Accepted Manuscript Title: An injectable, dual pH and oxidation-responsive supramolecular hydrogel for controlled dual drug delivery Author: Xinfeng Cheng Yong Jin Tongbing Sun Rui Qi Hanping Li Wuhou Fan PII: DOI: Reference:

S0927-7765(16)30034-0 http://dx.doi.org/doi:10.1016/j.colsurfb.2016.01.034 COLSUB 7611

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

27-8-2015 15-11-2015 19-1-2016

Please cite this article as: Xinfeng Cheng, Yong Jin, Tongbing Sun, Rui Qi, Hanping Li, Wuhou Fan, An injectable, dual pH and oxidation-responsive supramolecular hydrogel for controlled dual drug delivery, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2016.01.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

An injectable, dual pH and oxidation-responsive supramolecular hydrogel for controlled dual drug delivery Xinfeng Chenga,b, Yong Jin*c,d, Tongbing Suna,b, Rui Qia,b, Hanping Lic,d, Wuhou Fanc,d

a

Chengdu Institute of Organic Chemistry, Chinese Academy of Science, Center of

Polymer Science and Technology, Chengdu 610041, People's Republic of China b

University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049,

People's Republic of China c

National Engineering Laboratory for Clean Technology of Leather Manufacture,

Sichuan University, Chengdu 610065, People's Republic of China. E-mail: [email protected]; Tel: +86-28-85214963 d

Key Laboratory of Leather Chemistry and Engineering (Sichuan University),

Ministry of Education, Chengdu 610065, People's Republic of China

Graphical abstract

1

A novel injectable supramolecular hydrogel was successfully prepared based on PEGylated poly(ether-urethane) (PEU) nanoparticles and α-CD, which displayed a pH-induced reversible sol-gel transition and an oxidation-triggered degradation behavior, and showed promise as an ideal drug carrier for co-encapsulation and triggered release of hydrophobic and hydrophilic drugs.

Highlights 2

►Dual pH and oxidation responsive poly(ether-urethane) nanoparticles were developed. ►Injectable supramolecular hydrogels based on poly(ether-urethane) nanoparticles and α-CD were prepared. ►The hydrogels displayed a pH-induced reversible sol-gel transition and an oxidation-triggered degradation behavior. ►The hydrogels showed promise for co-encapsulation and triggered release of hydrophobic and hydrophilic drugs.

Abstract A novel pH and oxidation dual-responsive and injectable supramolecular hydrogel was developed, which was formed from multi-block copolymer poly(ether urethane) (PEU) and α-cyclodextrin (α-CD) inclusion complexes (ICs). The PEU copolymer was synthesized through a simple one-pot condensation polymerization of poly(ethylene glycol), di(1-hydroxyethylene) diselenide, dimethylolpropionic acid and 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate. In aqueous solution, the amphiphilic PEU copolymers could self-assemble into nanoparticles with dual pH and oxidation sensitivities, which can efficiently load and controllably release a hydrophobic drug indomethacin (IND). Then a dual-drug loaded supramolecular hydrogel was obtained by addition of α-CD and hydrophilic model drug (rhodamine B, RB) into the resulting IND-loaded PEU nanoparticle solution. The rheology studies 3

showed that the supramolecular hydrogels with good injectability underwent a pH-induced reversible sol-gel transition and an oxidation-triggered degradation behavior. The in vitro drug release results demonstrated that the hydrogels showed dual drug release behavior and the release rates could be significantly accelerated by addition of an oxidizing agent (H2O2) or increasing the environmental pH. Therefore, this injectable and dual stimuli-responsive supramolecular hydrogel based codelivery systems could potentially be a promising candidate for controlled drug delivery systems. Keywords: Supramolecular

hydrogel;

Polyurethane;

pH-responsive;

Oxidation-responsive; Dual drug delivery

1. Introduction Hydrogels have received considerable attention over the past decades in the field of biomedicine and bioengineering because of their hydrophilic character and biocompatibility [1-6]. The hydrogels can be crosslinked chemically (covalent interactions), physically (noncovalent interactions) or a combination of both [7-10]. Through inter-molecular covalent bonding, chemical cross-linking promotes the stability of polymeric hydrogels [2,5]. Compared with chemical cross-linking, supramolecular cross-linking, as a kind of physical interaction, exhibits unique convenience and flexibility, which has been widely used for fabrication of diverse functional hydrogels [11-14]. In recent years, the supramolecular hydrogels based on the self-assembly of the inclusion complexes (ICs) between cyclodextrins (CDs) with various polymers have been extensively studied as promising injectable biomaterials due to their shear-thinning property and excellent biocompatibility [15-18]. The in-situ encapsulation characteristic of such IC-based hydrogels not only enhances drug loading-levels, but also avoids structural changes of the drugs [19]. In addition, shear-thinning makes such hydrogels injectable simply under shear stress and recover 4

