pH-Responsive Nanocomposite Hydrogel as Long

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Injectable, NIR/pH-Responsive Nanocomposite Hydrogel as LongActing Implant for Chemo-Photothermal Synergistic Cancer Therapy Xiaoyu Xu, Ziyuan Huang, Zeqian Huang, Xuefei Zhang, Siyu He, Xiaoqi Sun, Yifeng Shen, Mina Yan, and Chunshun Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 24, 2017

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ACS Applied Materials & Interfaces

Injectable, NIR/pH-Responsive Nanocomposite Hydrogel as Long-Acting Implant for Chemo-Photothermal Synergistic Cancer Therapy

Xiaoyu Xu, Ziyuan Huang, Zeqian Huang, Xuefei Zhang, Siyu He, Xiaoqi Sun, Yifeng Shen, Mina Yan, Chunshun Zhao*

School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road, Guangzhou Higher Education Mega Center, GuangZhou 510006, PR China

*Corresponding author: Chunshun Zhao E-mail address: [email protected] Tel: +86 020-39943118

ACS Paragon Plus Environment


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Abstract In this study, Gold nanorods (GNRs) were incorporated into the hydrogel networks formed by the copolymerization of N-isopropylacrylamide (NIPAm) and methacrylated β-cyclodextrin-based

macromer

(MPCD)

to

fabricate

an

injectable

and

NIR/pH-responsive poly(NIPAm-co-MPCD)/GNRs nanocomposite hydrogel, which could serve as a long-acting implant for chemo-photothermal synergistic cancer therapy. The nanocomposite hydrogel showed superior mechanical and swelling properties, gelation characteristic and excellent NIR-responsive property. A hydrophobic acid-labile adamantane-modified doxorubicin prodrug (AD-DOX) was loaded into hydrogel efficiently by host-guest interaction. The nanocomposite hydrogel exhibited a sustained drug release manner and could sustain the slow and steady release of DOX for more than one month. The pH-responsive release of DOX from the nanocomposite hydrogel was observed owing to the cleavage of acid-labile hydrazone bond between DOX and adamantyl group in acidic environment. NIR irradiation could accelerate the release of DOX from the networks, which was controlled by the collapse of the hydrogel networks induced by photothermal effect of GNRs. The in vitro cytotoxicity test demonstrated the excellent biocompatibility and photothermal effect of nanocomposite hydrogel. Moreover, the in-situ forming hydrogel showed promising tissue biocompatibility in mouse model study. The in vivo antitumor test demonstrated the capacity of nanocomposite hydrogel for chemo-photothermal synergistic therapy with reduced adverse effects owing to the prolonged drug retention in the tumor region and efficient photothermal effect. Therefore, this injectable and NIR/pH-responsive nanocomposite hydrogel exhibited great potential as a long term drug delivery platform for chemo-photothermal synergistic cancer therapy.

Keywords: Gold nanorods, NIR/pH-responsive, long-acting implant, nanocomposite hydrogel, host-guest interaction, chemo-photothermal


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1. Introduction Recently, nanocomposite hydrogels have attracted great attention in the biomedical and biotechnological fields.1-5 Incorporating nanosized materials, such as polymeric, metallic, and carbon-based nanomaterials, into the hydrogel networks can acquire nanocomposite hydrogels with additional function and/or reinforced properties.6-7 Nanoparticles can promote the response of hydrogels to a desired stimulus, while the hybrid systems can release drug constantly and sustain high drug concentration at the injection site over a long period of time and avoids undesirable drug accumulation in normal tissues to diminish side effects. Among these nanocomposite hydrogels, near-infrared (NIR) light-responsive hydrogels are of particular interest, because NIR light is able to penetrate human tissues with sufficient intensity and minimal damage as well as used as a precise “on/off” trigger.8-12 Typically, NIR light-responsive nanocomposite hydrogels are fabricated by introducing NIR-absorbing nanomaterials such as gold nanoparticles,7, 13 carbon nanotubes,14-15 graphene oxide nanosheets8, 12 and upconversion nanoparticles11, 16-17

into thermo-responsive polymers such as poly(N-isopropylacrylamide) (PNIPAm)

that can respond to an external temperature, with the aim of inducing changes in the structure of hydrogel by photothermal effect and achieving remotely controlled pulsatile release of drugs. Among them, gold nanorods are attractive photothermal nanomaterial for localized cancer hyperthermia therapy.18-21 Once the gold nanorods incorporated into hydrogel absorb NIR light, it can efficiently convert NIR light into heat. The local photothermal effect can not only achieve cancer hyperthermia therapy but also trigger a destruction of the hydrogel networks resulting in the accelerated release of embedded drugs.13, 22-26

However, the way of drug loaded into the hydrogel matrix has a great influence on controlling drug release. The conventional hydrogels are 3D networks composed of cross-linked hydrophilic polymers, which typically tend to load hydrophilic drugs.



