Rapid Synthesis of Dual-Responsive Hollow Capsules with

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

Rapid Synthesis of Dual-Responsive Hollow Capsules with Controllable Membrane Thickness by Surface-Initiated SET-LRP Polymerization Xiaoling Liu,†,‡ Dietmar Appelhans,*,† Tao Zhang,‡ and Brigitte Voit*,†,‡ †

Leibniz-Institute für Polymerforschung Dresden e.V., Hohe Straße 6, D-01069 Dresden, Germany Organic Chemistry of Polymers, Technische Universität Dresden, D-01062 Dresden, Germany

‡

S Supporting Information *

ABSTRACT: We present a facile and highly effective route to construct a dual-responsive polymeric capsule with photo-crosslinkable property based on surface-initiated single electron transfer living radical polymerization (SI-SET-LRP) exploiting silica particles as templates, dimethylaminoethyl methacrylate (DMAEMA) as dual-responsive component, and 2-hydroxy-4(methacryloyloxy)benzophenone (BMA) as an effective photocross-linker. This approach is highly efficient with complete monomer conversion in 15 min at ambient temperature resulting in wall thickness of 55 nm and usable in technical applications. Hollow capsules are available after photo-cross-linking of polymeric shell and removing silica particle, of which the morphology and composition were confirmed by employing a range of techniques, such as FTIR, TGA, TEM, SEM, cryo-TEM, DLS, GPC, and UV−vis spectroscopy. Thus, it represents a significant advance in the development of complex polymeric capsules synthesis usable for various applications (e.g., biotechnology and systems biology).

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INTRODUCTION Hollow capsules have gained intense research interest for wide applications in many fields, including artificial cells, mimicking cellular functions, biomedicine, nanoreactors, controlled drugdelivery systems, catalysis, and photonic crystals.1−7 Very recently, there are existing various routes used to prepare hollow capsules,8−13 such as emulsion polymerization,14 phase separation,15 layer-by-layer technique,16 or a directed selfassembly method.17−19 Among them, the surface-initiated living radical polymerization (SI-LRP) using sacrificial particles as templates20,21 is especially popular because of its unexceptionable controllability of the molecular weight and dispersity of the grafted polymers.22 So, various surface-initiated LRP techniques have been applied very successfully in the synthesis of hollow capsules, mainly surface-initiated atom transfer radical polymerization (SI-ATRP),21,23,24 surface-initiated reversible addition− fragmentation chain transfer polymerization (SI-RAFT),25,26 surface-initiated nitroxide-mediated polymerization (SINMP),27 and surface-initiated distillation−precipitation polymerization.28,29 However, the limitations of current surfaceinitiated LRP techniques, such as complex and onerous process,22,23,25 strict reaction conditions,21 numerous purification steps,26,30 time-consuming (>15 h),21,23,26,31 poor yields (1.3 for 20 000 g/mol polymer,26,28 have a detrimental effect on a wide range of applications. A preferable alternative route to achieve attached polymers with a higher grafting density is to proceed via a continuous © XXXX American Chemical Society

polymerization of monomers from an easily modified initiator by single electron transfer living radical polymerization (SETLRP), pioneered and continuously further developed by Percec and co-workers.32−37 In this context a novel protocol from Haddleton’s group for the polymerization of acrylamide monomers in aqueous solution has to be mentioned, exploiting the in situ fast and complete disproportionation of Cu(I)Br in the presence of Me6Tren to yield insoluble Cu(0) and Cu(II)Br2 prior to the addition of monomer and initiator.38 Generally, the described SET-LRP method is not only rapid but also results in high control over the polymerization with extremely high livingness at quantitative conversions, while it is performed under mild reaction conditions and overall in a simple experimental setup.36−38 Inspired by the SET-LRP method, herein, we report for the first time the use of surface-initiated SET-LRP (SI-SET-LRP) to synthesize an intelligent polymer capsule with temperature and pH dual-responsiveness (Scheme 1). Thus, dimethylaminoethyl methacrylate (DMAEMA) monomer was selected as dual-responsive component in the final polymeric membrane. Because of their tertiary amine groups, it can be charged or uncharged at low or high pH, respectively, while it also resulted in a noticeable pH-dependent thermosensitivity behavior usable for our final capsules in aqueous solution.39 Received: November 20, 2017 Revised: January 3, 2018

