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Selective Release of Hydrophobic and Hydrophilic Cargos from Multi-Stimuli-Responsive Nanogels Ziquan Cao, Xiaoteng Zhou, and Guojie Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10360 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 7, 2016

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Selective Release of Hydrophobic and Hydrophilic Cargos from Multi-Stimuli-Responsive Nanogels Ziquan Cao, Xiaoteng Zhou, Guojie Wang* School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China

Corresponding author: Guojie Wang: Phone: +86-10-62333619 Email: [email protected]

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ABSTRACT. Highly stable multi-stimuli-responsive nanogels for selective release of simultaneously encapsulated hydrophobic and hydrophilic cargos in a spatiotemporally controlled manner are demonstrated here. The nanogel is composed of hydrophilic pH- and thermo-responsive poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and hydrophobic photocleavable o-nitrobenzyl (ONB) linkage. The hydrophobic cargos were noncovalently encapsulated into lipophilic interiors of the nanogels, while the hydrophilic cargos were chemically linked to the nanogel precursor polymer PDMAEMA through a redox-cleavable disulfide junction. For these dual-loaded nanogels, hydrophobic cargos can be released in response to temperature, pH, and UV light, while the hydrophilic cargos can be released in response to redox reagent. The stimuli-selective release of hydrophobic and hydrophilic cargos affords the system with great potential applications in combination chemotherapy, tissue engineering, anticorrosion, and smart nanoreactors. KEYWORDS. dual-cargo delivery, multistimuli responsiveness, nanogels, nanotechnology, selective release

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1. Introduction In recent years, multidelivery systems that are able to effectively encapsulate multiple functional cargos and to concurrently deliver them to the target location have been designed and abundantly investigated owing to their broad range of potential applications in biomedicine,1−7 anticorrosion,8−10 and water purification.11 It is noted that the codelivery of multiple functional cargos onto the same system could have superior efficacy compared to delivering a single cargo. For example, it has already been demonstrated that combination chemotherapy with multiple anticancer drugs provides a promising strategy to promote synergistic effects, overcome multidrug resistance, and reduce side effects.12−14 To date, some traditional cargo-delivery systems, such as liposomes,15−17 micelles,18−21 mesoporous silica nanoparticles,22−26 and polymer capsules,27−31 have shown the potential to codeliver multiple cargos. Among these, stimuliresponsive multidelivery systems are of particular interest because they offer an excellent means for delivering and releasing cargos in a spatiotemporally controlled manner.16, 19−21, 23, 25, 29−31 However, technological challenges in encapsulating multiple functional cargos in a sophisticated multidelivery system still exist. On the one hand, only the cargos with similar physicochemical properties (such as similar solubilities, charges, and molecular weights) can be conveniently loaded into a single delivery vehicle at the same time,32−34 it is difficult to simultaneously encapsulate multiple cargos with diverse physicochemical properties, particularly for hydrophobic and hydrophilic types.35−38 On the other hand, the precise control of different cargos released individually from a single multidelivery platform remains an unmet need. Typical multiple cargo release patterns include simultaneous release,39−41 sequential release,42, 43 and spatiotemporal release.44 Undoubtedly, the last one provides significant opportunities to control the administration order, timing, dosage, and duration of each individual cargo. In this

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case, it is anticipated that one cargo can be released in response to a specific stimulus while the other cargo can be released in response to the other stimuli. Meanwhile, these stimuli-responsive approaches for selective release of different cargos are independent. Most recently, Tsukruk and colleagues have just fabricated multicompartmental microcapsules based on star-graft quarterpolymers by layer-by-layer assembly, in which the programmable and sequential release of hydrophobic and hydrophilic molecules could be triggered independently by temperature and pH variations, respectively.45 However, the facile synthesis of highly stable nanocarriers for controlled release of multiple cargos with very different orthogonal solubilities is still a big challenge. As is well known, nanohydrogels are excellent nanocarriers in cargo delivery because of their higher stability for prolonged circulation, and their controlled release and site-specific targeting of loaded cargos modulated by external stimuli (like pH, temperature, UV light, etc).46−48 They normally have a hydrophilic interior network for loading of hydrophilic molecules, while the highly hydrated structures of nanohydrogels make them disadvantageous for entrapment of hydrophobic molecules. To counter the above problems, we may design and fabricate multi-stimuli-responsive nanogels for simultaneous encapsulation of hydrophilic and hydrophobic cargos, which can provide unique opportunity to precisely control release of these cargos under different stimuli in a spatiotemporally controlled manner. Herein, we report the synthesis of a novel type of highly stable multi-stimuli-responsive nanogels that allow for simultaneous encapsulation and stimuli-responsive selective release of hydrophilic and hydrophobic cargos for the first time. The formation of nanogels is based on linear polymer nanogel precursor by intra-/interchainly cross-linked in a stable oil-in-water miniemulsion. The nanogel precursor is pH-, temperature-, and redox-responsive hydrophilic polymer and the cross-linking agent is photocleavable hydrophobic bis-bromine compound. The