rapidly after removal of the shear stress, which breaks through the limits of the conventional implantable strategy for hydrogels [20,21]. More interestingly, some IC-based supramolecular micellar hydrogels have been developed in recent years [15,22,23], which could be serving as co-drug delivery systems for co-encapsulation and prolonged release of different therapeutic agents synchronously [24,25]. In this system, both the partial inclusion complexation and the hydrophobic aggregation act as supra-cross-links playing important roles in supramolecular hydrogel formation, dual drug encapsulation and controlled release. Compared to the conventional hydrogels, which typically tend to absorb hydrophilic drugs, this supramolecular micellar hydrogels have been designed as innovative solutions to overcome limitations as inefficient hydrophobic drug loading [26,27]. The drug release kinetics of such supramolecular micellar hydrogels are mostly determined by the dissolution of the hydrogels due to de-threading of PEG chains of micelles from the cavities of α-CDs [25,26]. However, most of these works focused on the linear PEGylated amphiphilic copolymers based supramolecular micellar hydrogels without external sensitivity. As a class of unique synthetic polymers, polyurethanes (PUs) with excellent mechanical properties and biocompatibility have been widely used in biomedical fields [28-31]. The biodegradability and sensitivity of PUs can be designed through a proper incorporation of corresponding functional blocks into the soft or hard segments of the polymer backbones [32,33]. Recently, a series of stimuli responsive polyurethanes-based nanoparticles have been developed as controlled drug delivery systems, which exhibited excellent biocompatibility and biodegradability, as well as the highly tunable composition and functionality [34-36]. For example, the pH-sensitive polyurethane nanoparticles containing ionizable groups (e.g. tertiary amine) in the backbones are especially interesting, as the variation of environmental pH may result in the swelling or disassembly of nanoparticles and triggered release of the encapsulated drugs [34,37]. However, for ordinary polyurethanes, the main

5

pathway of biodegradability is dependent on hydrolysis with degradation time ranging from weeks to months, limiting their applications as temporary biomaterials [32,33]. By incorporation of environmental-labile linkages [38-40], such as redox-cleavable disulfide or diselenide groups into the PU backbones, the PU based nanoparticles can be rapidly degraded into fragments accompanied by complete release of encapsulated model drugs [41-44]. Notably, in biological systems, there is a significant difference of redox potential existing between the extracelluar and intracellular compartments, and moreover elevated concentration levels of glutathione (GSH) or reactive oxygen species (ROS) are found in cancer cells [45,46]. So that the redox-degradable PU nanoparticles can be used as site-specific drug delivery systems for triggered release of encapsulated payloads due to their localized degradation characteristic [47]. Therefore, the combination of environment-sensitive and degradable PU nanoparticles with supramolecular hydrogel networks may provide the supramolecular hydrogels with unique self-assembly and stimuli-responsive characteristics, which will extend their practical applications in biomedical areas. To our knowledge, however, no work has been done for such an attempt. Herein, an injectable supramolecular hydrogel based on a dual pH and redox-responsive amphiphilic poly(ether urethane) (PEU) copolymer and α-CD was prepared and used to co-load and release of hydrophobic and hydrophilic drugs (Scheme 1). Firstly, an amphiphilic multiblock PEU copolymers consisting of PEG, dimethylolpropionic acid (DMPA) as pH sensitive goups, and di(1-hydroxyethylene) diselenide (DiSe) as redox sensitive goups were synthesized. They can self-assemble into nanoparticles in aqueous solution and load a hydrophobic drug indomethacin (IND). Then a dual-drug loaded supramolecular hydrogel was formed based on the inclusion complexes between α-CD and the PEG chains of IND-loaded nanoparticles by incorporating another hydrophilic model drug (rhodamine B, RB). Once hydrogels were exposed to a microenvironment of high pH or high H2O2 concentration, the PEU nanoparticles were swelling or destroyed, resulting in the relaxation or degradation of

6

the hydrogel and triggered release of the loaded dual drugs. Owing to this special pH or oxidation induced structural transformation, this injectable supramolecular hydrogel might be used as a candidate for on-demand dual drug delivery systems.

2. Experimental 2.1 Materials Poly(ethylene glycol) (PEG, Mn=2000), dimethylolpropionic acid (DMPA, 99%) and α-cyclodextrin

(α-CD,

98%)

were

purchased

from

Aldrich

Chemical.

Tetrahydrofuran (THF), dibutyltin dilaurate (DBTDL), hydrogen peroxide (H2O2), rhodamine B (RB) and indomethacin (IND) were analytical grade products purchased from Shanghai Aladdin Chemistry Co. Ltd. Di(1-hydroxyethylene) diselenide (DiSe) was

synthesized

and

purified

according

to

previous

report

[48].

3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate (IPDI, Analytical grade) was obtained from Shanghai Chemical Reagent Corporation (Shanghai, China) and used as received. PEG and DMPA were dried under vacuum at 80oC before use. All other reagents were of analytical grade and used as received. 2.2 Instrumentation The 1H NMR spectrum was recorded on a JEOL JNM-ECA 300 (300 MHz) spectrometer. Gel permeation chromatography (GPC) measurement was performed on a HLC-8320 GPC using polystyrene as a standard and THF as an eluent. Fourier transform infrared (FTIR) spectra were recorded on a ThermoFisher Nicolet 6700 spectrophotometer in KBr pellets. A Hitachi U-2010 spectrophotometer was used to record the transmittance and absorbance of samples. Atomic Force Microscopy (AFM) images were obtained using a Shimadzu Scanning Probe Microscope (SPM-9600). Sizes and distributions of polyurethane nanoparticles were measured with a Zetasizer Nano ZS dynamic light-scattering (DLS) instrument (Malvern, UK) at 25 oC at an angle of 90o. X-ray diffraction (XRD) measurements were conducted with powder