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DOI: 10.1021/acs.macromol.7b02347 Macromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Fabrication of Photo-Cross-Linked Dual-Responsive Hollow Capsules via Surface-Initiated Single Electron Transfer Living Radical Polymerization (SI-SET-LRP): (a) Silica Particle Modified with 3-(Ethoxydimethylsilyl)propylamine and 2Bromoisobutyryl Bromide Used as Surface Initiator; (b) Disproportionation of CuBr/Me6TREN Yields Insoluble Cu(0) and Cu(II)Br2 in DMSO/H2O; (c) Copolymer of Dimethylaminoethyl Methacrylate (DMAEMA) and 2-Hydroxy-4(methacryloyloxy)benzophenone (BMA) Grafted on Silica Surface with SI-SET-LRP; (d) Polymeric Membrane Stabilization by Photo-Cross-Linking and Core Removal for Hollow Capsule Formation

with 3-(ethoxydimethylsilyl)propylamine. In this process, the particles were first washed five times with ethanol and finally dispersed in anhydrous toluene. Then 3-(ethoxydimethylsilyl)propylamine (1 g) as a coupling agent was added to a round-bottom flask with a suspension (16 mL) of 5 wt % silica particles, and the suspension formed was agitated under 250 rpm and refluxed for 24 h at 70 °C.After centrifugation of the reaction mixture at 8000g for 3 min, aminofunctionalized silica particles were washed at least four times with ethanol via centrifugation/redispersion cycles followed by washing twice with anhydrous THF and dispersing directly into THF for subsequent use. Preparation of SET-LRP Initiator Anchored SP. The aminofunctionalized particles were reacted with 2-bromoisobutyryl bromide to attach the SET-LRP initiators via amide bond formation. In this process, the THF solution of the amino-functionalized silica particles (16 mL, 50 mg mL−1) and triethylamine (2.09 mL, 15 mmol) in dry THF (24 mL) was stirred under a N2 atmosphere in a round-bottom flask. Then 2-bromoisobutyryl bromide (1.24 mL, 10 mmol), diluted in 5 mL of dry THF, was finally added dropwise to the reaction solution over 15 min. The reaction mixture was stirred for 48 h at room temperature. The particles were separated from the reaction solution by centrifugation at 8000g for 3 min. Then the isolated particles were washed with ethanol. The centrifugation and washing step was repeated at least five times until the supernatant layer after centrifugation was colorless. Finally, the SET-LRP initiator anchored silica particles were washed twice with DMSO/H2O (v/v 15%) and dispersed in DMSO/H2O (v/v 15%) for subsequent use. An aliquot of the SET-LRP initiator-anchored silica particles was dried and subjected to thermal gravimetric analysis (TGA) and IR to determine the amount of SET-LRP initiator on the silica particles. From the TGA, the density of the SET-LRP initiator on the particle surface was calculated from eq 1:

The additional use of 2-hydroxy-4-(methacryloyloxy)benzophenone (BMA) as an effective photo-cross-linker only requires a short and mild UV irradiation to preserve the spherical shape of hollow capsules.16,40,41 Adapting the method of Haddleton et al.,38 we can achieve a precisely defined membrane structure with a wall thickness of 55 nm in a quantitative mannertypically full conversion within 15 min of reaction time with higher molecular weight and narrow molecular weight distributions at ambient temperature. We propose that this easy approach represents a significant step toward fundamental goals in the field of polymer structures and technology.

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EXPERIMENTAL SECTION

Materials. All reagents and solvents were purchased from commercial suppliers and used as received unless otherwise noted. 3-(Ethoxydimethylsilyl)propylamine, 2-bromoisobutyryl bromide, triethylamine, copper(I) bromide (CuBr), tris[2-(dimethylamino)ethyl]amine (Me6TREN), 2-(dimethylamino)ethyl methacrylate (DMAEMA), ethyl α-bromoisobutyrate (EBiB), monosodium phosphate, disodium phosphate, hydrofluoric acid (HF), ammonium fluoride (NH4F), D-maltose monohydrate (Mal), and rhodamine B isothiocyanate were purchased from Sigma-Aldrich. 2-Hydroxy-4(methacryloyloxy)benzophenone (BMA, 99%) was purchased from Alfa Aesar. Hyperbranched poly(ethylene imine) (PEI 5, Mw = 5000 g mol−1) and hyperbranched poly(ethylene imine) (PEI 25, Mw = 25 000 g mol−1) were obtained from BASF SE (Ludwigshafen, Germany). 500 nm diameter SiO2 particles (5 wt %) were obtained from Microparticles GmbH, Germany. Dialysis tubes made from regenerated cellulose (MWCO 5000 and MWCO 150 kDa) were purchased from Carl Roth. High purity and resistivity (>18 MΩ cm) deionized water (Milli-Q water) were obtained from an inline Milli-Q Reagent water purification system (Millipore Corporation) and was used in all the reactions, solution preparations, and polymer isolations. All other chemicals were obtained from Acros Organics, and anhydrous solvents were stored over molecular sieves. Preparation of Amino-Functionalized Silica Particles (SP). To attach the SET-LRP initiators onto the surface of the silica particles, the particles were first amino-functionalized through reaction

density of the initiator =

w × 106 Mn

(1)

where w is the mass of initiator grafted on the SP and Mn is the number-average molecular weight of the initiator. By calculating the values from eq 1, the density of the SET-LRP initiator on the particle surface was determined to be about 25 μmol per gram of SET-LRP initiator-functionalized SP. B