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hydrophilic cargos are covalently conjugated to the nanogel precursor through a redox-cleavable disulfide junction. The hydrophobic cargos are encapsulated into the hydrophobic interiors of the nanogels through hydrophobic interaction. The selective release of hydrophilic and hydrophobic cargo molecules under different stimuli is shown in Scheme 1. At higher temperature, the nanogels shrink to smaller ones, resulting in the release of hydrophobic cargo molecules. Under acidic condition, the hydrophobic cargo molecules can be released from the swelled nanogels. For above two stimuli-release, the hydrophilic cargo molecules cannot be released, owing to the intact conjugation of disulfide bond. Under UV light irradiation, the cross-linking agent undergo photocleavage reaction, triggering the degradation of the intra-/interchainly cross-linked nanogels, thus resulting in the rapidly release of hydrophobic cargo molecules. Correspondingly, the hydrophilic cargo molecules conjugated the linear nanogel precursor polymer can be also released. In response to redox environments, the disulfide-linked hydrophilic cargo molecules in the presence of DL-dithiothreitol (DTT) can be released from the nanogels. The hydrophobic cargo molecules are still encapsulated into the nanogels as DTT has no effect on the network structure and hydrophobic functionalities of the nanogels. As a consequence, the selective release of the two target molecules with different hydrophilic and hydrophobic properties can be achieved under different stimulation conditions.

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Scheme 1. Schematic Illustration of the Selective Release of Hydrophobic and Hydrophilic Cargos from Multi-Stimuli-Responsive Nanogels upon Different Stimuli (Temperature, pH, UV Light, and Redox Reagent).

2. Experimental Section Materials. 5-Hydroxy-2-nitrobenzyl alcohol (Aldrich, 99%), bromoacetyl bromide (Alfa Aesar, 98%), N, N’, N’, N’’, N’’-pentamethyldiethylenetriamine (PMDETA, Aldrich, 99%), copper(I) bromide (CuBr, Alfa Aesar, 99.998%), DL-dithiothreitol (DTT, Sigma-Aldrich, 99%), rhodamine B (RhB, J&K, 95%), coumarin 102 (Aldrich, 99%), dicyclohexylcarbodiimide (DCC, Alfa Aesar, 99%), and 4-dimethylaminopyridine (DMAP, Alfa Aesar, 99%) were used without further purification. 2-(N, N-Dimethylamino)ethyl methacrylate (DMAEMA, Aldrich, 99%) was