7

samples using a Philips X'Pert MPD

(40 kV, 30 mA) using Ni-filtered Cu Kα

radiation. Powder samples were mounted on a sample holder and scanned from 2θ = 3 to 5o at a speed of 20 min-1. The interior morphology of lyophilized hydrogels was observed by a Stereo Dissecting Microscope (XTL-340, Changfang Optical Instrument Co. Ltd., Shanghai, PR China) with a CCD camera (JVC TK-C921EC, Victor Co. of Japan Ltd.). The dynamic rheological measurements were performed on an Anton Paar MCR302 rheometer with parallel plate geometry (25 mm diameter) at a gap of around 1 mm. The oscillatory mode was applied in order to determine the variation of the storage and loss moduli (G’ and G”) as a function of frequency from 0.1 to 100 rad s-1 at 1% strain. The hydrogel viscosity was measured in steady mode as a function of shear rate sweeping from 0.01 to 100 s-1. All tests were performed at 25 oC. Digital photographs were taken by a Canon Power Shot A3000 IS digital camera. 2.3 Synthesis of poly(ether-urethane) (PEU) compolymers The poly(ether-urethane) (PEU) copolymers containing diselenide bonds and carboxyl groups were synthesized by a one-pot condensation of PEG, DiSe and DMPA-diol with IPDI, as shown in Scheme 2. The condensation reaction was conducted at a certain molar ratios of PEG:DiSe:DMPA:IPDI=1:2:3:6. Typically, 2.0 g of PEG (Mn=1000, 2.0 mmol), 1.0 g of DiSe (4.0 mmol), 0.8 g of DMPA (6.0 mmol) and a catalytic amount of DBTDL were dissolved at 50 oC in 60 ml of anhydrous THF in a flask under a dry nitrogen atmosphere. Then, 2.67 g of IPDI (12.0 mmol, the molar ratio of IPDI to (PEG+DiSe+DMPA) was 1:1) was added dropwise into the flask under stirring. The reaction mixture was stirred at 50 oC under a nitrogen atmosphere for 24 h. After that, an excess amount of methanol was added, and the mixture was reacted for another hour to eliminate the dibutyltin dilaurate residue and oligomers. The resulting products were collected by precipitation in diethyl ether, and further purified by redissolving in THF followed by precipitation in diethyl ether. The final product was dried under vacuum under 50 oC for 24 h, affording a yield of 80%. 8

1

H NMR (300 MHz, CDCl3): δ (ppm): 0.89-1.78 (trimethylcyclohexyl), 3.52-3.83

(-CH2CH2O-),

3.32-3.45

(-CH2-CH2-SeSe-),

3.83-3.95

(-O-CH2-CH2-SeSe-),

4.11-4.37 (-O-CH2-C(CH3)COOH-). 2.4 pH and oxidation responsiveness of the PEU nanoparticles Initially, the poly(ether urethane) (PEU) nanoparticles were prepared by direct dissolving method. No organic solvent was employed in the whole process of nanoparticles preparation. Typically, 0.30 g of the amphiphilic polymers was added into 20 mL of distilled water, followed by mild sonication for 5 min to accelerate dissolution of the polymers and then stirred slowly for 24 h at 25 oC to form the polymer nanoparticles solution (15 mg/mL). Then the PEU nanoparticles aqueous solution at pH~6.8 was prepared for the stimuli-responsiveness measurement. The PEU nanoparticles aqueous solution in response to pH was carried out by the stepwise addition of 0.1 N NaOH or HCl. The oxidation responsiveness of the PEU nanoparticles was investigated by adding an oxidizing agent H2O2 into the solution. In brief, 0.102 g of 30 wt% H2O2 was added into 10 mL of PEU nanoparticles solution to obtain a final concentration of H2O2 at 100 mM for the oxidation-responsiveness measurement. The particle size and distribution of the nanoparticles in response to pH and H2O2 were also recorded using a Zetasizer Nano ZS90 Malvern instrument. The morphology transformation of the particles was recorded by AFM measurement. 2.5 Preparation of PEU/α-CD based supramolecular hydrogels Supramolecular hydrogels were prepared by inclusion complexation of α-CD and PEU copolymers. The PEU/α-CD based supramolecular hydrogels were prepared by adding α-CD into the PEU nanoparticle solution. Typically, the PEU nanoparticle solution (15 mg mL-1) was prepared using the previous method prior to use. Then, 0.382, 0.436 or 0.491 g of α-CD was added into PEU nanoparticle solutions (5 mL, 15 mg mL-1) to reach a certain α-CD concentration of 7, 8, or 9 wt%, respectively. The mixture was stirred vigorously followed by sonication for 10 min to dissolve the 9

α-CD, then incubated in a 25 oC water bath for a few minutes. A gelation could occur due to the host–guest inclusion complexation between α-CD and the PEG chains of poly(ether urethane) copolymers in an aqueous system. The gelation time was visually observed by a vial inversion method when the sample solution did not flow. Rheological studies of the resultant hydrogels was measured on a rheometer in oscillatory or steady mode at 25 oC. A small amount of resultant hydrogel was freeze-dried as powders, and then analysed by XRD and FT-IR measurements. 2.6 Dual pH- and oxidation-responsive behaviors of the PEU/α-CD hydrogels The pH responsive behavior was studied by varying the pH values of the PEU/α-CD hydrogels. The pH of the hydrogel systems were adjusted to 6.8 and 9.2 using an appropriate buffer solution with the addition of 0.1 N HCl or NaOH. Then, a reversible transition between immobile and semifluid state was observed for the above hydrogels. The oxidation responsive behavior of the hydrogels was studied by using H2O2 as an oxidizing agent to cleave the diselenide linkages of hydrogels. The H2O2 solution with a concentration of 100 mM was added into the prepared hydrogels at room temperature to mimic the presence of an accelerated pathophysiologic oxidative microenvironment [49]. The gel with α-CD concentration of 9 wt% was chosen as an example for the oxidation-responsiveness measurements. Then a digital camera was used to record the gel-to-sol transition of hydrogels. 2.7 In vitro drug loading and release 2.7.1 In vitro release studies of indomethacin (IND)-loaded PEU nanoparticles IND was loaded into PEU nanoparticles as a hydrophobic model drug by a thin film hydration method. Briefly, IND (10 mg) and PEU copolymer (150 mg) were dissolved in 2.0 mL of ethanol and stood at room temperature for 2 h. Then the solvent was removed by rotary evaporation yielding a thin drug-containing polymer film on the sides of a round bottom flask. After that, 10 mL of phosphate buffer solution (PBS, pH=6.8, 5 mM) was added into the film, followed by mild 10