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Classically, drugs are conveniently mixed with the precursor solution followed by gelation. Drug molecules encapsulated physically are released from the hydrogel networks through passive diffusion or hydrogel degradation, which cannot meet the demands for on demand drug delivery with tunable release kinetics.2 The release of the loaded drugs shows a burst release and can’t be well controlled that result in a relatively rapid and irregular release within a short time.27 Moreover, these hydrogels are usually innately restricted because of quick degradation after injection.28 Therefore, the efficacies of these drug delivery systems are reduced and often cause multiple side effects.29-30 On the other hand, due to the innate incompatibility of hydrophilic hydrogel networks and hydrophobic drugs, it is still a great challenge to deliver hydrophobic drugs effectively and persistently using hydrogels as carriers.31-34 While most of the anticancer drugs used in clinical trials are hydrophobic drugs, the applications of conventional hydrogels for cancer therapy are inefficient due to deficient drug loading, drug aggregation or rapid release behavior which results in adverse effects.32, 35-36 Consequently, development of strategies aimed at localized and sustained delivery of anticancer drugs over a prolonged period with minimal side effects using hydrogels is imperative for cancer therapy.

In this study, we have prepared a “smart” nanocomposite hydrogel as a long-acting implant for hydrophobic anticancer prodrug delivery and chemo-photothermal synergistic cancer therapy. Firstly, we prepared a linear water-soluble β-CD-based polymer (PCD) possessing unique and excellent capability of forming inclusion complexes with various guest molecules by the hydrophobic cavity structures of β-CD, which was proved to be more effective compared to pristine β-CD.37 Then, methacrylated β-CD-based macromer (MPCD) was synthesized by the reaction of PCD with glycidyl methacrylate (GMA). Afterward, an acid-labile doxorubicin prodrug AD-DOX was synthesized. Adamantane was conjugated with anticancer drug doxorubicin through a hydrazone bond which was stable at physiological pH while labile at acidic pH. With the strong interaction between


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β-CD and the adamantyl group, the prodrug can be efficiently encapsulated into MPCD to form inclusion complex by both hydrophobic and host-guest interactions. Finally, using MPCD itself as a cross-linker and ammonium persulfate (APS) and ascorbic acid (AA) redox system as the initiator, nanocomposite hydrogels were prepared by copolymerizing drug-loaded MPCD and NIPAm in the presence of GNRs as shown in Scheme 1.

In this nanocomposite hydrogel, the hydrophilic networks containing β-CD finely offer specific affinity-based mechanism of drug loading and sustained/controlled release by conveying the ability of forming inclusion complexes.38 The nanocomposite hydrogel exhibited a sustained drug release manner and could sustain the slow and steady release of DOX for more than one month. The pH-responsive release of DOX from the nanocomposite hydrogel was observed owing to the cleavage of acid-labile hydrazone bond between DOX and adamantyl group in acidic tumor tissue environment. Moreover, the nanocomposite hydrogel combining GNRs and thermo-responsive PNIPAm networks exhibited excellent NIR light-responsive property. The NIR irradiation could not only achieve cancer hyperthermia therapy but also accelerate the release of DOX from the networks in an on demand way. In vitro cytotoxicity test demonstrated the favorable biocompatibility and excellent photothermal effect of nanocomposite hydrogel. Furthermore, the in-situ forming hydrogel showed promising tissue biocompatibility in mouse model study. In vivo antitumor test demonstrated the capacity of nanocomposite hydrogel for chemo-photothermal synergistic therapy with reduced adverse effects owing to the prolonged drug retention in the tumor region and efficient photothermal effect. The results demonstrated that this NIR/pH-responsive nanocomposite hydrogel might be a promising candidate as a long-acting implant for chemo-photothermal synergistic cancer therapy.



