used Nile Red; a completely water insoluble dye, as a model substance as well as (RS)-2-(4(2-methylpropyl)phenyl)propanoic acid (Ibuprofen) as an example drug that exhibits pH-dependent solubility in water. The loading capacity of the microgels with Nile Red and Ibuprofen is shown in Fig. 7 and Fig. 8 respectively. As reference sample, a microgel crosslinked only with BIS

Fig. 5. Variation of the swelling ratio with temperature for microgels crosslinked by starPCL, BIS or starPCL/BIS mixture. 290

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Fig. 6. Size distribution curves (DLS) for microgels at different degradation times (microgel sample synthesized with 0.3 g starPCL) (A). FESEM images of microgels (starPCL 0.3 g) after 11 days (B) and after 30 d (C) of degradation; microgels (BIS 0.035 g; starPCL 0.035 g) after 12 days (D) and microgels (BIS 0.07 g) after 40 days (E) of degradation.

starPCL-modified microgels after 90 min is in a similar time range like already shown in other studies [50–52]. Overall, we note that the incorporation of starPCL in the microgels reduces the release rate and increases the time needed until complete release. At pH 1.2, we observe a burst release of about 20% for each sample and afterwards a much slower release compared to pH 7.4. The burst release of the Ibuprofen within first 10 min is probably due to the faster dissolution of the drug molecules adsorbed at the microgel surface. For all samples after 4 h, almost 40% of the loaded Ibuprofen has been released. An extrapolation of the slope leads to a time of 10–14 h until complete release. The difference in the release behaviour between pH 7.4 and 1.2 can be explained by the difference in the solubility of the Ibuprofen [50]. The experimental data presented above indicate that synthesized microgels exhibit nanostructured interior and very good solubilizing activity towards hydrophobic organic molecules. In combination with the biocompatibility and degradability features, the synthesized microgels are interesting candidates for carrier systems in biomedical applications.

was used. UV–Vis spectroscopy allows monitoring of Nile Red solubilized in the microgels by following the peak intensity at 550 nm, which is a characteristic feature of this fluorescent dye (depending on the solvent system) (Fig. 7A). Microgels crosslinked with starPCL can be loaded with Nile Red and show the increase of saturation concentration depending on the starPCL concentration in the microgel (Fig. 7B). The efficient uptake of Nile Red and the dependency on the starPCL concentration are clear evidence that the starPCL crosslinker generates hydrophobic pockets in the microgel structure and they can be used to immobilise hydrophobic substances. In the case of Ibuprofen, the general behaviour is the same as for Nile Red. An increase of the saturation concentration with increasing starPCL concentration can be seen (Fig. 8A). The reference sample prepared with BIS (without starPCL crosslinker) shows also by far the lowest uptake, but in difference to Nile Red a small amount of Ibuprofen can be loaded, probably due to the fact that Ibuprofen is less hydrophobic compared to Nile Red. These experiments give clear evidence that the incorporation of starPCL in the microgel network allows the generation of hydrophobic pockets, which can immobilise hydrophobic substances like drugs or dyes. The release of Ibuprofen from microgels was studied at pH = 1.2 and pH = 7.4 and 37 °C to reflect the gastric and intestinal conditions. The solubility of Ibuprofen is pH dependent: at pH 1.2 it is approximately 10−4 mol/L and at pH 7.4 it is 10−2 mol/L [49]. The solubility is in both cases not very high, but also in both cases the saturation concentration was higher than the loaded amount of Ibuprofen in 15 mg of microgel, which have been used for the UV/Vis measurements. Fig. 8B shows the release of Ibuprofen from microgel samples with and without starPCL. It is obvious that at pH = 7.4 with an increasing amount of starPCL the release takes much longer compared to the microgel without starPCL. The complete release of Ibuprofen from

4. Conclusions In this work, we developed a new method to synthesize biocompatible degradable aqueous microgels able to solubilize hydrophobic organic molecules. We describe the synthesis of PVCL microgels with degradable hydrophobic pockets. The microgels are designed by integration of a star-shaped acrylate end-functionalised PCL crosslinkersin microgel networks. The microgels were synthesized with different amounts of starPCL as well as with various ratios of conventional crosslinker BIS and starPCL. We found that microgels solely crosslinked with starPCL do not form monomodal particles unless a high amount of starPCL of 0.3 g (1.5 mol%) is used. Also higher amounts than 50% of starPCL in crosslinker mixtures with BIS do not form particles with a 291

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Fig. 7. UV–Vis spectra of microgel sample loaded with different amounts of Nile Red (A). Loading capacity of various microgel samples with Nile Red (B) (The scheme shows the experimental procedure to determine the solubilisation capacity of microgels).

innovative nanocarrier can be presented as a promising material for further studies in the field of drug delivery.

very narrow particle size distribution. The hydrodynamic radii of the particles vary between 180 and 400 nm depending on the crosslinker concentration. The microgels show a temperature-sensitive behaviour in water. The VPTT of microgels is not strongly influenced by the hydrophobic crosslinker. The enzymatic degradation of the microgels crosslinked only by starPCL takes place in a period of 40 days, as proven by FESEM and DLS analysis. It has been shown that the uptake of Nile Red and Ibuprofen into to microgels can be controlled by the amount of hydrophobic domains generated by starPCL. In conclusion, this

Acknowledgement The authors thank Volkswagen Foundation and Deutsche Forschungsgemeinschaft with Collaborative Research Centre SFB 985 “Functional Microgels and Microgel Systems” for financial support of this research.

Fig. 8. Loading capacity of microgel samples with different xPCL concentrations (A) and release of Ibuprofen from the microgels at pH = 7.4 and pH = 1.2 at 37 °C (B). 292

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Appendix A. Supplementary data

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