ultra-sonication for 5 min and stirred slowly overnight at room temperature. Finally, IND-loaded nanoparticles were obtained by filtration to remove the free drugs. For determination of drug loading content, IND-loaded nanoparticles were lyophilized, and then dissolved in DMF and measured by recording the absorption spectra at 320 nm using a UV-Vis spectrophotometer (Hitachi U-2010), wherein calibration curve was obtained with IND/DMF solutions with different IND concentrations. The drug loading capacities (DLC) and drug loading efficiency (DLE) were calculated by the following formula: DLC = (weight of loaded IND/ weight of PEU copolymers)×100% DLE = (weight of loaded IND/ weight of IND in feed)×100% In vitro release profiles of IND from nanoparticles were investigated in 50 mL of PBS solution (pH 7.4 or 9.2) with or without 50 mM (or 100 mM) H2O2. Typically, 5.0 mL of the IND-loaded nanoparticles solution was added into a dialysis bags (MWCO 3500 Da), then sealed and immersed into the release medium under gentle stirring at 37 oC. At predetermined time internals, absorbance measurements of different samples at 320 nm were recorded using a Hitachi U-2010 UV-Vis spectrophotometer. 2.7.2 In vitro release studies of IND/RB co-loaded supramolecular hydrogels The general protocol for the preparation of IND/RB co-loaded supramolecular hydrogels is as follows. Briefly, 70 μL of RB aqueous solution (1 mg mL-1) was added to 0.5 mL of stirred IND-loaded PEU nanoparticles solutions (15 mg mL-1), and then certain amount of α-CD was added into the solutions to reach α-CD concentration of 7, 8, or 9 wt%. The mixed solution was stirred and sonicated for 10 min, and then placed into a 1.5 mL cuvette and incubated at room temperature for 48 h to allow the mixture to form the hydrogel. To study the in vitro dual-drug release behavior, the cuvette was placed upside-down in a test tube with 50 mL of PBS solution with different pH values (pH~7.4 or 9.2) and concentrations of H2O2 solution (50 or 100 mM). And then the 11

test tubes were conditioned in a shaking water bath at 37 oC. At desired intervals, 2 mL of release medium was taken out and an equal volume of fresh media was added into the release system. The amount of RB and IND released from the hydrogel was determined from the absorbance by UV-vis spectroscopy at 554 and 320 nm, respectively. Each release experiment was conducted in triplicate.

3. Results and discussion 3.1 Synthesis of poly(ether-urethane) (PEU) compolymers Scheme 2 shows the synthetic procedures of pH and oxidation-responsive poly(ether-urethane) (PEU) copolymer. Briefly, the PEU copolymer was prepared via one-pot condensation reaction between IPDI and the diols PEG, DiSe and DMPA. To confirm the PEU copolymer, 1H NMR, FTIR and gel permeation chromatography (GPC) analyses were carried out. Fig. 1 shows the representative 1H NMR spectrum of PEU. The obvious signals located at 0.89-1.78ppm and 3.52-3.83ppm were ascribed to the protons of trimethylcyclohexyl frame and -CH2CH2O- of PEG segments, respectively. The signals at 3.32-3.45ppm, 3.83-3.95ppm and 4.11-4.37ppm were attributed to the protons of -CH2-CH2-SeSe-, -O-CH2-CH2-SeSe- and -O-CH2-C(CH3)COOH-, respectively, which indicated the coexistence of Dise and DMPA segments. These typical signals confirmed that the copolymer composed of PEG, DiSe, DMPA and IPDI units was successfully synthesized. The contents of the composites in the PEU copolymer were estimated by the 1H NMR analysis. The mol ratio of PEG/DiSe/DMPA/IPDI determined by the contrast of integral values of their characteristic peaks was calculated to be 1/1.8/2.5/5.6, which is consistent with their initial feed ratio (i.e., 1/2/3/6). The FT-IR spectrum of PEU was shown in Fig. S1. The absorption peaks at 1710 cm-1 (C=O), 1540 cm-1 (N-H), 1350 cm-1 (C-N) and no absorption band at 2270cm-1 (-NCO) indicated that IPDI had been incorporated into the copolymer chain. The existence of PEG segment was evidenced by the absorption bands at 1460 cm-1 (-CH2) and 1110 cm-1 (C-O-C). On the other hand, the bands at 12