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Mal 5 (c = 2 mg mL−1) and PEI-Mal 25 solution (c = 6 mg mL−1), respectively. The mixture was shielded from light and stirred for 12 h in PBS buffer pH 6.5 at room temperature. The nonencapsulated PEIMal is removed by dialysis against a membrane with MWCO = 150 kDa for 3 days in phosphate buffer pH 8.5 at 45 °C (three times a day, a standard procedure for dialysis). After the time points, the samples were taken and analyzed using UV−vis (500 nm) according to the calibration curves of rhodamine B labeled PEI-Mal 5 and PEI-Mal 25 in phosphate buffer at pH 8.5 to confirm the successful removal of the free rhodamine B. The experiments were carried out in triplicate. The loading efficiency was calculated from eq 3:

SI-SET-LRP Polymerization from Initiator-Anchored SP. To a Schlenk tube fitted with a magnetic stir bar and a rubber septum, DMSO/H2O (1.5 mL, v/v 15%) and tris[2-(dimethylamino)ethyl]amine (Me6TREN, 0.0011 mmol) were charged, and the mixture was bubbled with nitrogen for 30 min. In an additional flask, CuBr (0.0022 mmol) was dried for 30 min in a vacuum and flushed with nitrogen before transferring the Me6TREN solution of the first flask to this flask. The mixture immediately became blue (CuII), and a purple/red precipitate (Cu0) was observed. Then the blue suspension with purple red color copper(0) powder was allowed to stir at 480 rpm for 30 min. At the same time, to another vial fitted with a magnetic stir bar and a rubber septum, DMSO/H2O (1.5 mL, v/v 15%), 2-(dimethylamino)ethyl methacrylate DMAEMA monomer (1.4 mmol), EBIB (32.0 μmol), and the photo-cross-linkable monomer BMA (0.14 mmol) were charged, followed by adding the DMSO/H2O (v/v 15%) solution of the SET-LRP initiator-anchored silica particles (5 mL, 20 mg/mL), and then the mixture was bubbled with nitrogen for 30 min. After that, the degassed monomer/initiator solution was transferred through the septum to the Schlenk tube with Cu(0)/CuBr2/Me6TREN catalyst under nitrogen protection. The Schlenk tube was sealed, and the mixed solution was allowed to set at room temperature for the desired period (3, 6, 9, 12, or 15 min). The polymerization was stopped by in air; then the copolymer grafted silica particles were separated from the reaction solution and free polymer by centrifugation and additionally washed with ethanol. Both last steps were repeated at least five times. An aliquot of the copolymer grafted silica particles was dried and subjected to thermal gravimetric analysis (TGA) and IR. After complete degradation of the silica template, the grafted copolymer was analyzed by 1H NMR and GPC. From the TGA, the graft density (σ) was estimated from eq 2: σ=