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dried over CaH2 and distilled under reduced pressure. Tetrahydrofuran (THF) was dried by refluxing over sodium shavings and distilled just prior to use. 2-Hydroxyethyl-2’(bromoisobutyryl) ethyl disulfide (HO-SS-iBuBr) was synthesized in our laboratory as described previously.50 Triethylamine (Et3N), dichloromethane, sodium dodecyl sulfate (SDS), diethyl ether, toluene, and hexane were purchased from Sinopharm Chemical Reagent Co. Ltd.. All other reagents were used as received without further purification. Synthesis of Photodegradable Br-ONB-Br cross-linker. 5-Hydroxy-2-nitrobenzyl alcohol (0.338 g, 2 mmol) and Et3N (2 mL, 14.36 mmol) were dissolved in dry dichloromethane (20 mL) in a 50 mL flask, which was then placed in an ice/water bath. A solution of bromoacetyl bromide (6 mL, 69 mmol) in dry dichloromethane (4 mL) was added dropwise into the flask over 15 min. After stirring for 72 h at 25 °C, white solids were removed by filtration. The reaction mixture was concentrated under reduced pressure via a rotary evaporation. The crude product was further purified by column chromatography with mixtures of 3:1 hexane/ethyl acetate. After dried in a vacuum at 50 °C for 24 h, the pure photodegradable Br-ONB-Br cross-linker was obtained (0.444 g, 1.08 mmol, yield 54%). 1H NMR (Bruker AM 400, CDCl3): δ (ppm) 8.23 (d, 1H, Ar-H close to nitryl), δ 7.45 (s, 1H), δ 7.32 (d, 1H), δ 5.63 (s, 2H), δ 4.33 (s, 2H), δ 4.17 (s, 2H) (Figure S1). Synthesis of PDMAEMA-SS-OH. In a dried 50 mL Schlenk flask with a magnetic stirrer, HO-SS-iBuBr (0.060 g, 0.2 mmol), CuBr (0.028 g, 0.2 mmol), the monomer DMAEMA (2.21 g, 10 mmol), and anhydrous THF (4 mL) were added, degassed and filled with nitrogen. Then, PMDETA (40 µL, 0.2 mmol) was added through a syringe. The mixture was deoxygenated five times using the freeze−pump−thaw procedure and sealed under vacuum, then the reaction mixture was purged with argon for approximately 30 min to eliminate the oxygen. The flask was

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placed in a preheated oil bath at 68 °C for 36 h. The reaction mixture was diluted with THF and passed through a neutral alumina column to remove the copper catalysts. The eluent was removed by rotary evaporation and then the polymer was precipitated four times from an excess of hexane, filtered. The pure product was obtained after dried in a vacuum at 50 °C for 24 h (1.771 g, yield 78%). 1H NMR (Bruker AM 400, CDCl3): δ (ppm) 4.1 (2H), δ 2.6 (2H), δ 2.3 (6H), δ 2.1−1.6 (2H), δ 1.3−0.7 (3H). GPC (THF): Mn = 1.8 × 104 g/mol, PDI = 1.13 (Figure S2 and S3). Synthesis of PDMAEMA-SS-RhB. A 50 mL round-bottom flask was charged with dichloromethane

(20

mL),

dicyclohexylcarbodiimide

(0.6

g,

3.09

mmol),

4-

dimethylaminopyridine (30 mg, 0.24 mmol), PDMAEMA-SS-OH (1 g, 0.06 mmol), and excess rhodamine B (0.2 g, 1.72 mmol). The reaction mixture was stirred at room temperature for 48 h. After removing the insoluble byproduct dicyclohexylcarbodiurea by suction filtration, the filtrate was concentrated by rotary evaporator and precipitated into an excess of cold diethyl ether. The crude product was purified further by dialysis (cellulose membrane, molecular weight cut-off 3500 Da). After removing water by lyophilization, the pure product PDMAEMA-SS-RhB was obtained as a fuchsia solid (0.61 g, yield 50%). Preparation of Multi-Stimuli-Responsive Nanogels. Nanogels were prepared by a modified oil-in-water (O/W) miniemulsion method. Briefly, at 70 °C, PDMAEMA-SS-RhB (100 mg) and Br-ONB-Br (25 mg) were co-dissolved in toluene (4 mL), and then the mixture was added to 30 mL of deionized water containing 40 mg of SDS. After being stirred for 10 min, the solution was sonicated at 70 °C for 2 min to form a stable oil-in-water miniemulsion. The cross-linking reaction was performed at 70 °C for 22 h. Subsequently, the solution was stirred for another 2 h at 70 °C in a hood to evaporate toluene. The prepared nanogels were purified by dialysis