1650 cm-1 representing the C=O stretching vibration bands of carboxyl group and 775 cm-1 ascribed to the stretching of C−Se groups implied the presence of DMPA and DiSe segments in PEU copolymer. In addition, GPC measurement was performed to determine the molecular weights and polydispersity index of the synthesized copolymer, which were determined to be 7910 (Mw) and 1.65 (PDI), respectively (Fig. S2). Based on these results, the random PEU copolymer composed of PEG segments as the hydrophilic parts, IPDI as the hydrophobic PU blocks, and the remaining components (DMPA and DiSe) serving as pH and oxidation-responsive segments, were successfully prepared. 3.2 The pH- and oxidation-responsive behaviors of the PEU nanoparticles The resulting poly(ether urethane) copolymers were composed of hydrophilic PEG chains and hydrophobic PU blocks, so they could self-assemble into nanoparticles in aqueous solution. Assembly of the PEU copolymers into nanoparticles in aqueous media was confirmed by dynamic light scattering (DLS)-based size measurements and atomic force microscopy (AFM)-based morphology tests (Fig. 2). As shown in Fig. 2a, the DLS data shows that the hydrodynamic diameter (Dh) of the nanoparticles is about 165 nm with a PDI of 0.206 at pH~6.8. Meanwhile, the AFM images reveal that the nanoparticles generally have a regular spherical morphology with an average diameter around 142 nm at pH~6.8 (Fig. 2b). The value of Dh measured using DLS is a little larger than the average radius (about 23 nm) of nanoparticles measured by AFM. The reason is that DLS was measured in aqueous solution, while AFM images were observed after the solvent evaporation, which may lead to the collapse, shrinkage, and even destruction of the nanoparticles. However, to some extent, the AFM method can directly observe the morphology of the nanoparticles. For this result, the aggregation of the nanoparticles is in agreement with the result of DLS measurement. To investigate the pH and oxidation-responsive aggregation behaviors of the PEU nanoparticles, the as-prepared nanoparticles solution at pH~6.8 was adjusted to

13

different pH values (i.e., pH~4.0 or 9.2) or treated with the oxidizing agent (H2O2). Upon increasing the pH to 9.2, a nearly transparent solution was observed immediately (Fig. 2c). While, when decreasing the pH from 9.2 to 4.0, the solution turned to be cloudy at once. Notably, further treated back to pH~9.2, the cloudy solution returned to the almost transparent state again. These features indicated that the copolymer solution has good reversible pH responsiveness. When adding H2O2 into the copolymer solution (100 mM), both of the clear and cloudy solutions became transparent after 10 h (Fig. 2c). For more insights into the pH and oxidation-responsive aggregation behaviors of the PEU nanoparticles, DLS and AFM analyses were carried out. As shown in Fig. 2a, the DLS results implied that the size of the nanoparticles increased at both higher or lower pH conditions compared with that in neutral condition (pH~6.8). Fig. 2b shows the corresponding AFM images of the nanoparticles, which also showed larger particles under the treatment of H+ or OH-. However, the mechanisms of particle size increase at these two conditions are different. When nanoparticles were in higher pH condition, the Dh of the nanoparticles increased from 165 nm (pH~6.8) to 187 nm (pH~9.2). This is because the carboxyl moieties of DMPA segments are deprotonated at basic condition and changed to be more hydrophilic, giving rise to the nanoparticles transforming from dense to swollen structures or even dissolving. In acidic condition, the Dh increased from 165 nm to 309 nm (pH~4.0) accompanied by a broadened PDI of 0.262. The probably explanation is that carboxyl moieties are completely protonated in an acidic medium, resulting in increasing of intermolecular hydrophobic interactions and aggregation of the nanoparticles. Additionally, an obvious size distribution and morphology transformation was observed for the PEU nanoparticles in response to H2O2. As depicted in Fig. 2a, the size distribution became bimodal with the occurrence of large aggregates (Dh >1 μm) after treatment with 100 mM H2O2 for 24 h. Further, a broader size distribution was observed as the population of the large aggregates increased over time. The increase in size of the aggregates may be

14

assigned to the degradation of PU micellar cores upon the cleavage of diselenide linkages in response to oxidizing agent and formation of larger hydrophobic aggregates of deionized polyurethane fragments. Moreover, large and irregularly shaped aggregates were found from the AFM images (Fig. 2b), agreeing well with the DLS results. Similar phenomenon can also be found in some other polyurethane based stimuli-degradable micelle systems [42,50]. All the above results indicate that the PEU copolymer nanoparticles have dual pH and oxidation-responsive behaviors, which can be used as a drug nanocarrier for triggered release of model payloads, especially hydrophobic drugs. 3.3 Preparation of PEU/α-CD based supramolecular hydrogels The PEU/α-CD based supramolecular hydrogels were prepared by adding α-CD molecules into PEU nanoparticles solutions (15 mg mL-1) directly, as shown in Fig. 3. The formation of the supramolecular hydrogel was based on the host-guest inclusion complexation of α-CD and the chains of the PEG block in PEU nanoparticles solutions. Due to the amphiphilicity of the PEU copolymers, they could self-assemble into micelles with a hydrophobic PU core and a hydrophilic PEG shell in aqueous solution [33,51]. After α-CD molecules were added into the nanoparticles solutions, the chains of the PEG shell threaded them to form crystalline inclusion complexes (ICs). The hydrophobic ICs tend to aggregate into microcrystals, which act as physical cross-links and then induce the formation of supramolecular polymer networks. In addition, the aggregated PEU nanoparticles may serve as the second physical crosslinks to maintain the hydrogel networks. Scheme 1 shows a schematic illustration for the formation of a supramolecular hydrogel based on PEU copolymers and α-CDs. Moreover, the gelation time for the three hydrogel samples with different 15

