Nanogel-Incorporated Injectable Hydrogel for Synergistic Therapy

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Biological and Medical Applications of Materials and Interfaces

Nanogel-incorporated injectable hydrogel for synergistic therapy based on sequential local delivery of combretastatinA4 phosphate (CA4P) and doxorubicin (DOX) Wen Jing Yang, Peng Zhou, Lijun Liang, Yanpeng Cao, Junqin Qiao, Xiaohui Li, Zhaogang Teng, and Lianhui Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04394 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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Nanogel-incorporated injectable hydrogel for synergistic therapy based on sequential local delivery of combretastatin-A4 phosphate (CA4P) and doxorubicin (DOX) Wen Jing Yang,a Peng Zhou,a Lijun Liang,a Yanpeng Cao,a Junqin Qiao,b Xiaohui Li,a,c Zhaogang Teng,d Lianhui Wang a* a

Key Laboratory for Organic Electronics and Information Displays (KLOEID), Jiangsu Key Laboratory for Biosensor, Institute of Advanced Materials (IAM), Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing, 210023, China b

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry & Chemical Engineering and Center of Materials Analysis, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China c

School of Geography and Biological Information, Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China d

Department of Medical Imaging, Jinling Hospital, School of Medicine, Nanjing University, 163 Xianlin Avenue, Nanjing 210002, China.

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ABSTRACT

Drug combination therapies employing dual drug delivery systems offer an effective approach to reduce disadvantages of single drug therapy, such as high dose and easy generation of drug resistance. Herein, a dual drug delivery system based on nanogel-incorporated injectable hydrogel (NHG) was designed for sequentially local delivery of combretastatin-A4 phosphate (CA4P) and doxorubicin (DOX) for antiangiogensis and anticancer combination therapy. The injectable hydrogel was prepared for loading and quick release of hydrophilic drug CA4P, while the pH and redox stimuli-responsive nanohydrogels were incorporated into the injectable hydrogel by pH-responsive boronate ester bond for sustained long-term DOX delivery. The dual drug loaded NHG system released CA4P and DOX sequentially and exhibits high inhibitory activities on the cancer cell proliferation in vitro. It displayed superior therapeutic efficacy in vivo with only one single injection. Immunohistochemistry analyses suggested a synergistic therapeutic effect through tumor vascular collapse caused by CA4P and tumor cell apoptosis induced by DOX. The combination therapy of antiangiogenic and cytotoxic drugs using NHG delivery system offers a promising approach for improved cancer therapeutic efficacy. The nanogel-embedded injectable hydrogel can be employed as a universal drug carrier for local dual drug delivery with sequential release behaviors by simple injection.

KEYWORDS: Injectable hydrogel, nanohydrogel, stimuli-responsive, localized drug delivery, combination therapy

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1. Introduction In the past few decades, various nanomaterials have been designed for biomedical applications.13

Particularly, a variety of excellent drug nano-carriers for the treatment of cancers are designed

to minimize the side effect and improve therapeutic effect.4-6 Although there are many encouraging achievements, drug delivery vehicles typically exhibit the poor ability to load one single anti-cancer drug. In order to overcome the disadvantages of single drug therapy, such as high dose and easy generation of drug resistance, drug combination therapies employing dual drug delivery systems (DDS) have been developed.7-12 The “co-entrapped” approach is one of the most common strategies for delivering two or more drugs. Different drugs are entrapped simultaneously in the formation process of drug vehicles.8,9 However, the release profiles of the entrapped drugs are very similar without controlled sequential release properties. Another strategy involves the synthesis of “prodrug”. One drug is coupled to the polymer chains by various responsive bonds to produce “prodrug” polymers, and the other drug is entrapped by the “prodrug” polymers in the self-assembly process.10,13,14 In this case, the designed dual drug delivery systems can exhibit different drug release profiles. Nevertheless, the “prodrug” approach is only suitable for certain drugs which contain special structures/functional groups as well as retain their therapeutic activities in the coupling and decoupling reactions. Accordingly, it is preferable to develop a versatile dual drug delivery vehicles to achieve controlled different release profiles for different drugs.