loading efficiency of PEI‐Mal =

(3) In Vitro Release Studies for PEI-Mal 5 and PEI-Mal 25. As a general rule, all experiments were carried out in triplicate, and the average values were plotted. For the release behavior of capsules, the PEI-Mal loaded capsules were immersed in solutions to study the effects of pH and temperature on release kinetics. Thus, 0.01 M PBS at pH 6.5 and 0.01 M phosphate buffer at pH 8.5 were used as release media. PEI-Mal loaded capsules solution (5 mL) was transferred to dialysis tube (MWCO 150 kDa). The dialysis tube was sealed and then allowed to stir (200 rpm) in beaker containing 2 L of the corresponding release medium at 45 and 25 °C, respectively. At selected interval times, samples were removed from the dialysis tubes and quickly analyzed by UV−vis spectroscopy (500 nm) and returned back into the dialysis tube. The amount of PEI-Mal macromolecules released was calculated from the amount of PEI-Mal macromolecules initially present in the capsules and the amount of PEI-Mal macromolecules retained in the capsules at each sampling point. Characterization Methods. The molar weight and their dispersity (Đ) of the copolymers were determined using a Polymer Laboratories PL-GPC50 Plus Integrated GPC system (Varian Inc., UK) equipped with a PL data stream refractive index detector, a Polymer Laboratories pump, a PL ResiPore column (300 × 7.5 mm), and a PL-AS-RT autosampler. The calibration was performed by using 12 polystyrene standards with Mn values ranging from 162 to 371 100 g mol−1 (Varian Inc., UK). The eluent was N,N-dimethylacetamide (DMAc) with 3 g/L LiCl, and the flow rate was 1 mL min−1. The data were analyzed using Cirrus GPC offline GPC/SEC software (version 2.0). 1 H NMR spectra were recorded on Bruker Avance III 500 spectrometer operating at 500.13 MHz using CDCl3 as solvent at room temperature. Monomer conversions were determined via 1H NMR spectroscopy by comparing the integrals of monomeric vinyl protons to polymer signals. Furthermore, the content of PBMA was calculated to be about 10% by the integration of the ratio between the specific signals of BMA (aromatic H, 6.3−7.7 ppm) and DMAEMA (O−CH2, 4.02 ppm). The lower critical solution temperature (LCST) of the polymers was measured on a Tepper TP1 photometer (Mainz, Germany). Transmittance of the polymer in Milli-Q water at 670 nm was monitored as a function of temperature (cell path length: 12 mm; one heating/cooling cycle at a rate of 1 °C min−1), and the critical temperature was determined at 50% of relative transmittance. Thermogravimetric analysis (TGA) of the samples was performed on a TGA Q5000 thermogravimetric analyzer (TA Instruments, USA). The exact amounts of samples were placed in a sample pan and were sealed. During measurements, the temperature was increased from 25 to 900 °C with a heating rate of 10 °C/min under a N2 atmosphere, and the masses of the samples were weighed. The functional groups of the bare and modified silica particles were investigated by Fourier transform infrared (IR) spectroscopy on a Bruker Equinox 55 Fourier transform infrared spectrophotometer. The samples were analyzed directly using attenuated total reflectance or pretreated with KBr pressing (by mixing the silica particles with KBr powder). The absorption over the range of 600−4000 cm −1 (resolution = 2 cm−1, 100 scans per measurement) was scanned.

wA v M nπR2

loaded amount of PEI‐Mal × 100 initial amount of PEI‐Mal

(2)

where w is the mass of polymer grafted on the SP, Mn is the numberaverage molecular weight of the grafted polymer, Av is Avogadro’s number, and R is the diameter of the silica core. By calculating the σ values, the grafting density was determined to be about 1.00 polymer chain per nm2 (assuming the density of the silica nanoparticle 2.07 g/ cm3). Preparation of Hollow Capsules. After the polymerization mentioned above, the copolymer grafted silica particles were separated from the solvent by centrifugation and placed in a flask with ethanol. The solution of particles has to be sonicated for 30 min. Then the solution was placed in the UV chamber equipped with a low intensity (0.1 W cm−2) iron lamp and irradiated for 20 min to cross-link the polymer shell. Thus, the particle suspensions were mixed well by vortexing and transferred to Eppendorf tubes. The particles were worked up by one centrifugation/redispersion cycles with Milli-Q water (100 μL). The template silica cores were then removed to fabricate hollow capsules by the addition of a hydrogen fluoride (HF) buffered to pH 7.3 with ammonium fluoride (NH4F). (Caution! Note that hydrogen fluoride and ammonium f luoride are highly toxic. Extreme care should be taken when handling HF solution, and only small quantities should be prepared.) The samples were tapped gently to dissolve the silica cores for about 30 min. The excess NH4F, HF, and SiF4 were separated from hollow capsules by three centrifugation/redispersion cycles with Milli-Q water, followed by resuspending in a suitable volume (typically 0.5 mL) of Milli-Q water. Reversible Swelling−Shrinking Behavior of the CrossLinked Hollow Capsules. The photo-cross-linked hollow capsules were incubated with phosphate buffer possessing pH 6.5, 7.4, and 8.5. At each pH state, hollow capsules were allowed to stand for 20 min. Then the temperature of the capsules was switched between 25 and 45 °C, and the particles diameters were determined by DLS. This process was repeated for several cycles as shown in the main text. No fewer than 20 measurements were taken on each temperature. Encapsulation of PEI-Mal 5 and PEI-Mal 25. A concentrated solution containing the capsules with 10 mol % cross-linkable monomer and photo-cross-linked for 20 min was added to a PEIC