concentrations of α-CD varying from 7, 8 to 9 wt% was recorded to be 32, 28 and 26 min, respectively, which was determined by a vial inversion method. The inclusion complex formation between PEG blocks of the PEU copolymer and α-CD in the hydrogels was confirmed by X-ray diffraction (XRD) studies of the hydrogels (Fig. S3). Two obvious peaks at 19.6° and 23.8° from the PEG crystalline phase were found for PEU copolymer. Nonthreaded α-CD cage structure had salient reflections occurring at 12.0, 14.2, and 21.6. Different from the diffraction patterns of either PEU copolymer or α-CD, the lyophilized hydrogel sample showed two typical diffraction peaks located at 2θ = 20.2° and 22.9° ascribed to the channel-type crystallinity of the ICs, as reported by other studies [16,25]. In addition, from the FT-IR analysis (Fig. S1), the characteristic absorption peaks derived from the PEU copolymer and α-CD can also be found in the FT-IR spectrum of the dried hydrogel sample, indicating the coexistence of the two components in the hydrogel network. The interior morphology of lyophilized hydrogels was also examined by a Stereo Dissecting Microscope. A porous structure was observed for the lyophilized samples due to their high water content (Fig. S4). 3.4 pH- and Oxidation-responsive characteristics of the PEU/α-CD supramolecular hydrogels The pH response of the PEU/α-CD hydrogels were studied in PBS solution by altering the pH values between acid and basic conditions. At pH~6.8, all the hydrogels exhibited a solid state and no fluid was observed (Fig. 3). When adjusting the pH value of the hydrogels to 9.2, the hydrogels became a viscous fluid but not a

16

aqueous solution, which can flow slowly when inverting the vial (Fig. 3). The probably reason is that the nanoparticles transformed from dense to swollen structures or even dissolving at basic condition due to the more hydrophilic structure of DMPA segments, so that the intermolecular interactions of the hydrophobic PU blocks serving as the second crosslinks were weakened to some extent, resulting in a slipping loosely network (Scheme 1). It was noteworthy that the viscous fluid could become a solid gel again when changing the pH back to 6.8. In additon, the hydrogels displayed an oxidation responsive gel-to-sol transition, as shown in Fig. 3. As studied previously, the PEU nanoparticles underwent a oxidation-induced dissolution process due to the introduction of degradable DiSe segments, which can be cleaved under oxidation condition. Thus, the PEU nanoparticles in the hydrogels can be completely destroyed under oxidation condition leading to the bulk degradation of the supramolecular hydrogel network, as shown in Scheme 1. Overall, the PEU/α-CD hydrogels showed a pH and oxidation dual-sensitive property as expected, which can be developed as a stimuli-responsive drug delivery carrier, as triggered by changes in pH and oxidation potential of the environment. 3.5 Rheological behaviors of the PEU/α-CD supramolecular hydrogels Rheological studies of the supramolecular hydrogel were carried out to determine the physical properties of the gels, i.e. mechanical strength and shear-thinning behavior. Fig. 4 reveals the dynamic and steady rheological behaviors of the supramolecular hydrogel with 9 wt% of α-CD under different pH or oxidation conditions. At pH~7.4, the storage modulus (G’) was 240-300 Pa in the linear viscoelastic region, which was approximately one order of magnitude greater than the loss modulus (G”), indicating solid gel status (Fig. 4a). When increasing the pH value of the hydrogel to 9.2, both the modulus decreased remarkably, especially for the G’

17

value. The decreased mechanical strength was caused by the loosely structure of the hydrogel under basic condition as explained above. When the pH value was returned to 7.4, the G’ value was recovered to be 144-194 Pa, which could be estimated to be about 65% of the initial G’ (240-300 Pa). It should be noted that the recovered sample exhibited a gel state that remain unchanged after several days. As for the incomplete recovery of strength, a possible explanation for this effect is that the partial structural breakdown under basic condition leads to an incomplete structure recovery. While, after H2O2 treatment over time, both of G’ and G” were almost zero over the entire range of testing frequencies, indicating a fluid sol status. Such irreversible gel to sol transition was ascribed to the bulk dissociation of the supramolecular hydrogel induced by the oxidation-degradation behavior of the PEU copolymer. Fig. 4b depicts the steady rheological behavior of the hydrogels as a function of shear rate. It was clearly observed that the initial viscosity of the hydrogels show a sensitivity to the environmental factors (i.e., pH and oxidation potential), which was similar to that of the G’ value. Moreover, the viscosity of the resultant hydrogels displayed a progressive decrease with increasing shear rate, suggesting that the hydrogels have a shear-thinning behavior. It was also observed that the hydrogel structure can recover immediately after extrusion of the supramolecular hydrogel using a syringe (Insert, Fig. 4b). Such a shear-thinning behavior seems to be advantageous when it is used as an injectable hydrogel matrix [20]. Bioactive molecules (e.g., drugs, proteins, cells) can be encapsulated into the hydrogel matrix

18

firstly at room temperature without using any organic solvent. And then these drug-loaded hydrogels can be readily injected into the specific tissues under pressure owing to the shear-thinning property, and hence serve as a local depot for controlled release. In comparison with the conventional implantable hydrogels, the injectable hydrogels for drug delivery or cell encapsulation would be much more attractive. 3.6 In vitro stimuli-responsive drug release of drug-loaded nanoparticles and supramolecular hydrogels Due to the highly hydrated and hydrophilic internal structures of the PEU/α-CD hydrogels, these injectable supramolecular hydrogels can be used as efficient drug-carriers for encapsulation and controlled delivery of some hydrophilic drugs. Moreover, compared with the conventional hydrogels, the proposed hydrogels here can be further utilized for encapsulating a water-insoluble hydrophobic drug due to the incorporated PEU nanoparticles which have hydrophobic inner cores. More importantly, these hydrogels displaying pH/oxidation dual responses may be able to release on demand, as triggered by changes in pH and oxidation potential of the environment. To investigate the in vitro dual-drug release behavior of the injectable supramolecular hydrogels, a hydrophobic drug indomethacin (IND) and a hydrophilic dye rhodamine B (RB) were chosen as model drugs to incorporate into the hydrogels by a simple two-step method as described in the experimental section. Then the release behaviors of the dual drug loaded hydrogels were investigated at different pH with and without H2O2 at 37 oC, respectively. In addition, the release profiles of the IND-loaded nanoparticles were also studied for a comparison. 3.6.1 In vitro release of entrapped model drug IND from the PEU nanoparticles IND was loaded into PEU nanoparticles via a thin film hydration method. The hydrodynamic diameter of IND loaded nanoparticles was 175 nm with a PDI of 0.227, as determined by DLS (Fig. S5). Notably, the diameter of IND loaded nanoparticles 19