Utilization of anticancer drugs with different antitumor mechanisms to realize synergistic treatment is of great importance when designing the combination therapy systems. As a first-line chemotherapeutic agent, doxorubicin (DOX) triggers cell apoptosis by intercalating in the DNA

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base pairs and blocking the replication activities.15,16 Apart from the cytotoxic chemotherapy, the antiangiogensis therapy has attracted increasing attention.17,18 The tumor neovasculature plays an important role in the metastasis process that is considered as the main reason for cancer-related death.18,19 In other words, blocking angiogenesis to impede the metastasis could be regarded as an alternative cancer treatment method. However, the single antiangiogenesis therapy may lead to tumor hypoxia, increasing tumor invasiveness and chemotherapy resistance.20,21 Accordingly, a promising strategy is the combination of cytotoxic drugs with antiangiogenic agents for enhancing therapeutic effect. The optimal scenario for this combination therapy is to deliver antiangiogenic drug inside of a tumor with rapid release profiles leading to the vascular shutdown, and then sustained slow release of cytotoxic drugs for killing tumor cells. The antiangiogenic drug combretastatin-A4 phosphate (CA4P) is a hydrophilic phosphate derivative from combretastatin-A4, which targets abnormal vascular system of the tumor and causes blood vessel collapse.13,22,23 Thus, it would be ideal to design a dual drug delivery system delivering antiangiogenic agent (CA4P) and cytotoxic drug (DOX) with sequential release behaviors for combination therapy.

Hydrogel is three-dimensional polymeric networks with good hydrophilicity.24,25 A variety of drugs can be incorporated and released from their loosely inner structures. As a result, hydrogel can serve as effective hydrophilic drug depots to afford local drug delivery with high-dose and sustained drug release behaviors.26-28 Particularly, injectable hydrogels not only possess the advantages of high drug delivery efficiency and drug utilization, but also reduce the systematic side effect with simple injection instead of surgery.29,30 Therefore, injectable hydrogels exhibit great potential for local drug delivery. Polymer nanohydrogels, a class of nanometer-sized

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hydrogels, have extensively employed as drug nano-carriers due to their hydrophilicity, flexibility, versatility, biocompatibility, tunable morphology and tailored surface properties.31-34 By employing the facile reflux-precipitation polymerization, the polymer nanohydrogels, with various stimuli-responsive properties, can be readily prepared for DOX delivery.35-37 Furthermore, it has been reported that nanogel-embedded hydrogel leads to improved kinetic release profile and sustained long-term release of drugs.38,39 Thus, incorporation of both drugloaded nanohydrogel and hydrophilic drugs into hydrogels could offer a promising platform for dual drug delivery with sequential drug release profiles.

In this current study, a dual drug delivery system employing nanogel-incorporated injectable hydrogel (NHG) was designed for local delivery of CA4P and DOX sequentially for antiangiogenesis and anticancer synergistic therapy. Firstly, the pH and redox stimuli-responsive poly(acrylic acid-co-4-vinylphenylboronic acid) nanohydrogels (P(AA-co-4-VPBA) NG) were synthesized via one-step reflux-precipitation polymerization to serve as effective nano-carriers for DOX delivery. Meanwhile, the injectable hydrogel was design for delivery of hydrophilic drug CA4P. Then the DOX loaded NG were incorporated into the injectable hydrogels through reversible chemical bonds (boronate ester) to produce a dual drug delivery systems (DOXCA4P@NHG). After a single injection of DOX-CA4P@NHG, the antiangiogenic drug CA4P would be released quickly from the hydrogel, leading to the vascular collapse in tumor areas. The subsequent slow release of cytotoxic DOX, caused by dissociation of nanogels from hydrogel and DOX release from nanogels, would then induce tumor cells apoptosis. Such DOXCA4P@NHG system with sequential drug release behaviors was evaluated by a series of assays,

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including in vitro and in vivo experiment, to reveal its potential as dual drug delivery system for synergistic therapy.

2. Materials and Methods 2.1 Materials The chemicals were purchased from different companies. The detailed information and purification procedures were described in the Supporting information.

2.2 Preparation of P(AA-co-4-VPBA) nanohydrogels (NG) The P(AA-co-4-VPBA) NG with pH and redox dual stimuli-responsive properties were prepared via reflux-precipitation polymerization, as follows: AA (304 mg, 4.21 mmol) and 4-VPBA (266 mg, 1.80 mmol), AIBN (10 mg, 0.61 mmol) and crosslinker BMOD (26 mg, 0.09 mmol) were dissolved in acetonitrile (AN, 40 mL) with the aid of ultrasound (with a frequency of 37 kHz at 100% power, P30H, Elma, Germany) at room temperature for 10 min to get a homogeneous mixture. Then the reaction mixture was heated with a reflux condenser and became turbidity after boiling for about 5 min. After 1 h, the resulted P(AA-co-4-VPBA) nanohydrogels were separated and washed thoroughly by centrifugation at 10000 rpm for 5 min and redispersed in AN under ultrasonication thrice. Finally, the purified P(AA-co-4-VPBA) NG were uniformly dispersed in deionized water and freeze-dried with a lyophilizer.