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Figure 1. Characterization of copolymer grafted SP. (a) IR spectrum of bare SP, initiator-anchored SP and P(DMAEMA-co-BMA) copolymer grafted SP. (b) TGA curves of bare SP, initiator-anchored SP and P(DMAEMA-co-BMA) copolymer grafted SP; weight loss (solid line) and derivative weight loss (dotted line) of SP as functions of temperature are recorded. After measurements, the spectra were processed with baseline correction and subtraction of KBr absorption. The UV irradiation was performed using UVACUBE100 (honle UV Technologies, Germany) equipped with a low intensity (0.1 W cm−2) iron lamp as UV source. Dynamic light scattering (DLS) studies of hollow capsules were done at 25 °C using a Zetasizer Nanoseries instrument (Malvern Instruments, UK) equipped with a 633 nm He−Ne laser at fixed scattering angle of 173°. The data were analyzed using software version 6.12. The diameters and morphologies of the capsules were carried out on using a transmission electron microscopy (TEM) Libra 120 equipped with a charge coupled device (CCD) camera at an accelerating voltage of 120 kV. 2 μL of the capsules or particles was dispersed in water with the concentration of 1 mg mL−1 and allowed to adsorb for 2 min onto a 300 mesh, copper grid coated with carbon film, and the specimen was dried at room temperature or the grid was blotted dry using filter paper. For the cryo-TEM measurement was performed on the same instrument, except the sample was frozen in liquid ethane at −178 °C. The blotting with the filter paper and plunging into liquid ethane were done in a Leica GP device (Leica Microsystems GmbH, Wetzlar, Germany). All images were recorded in bright field at −172 °C. The scanning electron microscopy (SEM) was carried out on a Zeiss Ultra 55 Gemini scanning electron microscope. 1 μL of a concentrated capsule solution was spin-coated on Si wafers which were sputter-coated with gold. UV−vis measurements were carried out using Specord 210 Plus double-beam UV−vis spectrophotometer (analytikjena, Germany). Samples were analyzed at desired wavelength range in quartz cuvettes.

functionalized SP, and the grafting density of SET-LRP initiator on the surface of SP was 3.10 groups/nm2. Surface-initiated single electron transfer living radical polymerization (SI-SET-LRP) onto SP was initially started using the in situ fast and complete disproportionation of Cu(I) Br in the presence of Me6Tren to yield insoluble Cu(0) and Cu(II)Br2 in DMSO/H2O (Scheme 1b), followed by adding both the SET-LRP initiator anchored SP and monomers dimethylaminoethyl methacrylate (DMAEMA) and 2-hydroxy4-(methacryloyloxy)benzophenone (BMA) (Scheme 1). The DMSO/H2O (v/v 15%) solvent mixtures was used for the polymerization because H2O favors the disproportionation of Cu(I)Br and DMSO enhances the solubility of monomers. Polymerization proceeded for 15 min at ambient temperature with the final molar ratio [DMAEMA]0/[BMA]0/[initiator]0/ [CuBr]0/[Me6Tren]0 of 540:54:1:0.8:0.4. This resulted in a core−shell structure with the copolymer P(DMAEMA-coBMA) as shell (Scheme 1c). After polymerization, the P(DMAEMA-co-BMA) grafted SP were separated from other reactants and free polymers by intensive washing of the particles with ethanol at least five times. To confirm the successful growth of polymer brushes, the grafted particles were characterized by several techniques (FTIR, TGA, and TEM). In the IR spectra, the stretching vibration of ester carbonyl group at 1724 cm−1 and phenyl ring bands at 1453 and 700 cm−1 confirmed well that the polymerization takes place on the surface (Figure 1a). From the TGA study (Figure 1b), compared with the precursor SP-initiator, these P(DMAEMAco-BMA) grafted particles undergo the additional 58% weight loss because of the presence of the copolymer chain, and the grafting density was calculated to be 1.00 polymer chain/nm2.42 In contrast to the grafting density of SET-LRP initiator (3.10 groups/nm2), there is a slight decrease for the graft density of P(DMAEMA-co-BMA) copolymer because of the steric hindrance between the polymer chains. Moreover, the formation of this core−shell structure is observed by TEM (Figure 3e), which clearly shows a very uniform polymer shell with a thickness of about 55 nm formed around the particle. After the dissolution of the silica core without cross-linking the polymer shell in advance, the originally grafted copolymer was isolated and analyzed by 1H NMR (Figure S1, Supporting Information) and GPC (entry 5 in Table 1). Each characteristic 1 H NMR signal of both monomers, DMAEMA and BMA, in the polymer chain was identified. It further indicates the

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RESULTS AND DISCUSSION Assembly of the Core−Shell Structure by SI-SET-LRP. Silica particles (SP, 500 nm diameter) were selected as the template material for surface-initiated SET-LRP techniques because the SiO2 particle surfaces can be easily functionalized by reaction with 3-(ethoxydimethylsilyl)propylamine. Then, the amino-functionalized SP were reacted with 2-bromoisobutyryl bromide to attach the SET-LRP initiators onto the surface of SP via amide bond formation. After this, the SET-LRP initiator anchored silica particles were washed with THF until the supernatant layer after centrifugation was colorless. The successful anchoring of the SET-LRP initiators onto SP was confirmed by FT-IR and TGA (Figure 1). From the TGA, the density of the SET-LRP initiator on the particle surface was calculated to be 25 μmol per gram of SET-LRP initiatorD