was larger than the IND free ones (165 nm), which may due to the co-assembly of the block copolymers and the IND molecules. The drug loading capacity (DLC) and drug loading efficiency (DLE) were calculated to be 18.1% and 68.2%, respectively. The pH and oxidation-induced drug release behaviors of the IND-loaded nanoparticles were shown in Fig. 5. A slow release of about 35% of the entrapped IND within 24 h was observed at pH~7.4. However, more than 40% of IND was released in 12 h, and about 50% was released within 24 h at pH~9.2, owing to that the swelling behavior of nanoparticles under basic condition facilitates drug release. When treated with 50 mM H2O2 at pH~7.4, a dramatic increase in the release rate of IND was observed and an almost complete release was obtained within 16 h. In addition, by increasing the concentration of H2O2 to 100 mM, a faster release rate was obtained (Fig. S6). The possible reason is that the oxidation-triggered cleavage of the diselenide bond provided a fast disassembly of nanoparticles, thereby causing an accelerated release of the enclosed drugs. Notably, the highest IND release rate was observed when dual stimuli (pH 9.2 and addition of 50 mM H2O2) were combined. The possible explanation is as follows, the pH-induced particle swelling allows more H2O2 to cleave the diselenide linkages, resulting in a rapid dissociation of the nanoparticles, thus providing an enhancement in the release kinetics of the encapsulated drugs. These results indicate that PEU nanoparticles could be considered as a class of highly efficient drug delivery system to realize on-demand release of the encapsulated hydrophobic drugs when triggered by pH and/or oxidative environment. 3.6.2 In vitro release of RB and IND from the dual drug-loaded PEU/α-CD hydrogels In order to evaluate the ability of the PEU/α-CD hydrogels to efficiently deliver hydrophilic drugs, in vitro rhodamine B (RB) release from the injectable supramolecular hydrogels in PBS (pH~7.4) at 37 oC was studied. A sustained release for about one month was observed for the hydrogels with different contents of α-CD (7~9 wt%), as shown in Fig. S7. Additionally, the RB release rate decreased with the increase of α-CD amount, owing to that a greater gelation extent and a denser 20

hydrogel network were formed in the case of a higher α-CD amount, which hindered the release of loaded drugs from the supramolecular hydrogel. These preliminary results suggest that the PEU/α-CD hydrogels can be used as efficient drug-carriers for encapsulation and sustained release of the hydrophilic drugs. To test the possibility of the resultant hydrogel system for dual delivery of hydrophilic and hydrophobic drugs, further investigation was dealt with the in vitro release profiles of loaded RB and IND from the resultant hydrogels at different pH with or without H2O2, as shown in Fig. 6. The hydrogel with higher concentration of α-CD (9 wt%) was chosen as an instructive example. The hydrogel exhibited a sustained release of RB and IND at pH~7.4, e.g., only 47% IND and 60% RB were released within 30 days, respectively. The prolonged release of the both drugs was mainly caused by the diffusion and partial breakup of supra-crosslinks. While at pH~9.2, a relative faster release of the both drugs (e.g., 59% IND was released within 30 days) was observed, due to the more loosely structure of the hydrogel at basic condition (Scheme 1). However, in the presence of 50 mM H2O2 at pH~7.4, the release rates of RB and IND was obviously faster than that in PBS without H2O2 (at pH~7.4 and pH~9.2), especially in the initial 10 days. It might be ascribed to the rapid erosion of the supramolecular hydrogel due to the oxidation-degradation behavior of the PEU nanoparticles-based crosslinks, as shown in Scheme 1. Moreover, under higher concentration of H2O2 (100 mM), the hydrogel shows a more enhanced release behavior of the dual drugs relative to the former condition (50 mM H2O2) due to its stronger oxidation environment. Obviously, at this oxidation condition, the hydrogel shows an almost complete release (~96%) of RB after 17 days (Fig. 6a). Notably, in all cases, the hydrogel shows a slower release rate of IND compared to that of RB during the measurement time. This phenomenon could be explained as follows, the dissociation of the supramolecular hydrogels lead to releasing free RB molecules and IND-loaded nanoparticles, and then the released nanoparticles sequentially release the loaded IND that prolongs the release of the IND (Scheme 1).

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To understand the release mechanism of encapsulated drugs, we fitted the accumulative drug release data using Ritger-Peppas equation [52,53], as follows: Mt / M∞ = ktn