2.3 Synthesis of poly(N-(3,4-dihydroxyphenethyl) methacrylamide-co-poly(ethylene glycol) methyl ether methacrylate) (poly(DMA-co-PEGMA))

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Firstly, monomer N-(3,4-dihydroxyphenethyl) methacrylamide (DMA) was synthesized by reaction of dopamine and methacrylic anhydride as previous reported method.40,41 The poly(DMA-co-PEGMA) was synthesized via conventional free radical polymerization of DMA and PEGMA. The experimental details were described in the Supporting information.

2.4 Preparation of injectable hydrogels The injectable hydrogels were obtained by the thiol-catechol Michael addition reaction between 4-arm PEG-SH and poly(DMA-co-PEGMA). About 0.5 mL of the copolymer solution (50 mg/mL, containing around 10 mmol catechol groups) was mixed with 4-arm PEG-SH (20 mg, containing 8 mmol thiol groups) in PBS (pH 7.4) solution with existence of NaIO4 (15 µL, 0.046 wt%). Then the mixture was gently oscillated to induce gel formation. Hydrogels with different polymer concentrations were prepared following the same method. The gelation was confirmed by tube-inversion method.

2.5 Characterizations The P(AA-co-4-VPBA) NG were characterized by transmission electron microscope (TEM), dynamic light scattering and fourier transform infrared spectra (FT-IR). The chemical structures of DMA and poly(DMA-co-PEGMA) were confirmed by 1H NMR. The molecular weight of poly(DMA-co-PEGMA) was estimated by gel permeation chromatography (GPC). The inner microstructures of injectable hydrogels in the freeze-dried state were analyzed by scanning electron microscopy (SEM). The details of the characterization were provided in the Supporting information.

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2.6 Preparation of dual drug loaded P(AA-co-4-VPBA) NG-incorporated injectable hydrogel (NHG) The fabrication of P(AA-co-4-VPBA) NG-incorporated injectable hydrogel for dual drug delivery included two steps. Firstly, DOX-loaded P(AA-co-4-VPBA) nanohydrogels (DOX@NG) were prepared due to the electrostatic interaction between carboxyl in NG and amine groups in DOX. Then the DOX@NG were dispersed in the 4 arm-PEG-SH solution together with the water-soluble anticancer drug CA4P. As described in the previous step, the drug-loaded 4 arm-PEG-SH solution was mixed with the oxidized poly(DMA-co-PEGMA) solution. A series of the hydrogels with different drug concentrations were prepared accordingly.

2.7 In vitro CA4P and DOX release Typically, the injectable hydrogel (200 µL, containing 100 µg of CA4P) was placed in PBS (1 mL) within the Transwell insert chamber. At predetermined time intervals, the PBS solution was removed for determination, meanwhile, an equal amount of fresh PBS was added. The amount of released CA4P in the PBS solution was determined by HPLC (Agilent 1200, USA) at a flow rate of 1.0 mL/min. The mobile phase consisted of methanol/20 mM ammonium acetate (50:50, v/v) at 290 nm. Furthermore, the PBS with different pH (7.4 and 6.5) as well as glutathione (GSH, 10 mmol) were employed for the drug release assays. Similarly, the DOX-loaded injectable hydrogels (200 µL, containing 200 µg of DOX@NG) were prepared and placed a 24-well Transwell insert chamber (0.4 µm, Corning, polycarbonate film) for the drug release experiment.8 PBS (1 mL) was added to each well and the samples were incubated at 37 °C. At predetermined time intervals, the solutions with released drugs (DOX) were obtained from the wells of 24-well plate, and the amount of released DOX in this solutions was determined by UV-

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vis spectrometer at 480 nm. Meanwhile, the same amount of PBS solutions were added into the wells to maintain a constant volume for following release process.

2.8 In vitro assays The MCF-7 cells and normal 3T3-L1 cells were cultured for in vitro assays. The cellular uptake behaviors of the DOX@NG and DOX@NG encapsulated hydrogels (DOX@NHG) were investigated on the MCF-7 cells using confocal laser scanning microscope (CLSM, FV1000IX81, Olympus, Japan). The sustained long-term release abilities of DOX@NHG were explored by the flow cytometry (Amnis Flowsight, USA). The analysis of apoptosis/necrosis on MCF-7 was performed by Annexin V-FITC Apoptosis Detection Kit (Keygen Biotech, Nanjing). In vitro cell cytotoxicities towards the normal 3T3-L1 cells and MCF-7 cells were evaluated by MTT test. All the experimental details were described in the Supporting information.

2.9 In vivo assays The in vivo assays were performed using five-week-old male mice (obtained from Nanjin Mutu Animals Co. Ltd, China) inoculated with HepG2 cells subcutaneously in the right anterior limb of the mice. All animal experiments were carried out according to the guidelines of the institutional animal care and use committee (IACUC) of Jinling Hospital. All the experimental details were described in the Supporting information.