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(cellulose membrane, molecular weight cut-off: 10k Da) against deionized water at room temperature. Fresh deionized water was replaced every 6 h. Then the nanogel solution was lyophilized. Encapsulation of Coumarin 102 in Multi-Stimuli-Responsive Nanogels. 5 mg of nanogels was added to 0.5 mL of coumarin 102 solution (1 mg/mL in THF) with constant stirring for 2 h, and then 0.5 mL of deionized water was added. After stirring for 24 h, the resulting solution was dialyzed (cellulose membrane, molecular weight cut-off: 3500 Da) against deionized water at 25 °C for 24 h with repeated change of water to remove THF. Excess insoluble coumarin 102 was removed by filtration. Thiol-Triggered Release of RhB from Multi-Stimuli-Responsive Nanogels. The release behavior of RhB from nanogels was carried out by dialysis. Briefly, 20 mg of nanogels was dispersed in 4 mL of deionized water. The suspension was divided into four equivalent aliquots. Each 1 mL of the aliquot sample (5 mg/mL) was placed inside a dialysis bag (cellulose membrane, molecular weight cut-off 3500 Da) and then immersed into 19 mL of an aqueous medium with different DTT concentrations of 0, 5, 10, and 20 mM. At desired intervals, 2 mL of the solution outside the dialysis bag was taken out followed by the addition of an equal amount of fresh solution to keep the constant volume of the medium. The amount of RhB released from nanogels was measured by UV−vis spectroscopy at 554 nm at 25 °C. A calibration curve was drawn by measuring a series of RhB solutions with known concentrations. Characterization. 1HNMR spectra of samples were recorded using a Bruker AM 400 spectrometer with CDCl3 as the solvent. The chemical shifts were relative to tetramethylsilane. The average molecular weight and molecular weight distribution (Mw/Mn) were measured by gel

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permeation chromatography (GPC) (Waters 1515) with styragel columns relative to polystyrene standards using THF as eluent at a flow rate of 1.0 mL/min. The morphologies of the nanogels were characterized with a JEM-2010 EX/S transmission electron microscope (TEM). The samples for TEM observation were prepared by a nanogels solution (10 µL, 0.01 mg/mL) onto 300-mesh copper grids. The solvent was evaporated by freeze−drying procedure for 24 h. Hydrodynamic diameter and size distribution of nanogels were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 instrument (Malvern Instruments) equipped with a multipurpose autotitrator (MPT-2). All the nanogel concentrations were fixed at 0.1 mg/mL. The samples for DLS determination were filtered through a 0.45 µm filter. Size measurements were performed at least three times on each sample to ensure consistency. UV−vis absorption spectra were obtained on a JASCO V-570 spectrophotometer. Fluorescence spectra of coumarin 102 were recorded on F-280 fluorescence spectrophotometer with an excitation wavelength of 390 nm. Fluorescence emission spectra were collected within a range of 400 ~ 700 nm. Excitation and emission slit widths were both maintained at 2 nm and spectra were accumulated with a scan speed of 1200 nm/min. UV light irradiation for the samples was carried out on a high-pressure mercury lamp (365 nm, 500 W nominal power).

3. Results and Discussion Design and Synthesis of the Multi-Stimuli Responsive Nanogels. The nanogel precursors and photodegradable bis-bromine cross-linker were obtained through a multi-step synthesis, shown in Scheme 2. First, the light-responsive Br-ONB-Br cross-linker was synthesized from 5hydroxy-2-nitrobenzyl alcohol and bromoacetyl bromide by esterification. Its chemical structure

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was confirmed by 1H NMR analysis (Figure S1). Then PDMAEMA-SS-OH was synthesized via atom transfer radical polymerization (ATRP), using 2-hydroxyethyl-2’-(bromoisobutyryl) ethyl disulfide as an initiator. The number average molecular weight (Mn) and polydispersity index of PDMAEMA-SS-OH were 1.8 × 104 g/mol and 1.13, respectively, determined by gel permeation chromatography (GPC) (Figure S3). The average degree of polymerization of DMAEMA was calculated to be 115. The lower critical solution temperature (LCST) of PDMAEMA-SS-OH was measured to be about 57 °C at pH 7.4 (Figure S4). The hydroxyl functional groups of PDMAEMA-SS-OH provide a convenient handle for attaching different agents. In this study, the hydroxyl groups were conjugated with hydrophilic molecules rhodamine B (RhB) via a carbodiimide coupling condensation reaction to afford the procargo precursor polymer, PDMAEMA-SS-RhB. The procedure of synthesizing nanogels is schematically illustrated in Figure 1a. At 70 °C, the toluene solution of photodegradable Br-ONB-Br cross-linker and PDMAEMA-SS-RhB polymer was emulsified in a water continuous phase with sodium dodecyl sulfate (SDS) as the surfactant by pulsed ultrasound sonication to form a stable oil-in-water miniemulsion. The mixture was allowed to stir for 24 h at 70 °C. Relative high temperature (70 °C) is helpful to confine PDMAEMA-SS-RhB polymers in the oil phase because the precursors could become hydrophobic above the LCST (57 °C). The PDMAEMA-SS-RhB polymer was intra/intermolecularly cross-linked by Br-ONB-Br (0.095 equiv of tertiary amine function, targeted cross-linking degree = 19%) via a nucleophilic substitution of the bromine groups by the tertiary amine functions of the PDMAEMA block. The photocleavable linker Br-ONB-Br could not react with the tertiary amine group in RhB for its low molar ratio to the amine group in PDMAEMA (1/115). The obtained nanogels were purified by dialysis against deionized water at room