(for Mt / M∞ ≤ 0.6)

where Mt and M∞ are the cumulative amount of the drug released at t and equilibrium, respectively, k is the rate constant relating to the properties of the hydrogel matrix and the drug, and n is the release exponent characterizing the transport mechanism. If n = 0.5, the drug release mechanism is Fickian diffusion, corresponding to Case I. If n = 1, Case II transport occurs, leading to zero-order release. When the value of n is between 0.5 and 1, anomalous transport is observed, corresponding to Case III, and the structural relaxation is comparable to diffusion. When n < 0.5, a pseudo-Fickian behavior of diffusion occurs. The corresponding drug release kinetic data obtained from fitting drug release experimental data to Ritger-Peppas equation were listed in Table S1. The n values for the initial several hours of the RB release profiles at pH~7.4 and 9.2 without H2O2 were 0.44 and 0.46, respectively, indicating that the kinetics of RB release corresponds to that of pseudo-Fickian diffusion. The release of IND follows a similar diffusion controlled kinetic, which has a value of n = 0.41-0.44 from pH~7.4 to 9.2. While, the n values for IND and RB release profiles at pH~7.4 with H2O2 were calculated to be from 0.62 to 0.72, suggesting an anomalous transport process (0.5 < n < 1, case III), where the drug release is controlled by diffusion and structural erosion caused by the oxidation-induced cleavage of the diselenide bonds. Based on these results, the injectable PEU/α-CD hydrogels could be served as a potential co-delivery system for both hydrophilic and hydrophobic drugs, and their dual pH and oxidation stimuli sensitivities would provide an opportunity to fine-tune various parameters to obtain the desired release profile. 4. Conclusions A novel dual pH and oxidation-responsive supramolecular hydrogel was successfully prepared based on PEGylated poly(ether-urethane) nanoparticles and α-CD, which show promise as an ideal drug carrier for co-encapsulation and release of hydrophobic 22

and hydrophilic drugs. The supramolecular structure can recover immediately after extrusion of the hydrogels using a syringe, indicating its injectable characteristic. Moreover, the release kinetics of the dual drugs could be controlled by pH-induced relaxation and/or oxidation-triggered degradation of the supramolecular hydrogel. Together with many other advantages, such as the good biocompatibility of PEG and α-CD, mild preparation condition (e.g. without using organic solvent) and ease of handling, in-situ drug loading and controlled drug release property, this supramolecular hydrogel can be potentially used as an injectable co-delivery system. Acknowledgements This work was financially supported by the National High-tech Research and Development Projects (863) (2013AA06A306), the National Natural Science Foundation of China (21474065), and the Sichuan Province Science and Technology Support Projects (2010FZ0093). References [1] Y. Qiu and K. Park, Adv. Drug Delivery Rev., 64, Supplement (2012) 49. [2] M. K. Nguyen and E. Alsberg, Prog. Polym. Sci., 39 (2014) 1235. [3] N. K. Singh, D. S. Lee, J. Control. Release, 193 (2014) 214. [4] Y. Li, J. Rodrigues and H. Tomas, Chem. Soc. Rev., 41 (2012) 2193. [5] P. M. Kharkar, K. L. Kiick and A. M. Kloxin, Chem. Soc. Rev., 42 (2013) 7335. [6] C. T. Huynh, M. K. Nguyen and D. S. Lee, Macromolecules, 44 (2011) 6629. [7] D. Das, P. Ghosh, A. Ghosh, C. Haldar, S. Dhara, A. B. Panda and S. Pal, ACS Appl. Mater. Interfaces, 7 (2015) 14338. [8] M. K. Gupta, J. R. Martin, T. A. Werfel, T. Shen, J. M. Page and C. L. Duvall, J. Am. Chem. Soc., 136 (2014) 14896. [9] K. L. Liu, Z. Zhang and J. Li, Soft Matter, 7 (2011) 11290. [10] C. Pradal, L. Grøndahl and J. J. Cooper-White, Biomacromolecules, 16 (2015) 389. [11] X. Liao, G. Chen and M. Jiang, Polym. Chem., 4 (2013) 1733. [12] M. r. Saboktakin and R. M. Tabatabaei, Int. J. Biol. Macromol., 75 (2015) 426. [13] S. Tan, K. Ladewig, Q. Fu, A. Blencowe and G. G. Qiao, Macromol. Rapid Commun., 35 (2014) 1166. [14] E. A. Appel, J. del Barrio, X. J. Loh and O. A. Scherman, Chem. Soc. Rev., 41 (2012) 6195. [15] J. Li, NPG Asia Mater., 2 (2010) 112. [16] Z. Tian, C. Chen and H. R. Allcock, Macromolecules, 46 (2013) 2715. 23

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Figure captions: Fig. 1 1H NMR spectrum of PEU copolymer in CDCl3. Fig. 2 DLS data (a), AFM images (b) and Photographs (c) of self-assembled PEU nanoparticles in aqueous solution at different pH values and oxidation condition. Fig. 3 Formation of supramolecular nanoparticlar hydrogels induced by the inclusion complexation between α-CD and the PEG chains of the PEU copolymer and their dual pH- and oxidation-responsive behaviors. The concentration of the copolymer and α-CD in the hydrogels was 15 mg mL-1and 105 mg mL-1 (~9 wt%), respectively. Fig. 4 (a) Storage modulus (G’) and loss modulus (G”) of the hydrogels as a function of frequency; (b) The viscosity of the hydrogels as a function of shear rate (Insert: photograph of the extrusion of PEU/α-CD hydrogel using a syringe). The concentration of the copolymer and α-CD in the hydrogels was 15 mg mL-1 and 105 mg mL-1 (~9 wt%), respectively. Fig. 5 In vitro IND release from drug loaded nanoparticles under different pH and oxidation conditions at 37 ˚C. Fig. 6 In vitro RB (a) and IND (b) release from the dual drug-loaded supramolecular hydrogels upon exposure to different pH and concentrations of H2O2 solution at 37 ˚C.

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Fig 1

Fig 2a

27

Fig 2b

28

Fig 2c

29

Fig 3

30

Fig 4a

Fig 4b

31

Fig 5

Fig 6a

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Fig 6b

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Scheme captions Scheme 1 Schematic of the drug loading and environment triggered release from drug-loaded dual pH- and oxidation-responsive supramolecular hydrogels. Scheme 2 Synthetic route of the poly(ether-urethane) copolymers.

Scheme 1

Scheme 2

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