3. Results and discussion 3.1 Fabrication of injectable P(AA-co-4-VPBA) nanogel-incorporated hydrogel

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The fabrication procedure of the nanogel-incorporated injectable hydrogel was illustrated in Scheme 1. Briefly, the pH and redox stimuli-responsive P(AA-co-4-VPBA) nanogels were prepared via reflux-precipitation polymerization of AA, 4-VPBA and disulfide cross-linker BMOD. Meanwhile, the injectable hydrogel was prepared via thiol-catechol Michael addition reaction between 4-arm PEG-SH and poly(DMA-co-PEGMA). Then the P(AA-co-4-VPBA) NG were incorporated into the injectable hydrogel through reversible boronate ester bonds between phenylboronic acid of 4-VPBA in NG and catechol from hydrogel. The final nanogelincorporated injectable hydrogel was expected to be functionalized as a local dual drug delivery system with controlled sequential release profiles. Specifically speaking, the hydrogel matrix was employed for local delivery of hydrophilic antiangiogenic drug CA4P, and P(AA-co-4VPBA) NG were served as effective nano-carriers for DOX delivery. Due to the pH sensitive properties of boronate ester bonds between NG and hydrogel, the DOX-loaded NG would be dissociated from the hydrogel at the acidic tumor areas. After NG entered tumor cells, DOX was able to be released in acidic environment with elevated glutathione (GSH) level in cancer cells. Accordingly, once the dual drug loaded hydrogel injected into tumor areas, the antiangiogenic drug CA4P would be rapidly released from the hydrogel and destroy the tumor neovasculature, while DOX slowly released and trigger cell apoptosis. Thus, the resultant hybrid hydrogel DDS could achieve high treatment performance based on the antiangiogenesis and anticancer synergistic therapeutic effect.

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Scheme 1 The preparation process of injectable nanogel-incorporated hydrogel (NHG) for sequential local delivery of CA4P and DOX.

3.2 Preparation and characterization of P(AA-co-4-VPBA) NG The P(AA-co-4-VPBA) NG were synthesized via one-stage reflux-precipitation polymerization of AA, 4-VPBA and BMOD. The TEM images and DLS results were presented in Figure 1. Obviously, the P(AA-co-4-VPBA) NG exhibited good monodispersity with average diameter of around 247 nm (Figure 1a, b). For comparison purpose, pure PAA nanogels were also prepared by reflux-precipitation polymerization. As for the FT-IR spectra for PAA and P(AA-co-4VPBA) nanohydrogels (Figure 1c), the absorption band at wavenumber of 1715 cm−1, associated with the stretching vibration of carboxylic acid group in AA, was present in both PAA and

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P(AA-co-4-VPBA) NG.33,36 Compared with the spectra of PAA NG, the appearance of peaks at 1610 cm-1 and 1510 cm-1 in the P(AA-co-4-VPBA), assigned to the benzene ring, as well as the peak at 1348 cm-1 associated with C-B vibration, indicated the presence of 4-VPBA in P(AA-co4-VPBA) NG.42 The boronic acid in 4-VPBA can be coordinated to catechol in the injectable hydrogel to form boronate ester bond, resulting improved stability of P(AA-co-4-VPBA) NG in the hybrid hydrogel. Furthermore, the reversible boronate ester can be dissociated at acidic pH environment,43,44 providing an effective approach for the controlled release of P(AA-co-4VPBA) NG from hydrogel.

Figure 1 (a) Representative TEM images and (b) size distribution of P(AA-co-4-VPBA) nanogels. (c) FT-IR spectra of PAA and P(AA-co-4-VPBA) nanogels.

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3.3 Synthesis and characterization of poly(DMA-co-PEGMA) Poly(DMA-co-PEGMA) was synthesized by conventional free radical polymerization of DMA and PEGMA, containing catechol groups for subsequent reaction with 4-arm PEG-SH via thiolcatechol Michael addition.45,46 Firstly, the functional monomer DMA was obtained by the reaction between biomimetic adhesives dopamine and methacrylic anhydride. The 1H NMR spectrum of DMA was shown in Figure 2a. The vinyl proton signals of chemical shifts at 5.62 and 5.29 ppm (b), together with the proton signals of chemical shifts at 6.25-6.65 ppm from benzene ring (f), confirmed the successful synthesis of DMA.40,41 Secondly, the polymer poly(DMA-co-PEGMA) was prepared and characterized by FT-IR spectroscopy, 1H NMR spectroscopy and gel permeation chromatography (GPC) (Figure 2b-d). Two strong characteristic absorption bands of PEGMA (1110 cm-1 for ether stretching and 1730 cm-1 for ester stretching) appeared in FT-IR spectrum.47 The absorption peaks at 1651 and 1516 cm-1, corresponding to amide I and II absorption bands, indicated incorporation of DMA in the polymer. In addition, all the relevant peaks were labeled in 1H NMR spectrum (Figure 2b), confirming the chemical structure of poly(DMA-co-PEGMA). By comparing the proton signals from benzene ring at 6.25-6.65 ppm (h) and methoxy groups at 3.45 ppm (e), the composition ratio m:n was calculated to be about 1:3. The number-average molecular weight (Mn) of poly(DMA-co-PEGMA) was 62335 with a polydispersity index (PDI) of 1.77 (Figure 2d). All the characterization results confirmed the successful synthesis of poly(DMA-co-PEGMA), which contains catechol functional groups for subsequent preparation of injectable hydrogel.