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temperature. The morphological structures of these prepared nanogels were characterized by transmission electron microscope (TEM) and dynamic light scattering (DLS). A representative TEM image shows that the nanogels in the dry state are generally in monodisperse spherical shape with diameter of 88 ~ 130 nm (Figure 1b). DLS studies reveal that the average hydrodynamic diameter of nanogels is ~ 160 nm with larger sizes than those observed in TEM, which is attributed to the possible swelling of the nanogels in water (Figure 1c, left). In order to further elucidate that cross-linking will maintain the integrity of the nanogels formed after quaternization reaction, the nanogels were analyzed by DLS and TEM in ethanol, a cosolvent for the PDMAEMA-SS-RhB polymer and Br-ONB-Br cross-linker. As shown in Figure 1c right, only a single population is observed, and the size of the nanogels is approximately 220 nm. The TEM image from ethanol solution shows an average diameter of about 200 nm (Figure S5). Compared with the size of the nanogels in water, the apparent size of the nanogels in ethanol becomes larger, since ethanol is a good solvent for both PDMAEMA segments and ONB linkers, causing nanogels to swell more in ethanol than in aqueous solutions. Benefiting from the fluorescent dye molecule RhB conjugated into nanogels, the chemical structure of nanogels was further investigated by UV−vis spectra and fluorescence emission spectra. In comparison with PDMAEMA-SS-OH, the absorption of the nanogels becomes much stronger from 490 nm to 600 nm (Figure 1d), which represents the characteristic absorption of RhB. Moreover, the color of nanogels became red brown compared with colorless PDMAEMA-SS-OH (inset Figure 1d). Figure 1e presents the fluorescence emission spectra of RhB in the polymers, from which it can be seen that emission band centered at λmax = 583 nm appeared in nanogels. Taken together, these results clearly show that a new generation of nanogels were successfully fabricated.

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Scheme 2. Synthesis Route of the Multi-Stimuli-Responsive Nanogels

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Figure 1. (a) Schematic illustration of the preparation of the multi-stimuli-responsive nanogels in water. In the oil phase, PDMAEMA-SS-RhB and Br-ONB-Br were co-dissolved in toluene at 70 °C. In the water phase, sodium dodecyl sulfate (SDS) serving as the surfactant was used to stabilize oil-in-water (O/W) interfaces. After these two phases were emulsified by sonication to form a stable oil-in-water miniemulsion, the nanogels were obtained via quaternization reaction at 70 °C for 24 h. (b) Typical TEM image of nanogels in water before stimuli. (c) Hydrodynamic radius distributions of nanogels in water (left) and in ethanol (right), measured by DLS, respectively. The concentrations of nanogels for TEM and DLS are 0.1 mg/mL. (d) UV−vis

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spectra of PDMAEMA-SS-OH, PDMAEMA-SS-RhB, and nanogels in water. Inset: the photographs of PDMAEMA-SS-OH (left) and nanogels (right) in aqueous solution. (e) Fluorescence emission spectra of PDMAEMA-SS-OH, PDMAEMA-SS-RhB, and nanogels in water, excited at 554 nm.

The Multi-Stimuli-Responsive Properties of the Nanogels. The nanogels are responsive to temperature, pH, UV light, and redox reagent because of the corresponding properties of individual functional groups. Here, the multi-stimuli-responsive behaviors of the nanogels were confirmed and investigated by TEM and DLS measurements. Figure 2a shows the morphology of the nanogels when the temperature of nanogel solution was increased to 60 °C, the size of which was 30 ~ 55 nm, considerably smaller than that at 25 °C. The reason is that the PDMAEMA chains could transform from hydrophilic to hydrophobic under high temperature above LCST, leading to shrinkage of the nanogels. It is noted that the reversible collapse/swelling of the nanogel could be realized upon changing the temperature (Figure S6). Figure 2b shows the average diameter of nanogels increased to approximately 177 nm when the pH value of the nanogel solution was adjusted to 4, since the tertiary amine groups of the unquaternized PDMAEMA segments were protonated under acidic environment, thus the nanogels expanded to larger ones.49,