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shell increases correspondingly from 13.2 to 55.0 nm. This points to a relatively high grafting density leading to mushroom-type to stretched-out brushes, since the first theoretical value of fully expanded polymer chain length for P(DMAEMA-co-BMA) copolymer with Mn = 97 400 g mol−1 was calculated to be about 145 nm from Chem3D. Combining these results with the GPC analysis, the longer reaction time leads to a higher molecular weight of the grafted copolymer. This subsequently gives the desired rise to increasing the shell thickness. Assembly of the Dual-Responsive Hollow Capsules. To obtain stable polymeric capsules after template removal (Scheme 1, step d), final cross-linking of the shell is absolutely necessary. The photo-cross-linkable monomer (BMA) had been reported in our group’s previous work for effectively photo-cross-linking the capsules16 and therefore exploited in our study. The P(DMAEMA-co-BMA) grafted copolymer brushes on the surface of SP were cross-linked under the unfiltered light of a mercury lamp for 20 min. Then, the silica core was removed using NH4F/HF buffer at pH 7.5 to give the hollow capsules (Scheme 1d). TEM (Figure 4a) and SEM (Figure S2, Supporting Information) images clearly demonstrate the formation of the hollow capsules structure. The collapsed capsules structures with uniform diameters of about 660 nm can be clearly observed because the flexible polymer shell is not stable enough to remain a spherical structure in the dried state. Finally, the real morphology of the capsules was confirmed by cryo-TEM study (Figure 4b). The globular structure of capsules is very uniform, showing a diameter of about 780 nm at about pH 7.4. This is consistent with the results (ϕ ∼ 790 nm at pH 7.4 and 25 °C) obtained from dynamic light scattering (DLS) (Figure 5a) and points to a swelling of the capsules under the condition of dissolution of the 500 nm SP templates. Also, the membrane thickness of capsules after final monomer conversion within 15 min is in the range of 58 nm (Figure 4b). This is accordance with the TEM images obtained for the SP templated core−shell structures (Figure 3e). To confirm the pH-dependent thermosensitive characteristics and the stability of the hollow capsules, DLS study was used to investigate their cyclic swelling and shrinkage in aqueous solution at different pH values (6.5, 7.4, and 8.5) and temperatures between 25 and 45 °C (Figure 5a). In essence, the membrane of the hollow capsules, consisting of P(DMAEMA-co-BMA) copolymer photo-cross-linked for 20 min, undergoes the desired on/off switches for at least four cycles on stimuli, pH, and temperature. This implies that

Table 1. Characterization of the P(DMAEMA-co-BMA) Grafted Copolymers in This Study reaction time (min)

shell thicknessa (nm)

Mn of grafted polymerb (g/mol)

Đb

monomer convc (%)

3 6 9 12 15

13.21 23.75 33.51 45.81 55.00

23300 42000 59300 81000 97400

1.08 1.11 1.29 1.20 1.30

22 43 61 84 >96

a

The thickness of the core−shell structure was measured by TEM. Number-average molecular weight (Mn) and dispersity (Đ) of the grafted polymer were determined by GPC (polystyrene calibration). c Monomer conversion was measured by 1H NMR. b

successful growing of both monomers on the surface of SP. Moreover, by calculating the integration ratio between the specific 1H NMR signals of BMA (aromatic H, 6.3−7.7 ppm) and DMAEMA (O−CH2, 4.02 ppm), the content of PBMA in the copolymer chain was determined to be 10%. From GPC, the number-averaged molecular weight of the grafted copolymer P(DMAEMA-co-BMA) has been shown to be about 97 400 g/mol with a dispersity of 1.30. Moreover, to control the molecular weight and dispersity of grafted copolymer by the SI-SET-LRP polymerization, a kinetics study for DMAEMA and BMA copolymerization was carried out. After analysis of data it exhibited a linear relationship of monomer consumption versus time (Figure 2a). This implies the desired controlled radical polymerization. Furthermore, results of GPC (Figure 2 and Table 1) provide that the polymerization was fast, and the conversion reached 22% in 3 min, 43% in 6 min, 61% in 9 min, and 84% in 12 min, with full conversion attained in 15 min with a final very high number-average molar mass of Mn = 97 400 g mol−1 with a dispersity of 1.30. In detail, the molecular weights increased linearly during the polymerization, while the molecular weight distributions maintained narrowly (Đ ≤ 1.30) (Figure 2b). GPC further reveals an excellent agreement between the theoretical and the experimental molecular weights and a symmetrical molecular weight distribution. Thus, we can assume that the thickness of the shell for the copolymer grafted core−shell structure (Scheme 1, step c) is smoothly controllable by the polymerization time of both monomers. In Figure 3, different thicknesses of the shell (13.2, 23.7, 33.5, 45.8, and 55.0 nm) for the copolymer grafted core−shell structure confirm our working hypothesis. As the polymerization time is increased from 3 to 15 min, the thickness of the