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Figure 2 (a) 1H NMR spectrum of DMA. (b) 1H NMR spectrum, (c) FT-IR spectrum and (d) GPC curve of poly(DMA-co-PEGMA)

3.4 Fabrication and characterization of the injectable hydrogel The injectable hydrogel system was prepared by the previously synthesized poly(DMA-coPEGMA) polymer and water-soluble macromolecular crosslinker 4-arm PEG-SH via catecholthiol Michael addition reaction. As is known to all, the catechol moiety provides a versatile platform for coupling to a large amount of reagents and material surfaces.45 Especially, the catechol can be oxidized into quinone that exhibited high reactivity with a variety of chemical groups.48,49 The oxidation process of poly(DMA-co-PEGMA), together with the cross-linking to 4-arm PEG-SH, was schematically illustrated (Supporting information, Figure S1) and investigated by UV-vis spectroscopy (Supporting information, Figure S2). The absorption band

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located at 280 nm, corresponding to catechol,49 decreased obviously after oxidation. Meanwhile, a new absorption band at 400 nm associated with quinones appeared,49 confirming the occurrence of oxidation reaction. The following Michael addition reaction of oxidized poly(DMA-co-PEGMA) with 4-arm PEG-SH resulted in reduction of absorption band for quinones in the UV-vis spectrum of hydrogel.

A series of hydrogels with different polymer concentrations as well as ratios of catechol to thiol were synthesized. As shown in Table S1 (Supporting information), the formed hydrogel exhibited suitable injectability with the molar ratio of catechol and thiol groups of 1:0.8 and polymer concentrations of 50 mg/mL for poly(DMA-co-PEGMA) and 40 mg/mL for 4-arm PEG-SH, respectively. This formulation was employed for all the assays unless otherwise noted. Once the molar ratio of catechol and thiol functional groups was proper, the hydrogel could be readily obtained in 13-20 seconds. Therefore, it is more feasible to control the hydrogel injectabilities through adjusting the molar ratio of polymers than tuning the gelation time. The prepared hydrogel was observed by the tube-tilting method (Figure 3a, for visualization purpose, the pre-solution and hydrogel were stained with rhodamine B). The hydrogel could pass through a 1 mL-gauge needle without clogging, indicating its good injectability. The inner microstructures of the hydrogel were observed by scanning electron microscopy (SEM) (Figure 3a). The hydrogel was highly porous with interconnected polymer network, enabling it to be a good depot for hydrophilic drugs. Furthermore, the P(AA-co-4-VPBA) NG with diameter of about 250 nm were uniformly distributed in the hydrogel matrix, which was utilized for following DOX delivery. The FT-IR spectrum of hydrogel was provided (Figure 3b). As mentioned above, the typical ester and ether absorption bands at 1730 cm-1 and 1110 cm-1 of

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PEGMA from poly(DMA-co-PEGMA) were presented. The peak at 1348 cm-1 was associated with stretching vibration of C-B in P(AA-co-4-VPBA) NG. These results confirmed that the NGincorporated injectable hydrogel was prepared successfully.

Figure 3 (a) Photographs and SEM images of the injectable hydrogel. (b) FT-IR spectrum of the injectable hydrogel. (c) Degradation behaviors of injectable hydrogel at different pH environment at 37 ºC.

It is known that the microenvironment in tumor areas is slightly acidic.50 Accordingly, the degradation behaviors of the hydrogel were examined both at pH 6.5 and pH 7.4 (Figure 3c). The hydrogel had yet to completely decompose after 21 days, with the remaining mass of 9.4% at pH 6.5 and 20.5% at pH 7.4. The increased degradation rate from pH 7.4 to 6.5 indicated pHdependent degradation property of the hydrogel. This phenomenon was probably caused by the faster hydrolytically degradation behavior of the ester linkages in poly(DMA-co-PEGMA) and

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4-arm PEG-SH in acidic environment.51 The relatively long degradation time is quite preferable for local drug delivery applications with minimal injection times.