50

Figure 2c shows that the original spherical nanogels

disappeared and some small and irregularly shaped unimers were formed after UV light irradiation (365 nm, 24 mW/cm2), indicating that the polymer network structure was disassembled due to the photocleavage of chromophore of o-nitrobenzyl groups (Figure S7). Figure 2d shows the TEM image of the nanogels treated with 10 mM DTT over 48 h, from which it can be seen that the size slightly decreased to 80 ~ 95 nm after the removal of

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hydrophilic RhB molecules from the nanogels. Unlike other redox-responsive nanogels crosslinked by the disulfide bonds that can be disintegrated,51 our nanogels will remain the original spherical nanostructures in response to redox reagent. DLS data on the responsive properties of the nanogels upon different stimuli are shown in Figure 2e. Note that number-average size distributions of the multi-stimuli-responsive nanogels were selected in this study, owing to the smallest effects of errors in calculating or estimating the size distribution.52 At 60 °C, the average size decreased to 70 nm, due to the shrink of the nanogels as the hydrophilicity of PDMAEMA segments decreased above the LCST. At pH 4, the particle size of nanogels increased to about 190 nm, corresponding to the swelled nanogels. Under UV light irradiation, the nanogels were dissociated because of the photoinduced cleavage reaction. Thus the diameter detected by DLS decreased to about 8 nm. When the disulfide bonds linking the PDMAEMA and RhB were broken in the presence of 10 mM DTT for 48 h, the size of naogels changed to 150 nm in diameter. The size changes revealed by DLS are in good agreement with those revealed by TEM.

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Figure 2. TEM images of the multi-stimuli-responsive nanogels under different stimuli: (a) at 60 °C, (b) at pH 4, (c) after UV light irradiation (365 nm, 24 mW/cm2), (d) with 10 mM DTT for 48 h. (e) Size distributions for the multi-stimuli-responsive nanogels in water obtained by DLS under different stimuli: (1) at 60 °C, (2) at pH 4, (3) after UV light irradiation (365 nm, 24 mW/cm2), (4) with 10 mM DTT for 48 h.

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The Stimuli-Selective Release of Hydrophobic and Hydrophilic Cargos from the Nanogels under Different Stimuli. The multi-stimuli-responsive nanogels are expected to enable stimuli-selective release of two different molecules, namely, hydrophobic cargos by temperature, pH, and UV light, and hydrophilic cargos by redox reagent. To demonstrate this potential, the release kinetic for each cargo was quantified. For the controlled release of hydrophobic cargos, we selected coumarin 102 (a hydrophobic fluorescent dye) as the model molecule to trace the independent release process, since it will exhibit a relative high fluorescence intensity when solubilized by the hydrophobic environment, such as the interior of these nanogels, but the fluorescence emission intensity deceases significantly in the aqueous medium.53 As shown in Figure S8, the characteristic absorption peak of coumarin 102 at the wavelength around 390 nm appeared, indicating that the hydrophobic cargo was successfully encapsulated into the nanogels. Determined by UV−vis spectra, the loading content of hydrophobic cargos was 17.3% (Figure S9). Owing to the chemically crosslinked structure, the nanogels could maintain the capability of encapsulating the hydrophobic molecules even though the concentration was diluted to 0.00625 mg/mL (Figure S10), suggesting the high stability of the nanogels. To examine the temperature-dependent cargo release, the coumarin 102-loaded nanogel solutions (0.025 mg/mL) were heated to 60 °C (higher than the LCST of PDMAEMA) (Figure 3a). It can be seen clearly that the cargo release was increased with increasing temperature, since the PDMAEMA block would be transformed to a hydrophobic and collapsed state above its LCST and thus more cargos could be squeezed out from the nanogel.54 Figure 3b shows the cumulative release profiles of coumarin 102 from the nanogels upon UV light irradiation at different power densities. In the control experiment, the loaded hydrophobic cargo