Figure 2. Characterization of grafted copolymer P(DMAEMA-co-BMA) via SI-SET-LRP polymerization. (a) Polymerization kinetic plots for monomer conversion versus time. (b) Number-average molecular weight (Mn) determined by GPC and dispersity (Đ) versus conversion. E


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Figure 3. TEM images of P(DMAEMA-co-BMA) copolymer grafted SP after reaction times of 3 (a), 6 (b), 9 (c), 12 (d), and 15 min (e). Scale bar is 100 nm in each image.

Figure 4. TEM (a) and cryo-TEM (b) image of P(DMAEMA-co-BMA) hollow capsules prepared using 500 nm diameter SiO2 particles (silica templates were dissolved with NH4/HF buffer).

Figure 5. (a) Reversible swelling−shrinking of P(DMAEMA-co-BMA) hollow capsules photo-cross-linked 20 min, upon switching between 25 and 45 °C at pH 6.5, pH 7.4, and pH 8.5 buffer (open symbol = 25 °C; solid symbol = 45 °C) (n = 3). (b) Diameter of P(DMAEMA-co-BMA) hollow capsules at different temperatures and also in different pH media. F


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Figure 6. UV−vis monitoring of the dialysis process. Capsule membrane allows for transmembrane diffusion at pH 6.5 and 25 °C. When cleaned afterward at pH 8.5 and 45 °C using dialysis (dialysis tube MWCO 150 kDa), rhodamine B labeled PEI-Mal 5 (a) and PEI-Mal 25 (b) were still detectable and remained at a stable level, indicating a successful enclosure.

Figure 7. Cumulative release profile of rhodamine B labeled PEI-Mal 5 (a) and PEI-Mal 25 (b) from P(DMAEMA-co-BMA) hollow capsules, photocross-linked for 20 min, at different temperatures and in various pH medium (n = 3). The maximal standard deviations of (a) and (b) are ±1.02 and ±0.77, respectively.

with ϕ 5 nm and PEI-Mal 25 with ϕ 11 nm) were selected as model cargos, which mimic proteins in diameters and structures. Generally, the cargo solution was mixed with the concentrated hollow capsules at pH 6.5 and room temperature. Thus, the capsules switched to the “swelling” state for desired cargo loading (Figure 5a). After cargos postencapsulation for 12 h, the nonencapsulated cargos were removed by dialysis for 3 days in phosphate buffer at pH 8.5 and 45 °C. Under these dialysis conditions the capsules completely switched to the “deswelling = closed” state to remove the unloaded cargo as much as possible (Figure 5a). Of special importance is the stable level of UV/vis spectra recorded after 2 and 3 days of dialysis, indicating a successful separation of nonencapsulated cargo (Figure 6). According to the calibration curves of rhodamine B labeled PEI-Mal 5 and PEI-Mal (Figure S4), the loading efficiency of PEI-Mal 5 and PEI-Mal 25 was determined to be about 35 ± 5% and 40 ± 5%, respectively. This also means that most cargo macromolecules are captured in the cavity of capsules, while few captured cargo macromolecules will be integrated in the membrane and fixed at the interface of membrane and outer shell of the capsules, too. For the release behavior of capsules, the PEI-Mal-containing capsules were immersed in solutions to study the effects of pH and temperature on the release kinetics of cargo (Figure 7). The samples were taken and analyzed using UV−vis at regular intervals. Generally, the release profiles of both PEI-Mal cargos showed similar results. In the completely “swelling = open” state (completely protonated) of capsules for PEI-Mal 5 at pH 6.5 over 96 h, the amount of released PEI-Mal 5 is ≤63%, while the temperature influence at 25 or 45 °C plays no significant

successfully hollow capsules with tunable membrane permeability characteristics have been achieved. Thus, they are possibly suited for controlling the traffic of biomacromolecules from or into the capsule lumen depending on the pH and temperature stimulus. In detail, at pH 6.5 there is no cloud point of the random PDMAEMA segments in P(DMAEMA-coBMA) copolymer because of the full solubility of PDMAEMA units to offset the aggregation of hydrophobic temperaturesensitive components (Figure S3). This results in no obvious change of the capsules diameters when the temperature is continuously changed. However, above pH 6.5, PDMAEMA in capsules becomes partially deprotonated and less hydrophilic.39 Therefore, the capsules show the desired temperature sensitivity over four cycles of swelling and shrinking with defined changes in their diameters. Especially, the LCST of PDMAEMA at pH 8.5 decreases to 37.5 °C. This causes a higher shrinking power for the hollow capsules which shrink from about 765 nm at 25 °C to about 645 nm at 45 °C (Figure 5b). The observed change of the capsules diameters impressively indicates that the hollow capsules exhibit the desired pH-dependent thermosensitivity behavior at neutral and basic pH. Cargo Postloading and Controlled Release by Hollow Capsules. We further evaluated cargo release experiments to demonstrate pH-dependent temperature-controlled permeability of the established hollow capsules. Considering the potential application of the capsules as nanocarriers and nanoreactors, postencapsulation of larger biomacromolecules could be envisioned. In order to study this option, nanometer-sized rhodamine B labeled dendritic glycopolymers43 (PEI-Mal 5 G