3.5 Encapsulating and releasing behaviors of CA4P in hydrogel As a hydrophilic drug, CA4P could be readily incorporated into the injectable hydrogel and released in physiological environment. The CA4P release behaviors at different pH were explored (Figure 4a). The cumulative releases were about 57.2% at 9 h and almost 90.7% after 48 h at pH 6.5. In contrast, the CA4P release behaviors at pH 7.4 and 6.5 were similar, indicating the releasing was a swelling-diffusion controlled process. Therefore, the injectable hydrogel could be employed as an effective depot for short-term local delivery of antiangiogenic CA4P.

3.6 Loading and releasing behaviors of DOX in P(AA-co-4-VPBA) NG The P(AA-co-4-VPBA) NG possess pH and redox stimuli-responsive properties, enabling them to be competitive alternatives as intelligent DDS. The large amount of carboxyl functional groups in PAA are capable of loading drugs containing amino groups, such as DOX, through the strong electrostatic interactions.33,36 Due to the reversibly ionization of carboxylic and amino groups, DOX could be released from P(AA-co-4-VPBA) NG in the acidic tumor cells and tissues. Moreover, the disulfide linkages in the P(AA-co-4-VPBA) NG exhibit good response in the reducing environment, such as the high glutathione (GSH) concentration in the tumor cells, leading to drug release as well as degradation of NG.33,36 The potential application of P(AA-co4-VPBA) NG as effective drug nanocarriers was evaluated by releasing the anticancer drug DOX under both pH and redox external stimuli. First of all, a series of experiments with different mass ratios of NG to DOX were carried out. The drug loading capacity (DLC) and drug loading efficiency (DLE) were determined by UV-vis (Supporting information, Table S2). With the

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feeding ratios from 0.5 to 5.0, the DLC of DOX in NG ranged from 14.3 wt% to 96.3 wt%, and DLE from 48.4% to 92.5%. The high values for DLC and DLE indicated good loading ability of P(AA-co-4-VPBA) NG for DOX, indicating their great potential as drug nano-carriers.

Figure 4 (a) CA4P release profiles from injectable hydrogel in PBS at pH 7.4 and 6.5. (b) DOX release profiles from DOX@NG pH 7.4 and 5.0 with/without glutathione (GSH, 10 mM). (c) DOX release profiles from DOX@NHG at pH 7.4 and 6.5 with/without glutathione (GSH, 10 mM).

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Secondly, DOX release profiles from P(AA-co-4-VPBA) NG were explored (Figure 4b). The DOX-loaded NG (DOX@NG) displayed quick release of 24.7% without GSH and 64.1% with GSH presence at pH 7.4 in 84 h. This result indicated the redox stimuli-responsive properties of P(AA-co-4-VPBA) NG, which was attributed to the presence of disulfide linkages from BMOD. Moreover, the DOX cumulative releases at pH 5.0 (simulated cancer cytoplasm acidic environment) were also explored to investigate the pH stimuli-responsive properties in drug release process. About 83% of DOX released after only 80 h with the existence of reducing agent GSH at pH 5.0. It was much larger than the value of 64.1% at pH 7.4. Therefore, the P(AA-co-4VPBA) NG exhibited controlled drug release abilities in the acidic and reducing cellular environment.

After DOX@NG were incorporated into the injectable hydrogel via boronate ester linkages by boric acid in NG and catechol groups in hydrogel, the release behavior of DOX could be divided into two steps. Firstly, DOX@NG were released from hydrogel at the acidic tumor areas because of boronate ester dissociation at low pH.43,44 Then, DOX was expected to be released from NG due to low pH and GSH existence once entered the tumor cells. Due to this two-step release process, a sustained release behaviors of DOX should be observed. The delayed drug release behaviors of DOX-loaded P(AA-co-4-VPBA) NG-incorporated hydrogel (DOX@NHG) were explored at different pH (6.5 and 7.4) and reducing environment (10 mM GSH) (Figure 4c). At pH 7.4, a small amount of DOX (15.8%) was released over a long time (14 days) in the absence of GSH, indicating the good stability of the NHG in a physiological pH environment. At the same pH, the cumulative release increased to about 22.9% with the presence of GSH (10 mM), indicating the redox-responsive properties of the NG in the reducing environment. Notably,

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relatively fast DOX release profiles were observed at weak acidic environment. At pH 6.5, more than 66.3% of DOX was released from the NHG in 14 days without GSH, while it increased to 83.9% with GSH existence eventually. The accelerated releases in acidic environment (pH 6.5) indicated the pH stimuli-responsive properties of the NHG delivery systems. It could be attributed to the detachment of NG from hydrogel caused by the dissociation of boronate ester linkages at low pH and the decrease of electrostatic interactions between DOX and NG. It also confirmed the redox stimuli-responsive properties of the NG. Therefore, the DOX@NHG delivery system, with pH and redox dual stimuli-responsive properties, exhibited excellent sustained slow release behaviors, offering an effective means in the long-term therapeutic applications. In contrast, the DOX was released much slower from DOX@NHG than CA4P from hydrogel. The different release profiles of two drugs confirmed the controlled sequential release capability, indicating that NG-incorporated hydrogel exhibited great potential as dual drug local delivery system.