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molecules were hardly released without UV light irradiation. Upon UV light irradiation at a power density of 3 mW/cm2, 41% of the loaded hydrophobic cargos was released within 23 min. Increasing the UV light power densities to 12 and 24 mW/cm2 resulted in a sharply increased cumulative release of hydrophobic cargos to 86% and 97%, respectively, within the same period. The more intense UV light could make the photocleavage reaction faster, leading to faster dissociation of the nanogels and faster concomitant release of the hydrophobic cargos. Figure 3c displays the cumulative release profiles of coumarin 102 from the hydrophobic interior regions of the nanogels at different pH values, from which it can be seen that the release rate and the amount of the loaded hydrophobic molecules from the nanogels increase rapidly with increasing acidity. For example, only 8% of hydrophobic cargo molecules was released at pH 7.4 over a period of 16 h, while 35% and 59% of hydrophobic cargo molecules were released at pH 5.5 and 4.0, respectively. After incubated for 23 h, the nanogel solution was exposed to UV light (12 mW/cm2) for only 11 min to realize the combined stimulation of the pH and UV light. Interestingly, a burst release (96% and 81% at pH 4.0 and 5.5, respectively) of hydrophobic cargo molecules were observed (Figure S13). Furthermore, the nanogel solutions after UV irradiation for 23 min at 3 mW/cm2 were treated with dilute hydrochloric acid to pH 5.5, where the loaded hydrophobic cargos could be further released and the release level increased to 90% within 14 h (Figure S12b). Compared to the light- and pH-triggered release, the total released quantities in response to temperature are significantly less, which could be attributed to the more collapsed state to impede cargo diffusion. Together, these results demonstrate the ability of this nanoplatform to supply hydrophobic cargos in a temporally controlled fashion by either burst release upon UV light or slow release upon temperature and pH, which would be very valuable to generate on-demand release rate profiles.55

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The controlled release of hydrophilic cargo RhB from the multi-stimuli-responsive nanogels under different DTT concentrations was performed in a dialysis bag (molecular weight cut-off 3500 Da) and measured by UV−vis spectra. The loading content of RhB in the nanogels determined by UV−vis spectra was 0.1%. The hydrophilic model molecules are covalently linked to PDMAEMA chains of the nanogels by relatively stable disulfide bond, where the disulfide linkage can be cleaved under reducing condition.56 As shown in Figure 3d, with an increase in DTT concentration, the release rate and amount of hydrophilic cargos from the nanogels drastically increased. At low DTT concentrations (5 mM), only 5% of hydrophilic cargo molecules was released after 16 h. When the concentration of DTT increased to 10 mM and 20 mM, the hydrophilic cargo release significantly increased to 12% and 18.5%, respectively. The higher the concentration of DTT, the higher the extent of reaction between the disulfide and reducing agents, and the greater the release amount of RhB.

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Figure 3. Cumulative release profiles of hydrophobic coumarin 102 encapsulated in 0.025 mg/mL of nanogels: (a) at different temperatures, (b) upon UV light irradiation with different power densities, (c) at different pH values, the arrows indicate UV light irradiation (365 nm, 12 mW/cm2). (d) Redox-responsive release profiles of hydrophilic RhB from nanogels at room temperature under different DTT concentrations.

The effects of increased temperature, UV irradiation, and low pH on the release of hydrophilic cargos and the effects of the reducing agent DTT on the release of hydrophobic cargos were also investigated. For the study on the effects of increased temperature, UV irradiation, and low pH on the release of hydrophilic cargos, the nanogels linked with RhB (20 mg) were dispersed in 4 mL of deionized water and the dispersion was divided into four equivalent aliquots. One aliquot was directly diluted with 19 mL of deionized water and characterized by fluorescence spectroscopy. The other three aliquots were transferred into dialysis bags (cellulose membrane, molecular weight cut-off 3500 Da) and immersed into aqueous mediums (19 mL for each sample) separately. Then the samples were stimulated by high temperature of 60 °C, UV light irradiation for 1 h (365 nm, 24 mW/cm2), and pH 4.0, respectively. As desired intervals, the fluorescence of the solution outside the dialysis bag was characterized. Figure 4a, b and c show the fluorescence spectra of RhB in the nanogels diluted with 19 mL of water and in the solutions outside the dialysis bags after stimulation of increased temperature, UV irradiation, and low pH, respectively. Compared with the total fluorescence of RhB loaded in the nanogels (black dash line), the fluorescence of RhB released from nanogels could be hardly detected, indicating that little of the hydrophilic cargos could be released. For the study on the effects of the reducing agent DTT on the release of hydrophobic cargos, the nanogels loaded with coumarin 102 were