Article

Macromolecules rule (Figure 5a). However, the cargo PEI-Mal 25 has a slower release rate (≤45%) in the swollen state (Figure 7b) as found for PEI-Mal 5 (Figure 7a). Therefore, the phenomena for the different cargo release from protonated, non-temperatureresponsive hollow capsules with similar diameters (Figure 5a) are mainly triggered by the diameter match between cargos diameter and the permeability of protonated capsules membrane during the transmembrane diffusion. Considering the smaller diameter of deprotonated hollow capsules at pH 8.5 and their resulting temperature-stimulus responsive characteristics (Figure 5a), this causes an obvious variation of the release quantity and the release rate between 45 and 25 °C (Figure 7). When temperature is decreased from 45 to 25 °C, the amount of released PEI-Mal 5 and PEI-Mal 25 over 96 h increases from 27% to 55% and from 20% to 38%, respectively. This behavior subsequently indicates that the permeability of the capsules and consequently the rate of cargo release can be controlled by the temperature at pH 8.5. Overall, the present membrane properties of these capsules can be further optimized for better controllable pH- and temperaturedependent cargo postencapsulation and release by introducing block or triblock copolymer structures on silica particles, at which tunable cargo release is also possible at acidic pH.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected]; Tel 49-351-4658353; Fax 49-3514658565 (D.A.). *E-mail [email protected]; Tel 49-351-4658590; Fax 49-3514658565 (B.V.). ORCID

Dietmar Appelhans: 0000-0003-4611-8963 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Xiaoling Liu is grateful for a scholarship under the Chinese government award for outstanding students abroad by the China Scholarship Council (CSC). We thank Dr. Hartmut Komber for samples of NMR, Dr. Mikhail Malanin for samples of IR, Kerstin Arnhold for samples of TGA, and Dr. Petr Formanek for samples of SEM and cryo-TEM.

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CONCLUSIONS In this study we reported a facile and highly effective process for the successful synthesis of multiresponsive capsules. For this, surface-initiated SET-LRP polymerization of pH-dependent thermosensitive monomer DMAEMA and photo-crosslinkable monomer BMA from silica particles at room temperature was used to fabricate core−shell structures. The chemical composition and morphology of the core−shell structures were well confirmed using IR, TGA, 1H NMR, GPC, and the TEM method. Importantly, the polymerization proceeded homogeneously and in a controlled manner. Full conversion of both monomers was achieved within 15 min, yielding a high number-average molar mass of Mn = 97 400 g mol−1 and very low Đ (≤1.30) for copolymers of that molar mass as well as a final shell thickness of 55 nm. To the best of our knowledge, the current contribution is the first example of such well-defined polymers by a “graft from” polymerization. A more significant impact of this contribution may ultimately be the success to fabricate polymeric structures with defined shell thickness on silica particles. Furthermore, well-defined hollow capsules were smoothly obtained after photo-cross-linking of shell as future membrane structure of hollow capsules and template removal. Hollow capsules structures were confirmed by (cryo-)TEM, SEM, and DLS study. The uniform photocross-linked hollow capsules exhibit the desired pH-dependent thermosensitivity. The uptake and release properties for cargos in solution of the resulting hollow capsules were highly affected by changes in the external stimuli temperature and pH. Overall, this approach provides synthetic routes to a wide variety of polymeric nano-objects with an equally wide range of potential applications in fields as diverse as nanomedicine (e.g., drug delivery and synthetic biology), advanced materials, and electronic applications.

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Synthesis procedure of the rhodamine B labeled maltosedecorated hyperbranched poly(ethylene imine) macromolecules (PEI-Mal 5 and PEI-Mal 25); NMR data for the grafted copolymer P(DMAEMA-co-BMA); LCST data for the grafted copolymer P(DMAEMA-co-BMA); SEM images of hollow capsules and calibration curves of the rhodamine B labeled PEI-Mal 5 and PEI-Mal 25 (PDF)

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b02347. H


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Rapid Synthesis of Dual-Responsive Hollow Capsules with

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