3.7 Cellular uptake studies The cellular uptake behaviors of DOX@NG were investigated by CLSM analysis (Figure 5). The cells treated by DOX@NG showed weak red fluorescence after 1 h. After 5 h of incubation with the DOX@NG, a much stronger red fluorescence was observed, indicating the progressive internalization of DOX@NG and subsequent rapid release of DOX from the nanogels. As time went on, the red fluorescence in the cells gradually weakened after incubation of 8 h. It was because that the cancer cells became crimpy and shrunk due to toxicity of DOX. This result indicated that the P(AA-co-4-VPBA) NG could be considered as effective nano-carriers for DOX delivery.

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Figure 5 CLSM images of MCF-7 cells incubated with DOX-loaded P(AA-co-4-VPBA) NG after 2, 5 and 8 h.

Furthermore, the different uptake rates of DOX@NG and DOX@NHG were explored. The flow cytometric histograms of MCF-7 cells incubated with DOX@NG and DOX@NHG for 5 h were shown in Figure 6. The MCF-7 cells without any drug incubation were defined as the control group. In comparison to control group, MCF-7 cells treated by DOX@NG and DOX@NHG exhibited higher DOX fluorescence intensities, indicating the efficient cellular uptake of both delivery approaches. Due to the delayed release effect of the DOX@NHG system, a lower fluorescence was detected after treatment by DOX@NHG than that with DOX@NG for the same time. Thus, the NG-embedded injectable hydrogel showed excellent performances in longterm sustained drug release.

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Figure 6 Flow cytometric histograms of MCF-7 cells treated by DOX@NG and DOX@NHG.

3.8 In vitro cell apoptosis and cytotoxicities The apoptosis of MCF-7 cells incubated with hydrogel, DOX@NG, CA4P@NHG, free DOXCA4P, DOX-CA4P@NHG were further investigated by flow cytometry (Figure 7). The cells were double stained by PI and Annexin V-FITC for viability and apoptosis analysis. The PIAnnexin V-FITC+ cells represented the apoptotic cells, while PI+ annexin V-FITC+ cells were necrotic cells.8 The apoptosis and necrosis of cells incubated with blank hydrogel were very low and comparable to the cells incubated with PBS, suggesting non-cytotoxicity of the hydrogel. As for the DOX@NG and CA4P@NHG, much higher apoptosis and necrosis of the cells were observed. Moreover, the DOX-CA4P@NHG induced the highest apoptosis/necrosis of the cells. These results confirmed that the nanogels-embedded hydrogel provided a potential approach for co-delivery of dual drugs for improved therapy efficiency.

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Figure 7 Flow cytometry analysis of MCF-7 cell apoptosis induced by PBS, injectable hydrogel (HG), free drugs (DOX, CA4P), DOX@NG, CA4P@HG and DOX-CA4P@NHG using Annexin V-FITC/PI staining.

Moreover, the cellular cytotoxicites of P(AA-co-4-VPBA) NG and hydrogel for 3T3-L1 normal cells were evaluated by MTT assays for a preliminary assessment purpose (Figure 8a, b). The blank P(AA-co-4-VPBA) NG showed good cytocompatibility to 3T3-L1 normal cells over a wide range of concentrations, particularly in 10-200 µg/mL, for both 24 and 48 h. As for the hydrogel, they were also non-toxic to the 3T3-L1 normal cells with the polymer concentrations ranged from 0.33 wt % to 1.3 wt%. More importantly, the blank NHG system for the dual drug delivery in all the evaluation assays, with NG concentration of 100 µg/mL and polymer concentration of 0.65 wt%, did not have significant inhibitory effect on the 3T3-L1 normal cells. These results indicated that both NG and hydrogel exhibited no obvious cytotoxicity over a large range of concentrations and were suitable as DDS.

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Figure 8 (a) Cell viabilities of 3T3-L1 cells incubated with P(AA-co-4-VPBA) NG for 24 and 48 h. (b) Cell viabilities of 3T3-L1 cells incubated with hydrogel and P(AA-co-4-VPBA) NG@hydrogel for 24 and 48 h. (c) Cell viabilities of MCF-7 cells incubated NHG and DOX-CA4P@NHG with different drug concentrations for 24 and 48 h. (d) Cell viabilities of MCF-7 cells incubated with NHG, free DOX, free CA4P, DOX@NHG, CA4P@NHG and DOX-CA4P@NHG for 48 h and 72 h.