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treated with 10 mM DTT. The fluorescence emission spectra of coumarin 102 changed little upon the addition of DTT (Figure 4d), indicating that the redox reagent had very little effect on the release of hydrophobic cargos. These results indicate that the selective release of different cargos with various hydrophilic/hydrophobic properties can be achieved under different stimulation conditions. The synthesized multi-stimuli-responsive nanogels, which can provide opportunity to selectively release hydrophobic and hydrophilic cargos, have the prospect of broadly impacting fields such as tissue engineering, anticorrosion, smart nanoreactors, especially for biomedicine, since it has been proven that simultaneous administration of hydrophilic drug gemcitabine and hydrophobic paclitaxel to biliary cancer patients is an effective way to suppress the proliferation of cancer cells.38

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Figure 4. Fluorescence emission spectra of RhB released in the solution outside the dialysis bag: a) at 60 °C, b) after UV light irradiation for 1 h (365 nm, 24 mW/cm2), c) at pH 4.0, respectively (λex = 554nm). The black dash line (total) represents the total amount of hydrophilic cargo loaded in multi-stimuli-responsive nanogels. d) Fluorescence emission spectra of coumarin 102 loaded in the multi-stimuli-responsive nanogels in the presence of 10 mM DTT.

4. Conclusions In summary, we have designed and synthesized novel multi-stimuli-responsive nanogels allowing for selective release of multiple target cargos with distinct solubility characteristics (hydrophobic and hydrophilic). The hydrophobic cargos were noncovalently encapsulated into hydrophobic interiors of the nanogels. The hydrophilic cargos were covalently linked to the nanogels through redox-cleavable disulfide junction. Our results demonstrate that the release of hydrophobic cargos could be triggered by temperature, pH, and UV light whereas the hydrophilic cargos would be released in the presence of redox reagent. The multi-stimuliresponsive dual-cargo delivery system holds great potential for tissue engineering, anticorrosion, smart nanoreactors, especially for combination chemotherapy.

ASSOCIATED CONTENT Supporting Information. GPC, TEM image, UV−vis absorption spectra, and fluorescence spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; phone 86-10-62333619 (G.W.). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This study is supported by the National Natural Science Foundation of China (Grants 51373025 and 21074010), the Program for New Century Excellent Talents in University (NCET-11-0582), and the Fundamental Research Funds for the Central Universities (FRE-TP-12-004B). REFERENCES (1) Sengupta, S.; Eavarone, D.; Capila, I.; Zhao, G. L.; Watson, N.; Kiziltepe, T.; Sasisekharan, R. Temporal Targeting of Tumour Cells and Neovasculature with a Nanoscale Delivery System. Nature 2005, 436, 568−572. (2) Wang, Y.; Gao, S. J.; Ye, W. H.; Yoon, H. S.; Yang, Y. Y. Co-Delivery of Drugs and DNA from Cationic Core-Shell Nanoparticles Self-Assembled from a Biodegradable Copolymer. Nat. Mater. 2006, 5, 791−796. (3) Lehár, J.; Krueger, A. S.; Avery, W.; Heilbut, A. M.; Johansen, L. M.; Price, E. R.; Rickles, R. J.; Short III, G. F.; Staunton, J. E.; Jin, X. W.; Lee, M. S.; Zimmermann, G. R.; Borisy, A. A. Synergistic Drug Combinations Tend to Improve Therapeutically Relevant Selectivity. Nat. Biotechnol. 2009, 27, 659−666.

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For Table of Contents Use Only: Selective Release of Hydrophobic and Hydrophilic Cargos from Multi-Stimuli-Responsive Nanogels Ziquan Cao, Xiaoteng Zhou, Guojie Wang Here, we report a novel type of highly stable multi-stimuli-responsive nanogels for simultaneous encapsulation and selective release of hydrophilic and hydrophobic cargos. Due to the change of the morphology and chemical structures of the nanogels in response to temperature, pH, and UV light, the hydrophobic cargos would be released. In the presence of redox reagent, the hydrophilic cargos would be released via cleavage of the disulfide bonds.

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