To further evaluate whether NHG can be employed as potential carriers for cancer therapy, the anti-cancer cell performances of the dual drug loaded NHG system were investigated. Initially, a series of NHG loaded with different drug concentrations were used for concentration screening (Figure 8c). The cell viabilities decreased as drug concentrations increased after incubation with DOX and CA4P loaded NHG system (DOX-CA4P@NHG). For the cells treated by DOX-

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CA4P@NHG (DOX: 100 µg/mL, CA4P: 70 µg/mL), the cell viabilities were 29.2% and 19.9% after 24 and 48 h. Subsequently, in vitro cytotoxicities of NHG, free drugs, single drug-loaded NHG (DOX@NHG and CA4P@NHG) and DOX-CA4P@NHG against MCF-7 cancer cells were examined (Figure 8d). The NHG were also non-cytotoxicity to MCF-7 cells for 48 h and 72 h, suggesting its good cytocompatibility. After treatment by the free drugs and drug-loaded hydrogels, the growth and proliferation of MCF-7 cells were greatly inhibited. The cell viabilities for the DOX@NHG were similar to that of free DOX, around 46.2% and 22.8% after 48 and 72 h of incubation, respectively. Similarly, the CA4P@NHG displayed comparable cell proliferation inhibition to the free CA4P drug, around 70.1% for 48 h and 37.7% for 72 h. As expected, the DOX-CA4P@NHG significantly reduced the cell viability in comparison with the single drug loaded hydrogels, around 23% and 14% after 48 and 72 h of incubation, respectively. The results confirmed that the therapeutic efficiency could be greatly enhanced by co-delivery of the drugs using NHG system. Moreover, the combination index (CI) plots were determined to confirm the synergistic therapeutic effect. The combination index that is smaller or larger than 1.0 denotes synergism or antagonism, respectively, and it is equal to 1.0 for additivity.52 As show in Figure 9, the CI values were determined to be less than 1.0 at any drug effect level for 48 h of incubation. The half-maximal inhibitory concentration (IC50) of DOX@NHG and CA4P@NHG were 105.0 µg and 177.5 µg, respectively. In comparison, the IC50 values for DOX and CA4P in DOX-CA4P@NHG system were much lower, with the value 44.4 µg and 34.2 µg, respectively. Accordingly, the value of CI50 was calculated as 0.615, indicating an obvious synergistic effect of DOX and CA4P. Additionally, the values of combination index for free drugs (DOX and CA4P) were 0.709-0.804 in the drug effect level range of 30%-70% (Supporting information, Figure S3). In contrast, the lower values of combination index (0.417-0.742) for DOX-

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CA4P@NHG system, at the drug effect level of 40-60%, suggested better synergistic performance that was achieved by NHG delivery system. Hence, the nanogels-incorporated hybrid hydrogel provides potentially ideal candidate for dual-drug delivery, equipped with good biocompatibility, biodegradability and synergistic therapeutic efficacy.

Figure 9 CI plot for DOX-CA4P@NHG against MCF-7 cells.

3.9 In vivo antitumor efficacy The in vivo antitumor efficacies of the dual drug-incorporated hydrogel were carried out by single peritumoral injection of hydrogel, DOX@NG, CA4P@NHG, free DOX and CA4P, and DOX-CA4P@NHG, respectively, on the tumor-bearing mice. Then the tumor volume and body weight were measured every other day (Figure 10a). The tumors increased quickly for the PBStreated group (defined as control group) and hydrogel-treated group in 21 days, while the growth of tumor was suppressed for all the chemotherapy groups at different levels. Either DOX@NG or CA4P@NHG showed effective inhibition for tumor growth. In contrast, the dual drug-containing hydrogel system (DOX-CA4P@NHG) showed the highest therapeutic efficacy, as evidenced by

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the much lower average tumor sizes compared with the single drug delivery systems-treated groups after 21 days. These results confirmed the synergistic anticancer effect of DOX and CA4P combination therapy. In addition, the body weight of mice is a direct reflection of systemic toxicities (Figure 10b). The mice treated with single and dual drugs exhibited almost no weight loss, while the mice treated with PBS and blank hydrogel lost their body weight slightly, implying the effectiveness of chemotherapy. Particularly, the mice injected by DOXCA4P@NHG were alive with increased body weights, indicating their non-toxicities.

Figure 10 Tumor regression study of tumor-bearing mice treated with different materials. (a) Tumor volumes and (b) body weight changes of the mice as a function of time. The data were shown as mean±SD (n=5). ***P