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Scheme 1. Schematic Illustration of the Formation of Injectable, NIR/pH-Responsive Nanocomposite Hydrogel and Chemo-Photothermal Synergistic Cancer Therapy as Long-Acting Implant.

2. Experimental Section 2.1. Materials Tetrachloroauric acid (HAuCl4⋅3H2O), cetyltrimethylammonium bromide (CTAB), AA, silver nitrate (AgNO3) and hydrochloric acid (HCl, 36.0-38.0 wt%) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Methoxypoly(ethylene glycol) thiol (mPEG-SH) with a molecular weight of 2000 was purchased from Ponsure Biotechnology

(Shanghai,

China).

Sodium


borohydride

(NaBH4),

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3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide (PI) were obtained from Sigma-Aldrich (Shanghai, China). β-CD, NIPAm, epichlorohydrin (EPI), toluene, hydrazine monohydrate, 1-Adamantanecarbonyl chloride, 4-(dimethylamino)pyridine (DMAP), trifluoroacetic acid, APS and fluorescein diacetate (FDA) were purchased from Aladdin reagent Co. Ltd. (shanghai, China). GMA was obtained from J&K Chemical Ltd. (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl, 99.0%) was purchased from Beijing ZhongShuo Pharmaceutical Technology Development Co. Ltd. (Beijing, China). Tetracarboxyl phenoxy Zn-phthalocyanine (TPZnPc) was prepared previously by our laboratory. All reagents for the cell culture were purchased from Gibco. Deionized water was used to prepare all of the aqueous solutions. All the chemicals were analytical pure and used directly without further purified.

2.2. Preparation of PEG-modified Gold Nanorods (GNR-PEG) CTAB-coated gold nanorods (GNR-CTAB) were synthesized by a seed-mediated growth method with minor revisions.39-40 Briefly, 5 mL of 0.5 mM HAuCl4 was mixed with 5 mL of 0.2 M CTAB solution. Then, 0.6 mL of fresh ice-cold 0.01 M NaBH4 was added under vigorous oscillation. The oscillation was stopped after 2 min. After 2 h standing in 25 °C, the brown-yellow solution was further used as seed solution. The growth solution was prepared by mixing 5 mL of 1 mM HAuCl4, 5 mL of 0.2 M CTAB, 100 µL of 10 mM AgNO3, 55 µL of 0.1 M ascorbic acid and 40 µL of 1 M HCl solution together. Then, 12 µL of seed solution was added to the growth solution at 30 °C. The mixture was homogenized by shaking gently for 30 s and left undisturbed at 30 °C overnight to obtain GNRs.

For further surface modification, the as-prepared solution of GNRs (10 mL) was centrifuged twice at 12000 rpm for 15 min to remove the excess CTAB. The purified



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GNRs were redispersed in 10 mL of deionized water. Then, 2 mL of mPEG-SH (5 mg/mL) water solution was added slowly into 10 mL of the purified GNRs in deionized water and stirred for 24 h at room temperature. Finally, GNR-PEG were purified by repeated centrifugation and redispersed in deionized water for further use. For photothermal conversion study, 1 mL of GNR-PEG aqueous solution at various concentrations in a quartz cell was irradiated with a NIR laser (785 nm, 1 W/cm2) (Changchun New Industries Optoelectronics Technology, Changchun, China) for 10 min. The temperature of the solution was monitored using a submerged digital thermometer. The zeta potentials of the GNRs were determined using dynamic light scattering (Nano ZS90, Malvern Zetasizer, UK) and the morphology was studied using transmission electron microscopy (TEM, JEM-1400, JEOL, Japan).

2.3. Synthesis of Methacrylated Poly-β-cyclodextrin Macromer (MPCD) PCD was synthesized by cross-linking β-CD with EPI under strong alkaline condition, according to the method described previously.41-42 Briefly, 5 g of β-CD was dissolved in 25 mL of a 20 wt% aqueous sodium hydroxide solution and stirred vigorously for 2 h at 35 °C. Then 1 mL of toluene was added and continued stirring for 2 h at 35 °C. Subsequently, 5 mL of epichlorhydrin was added to the mixture dropwise. After stirring for 3 h, the pH value of the solution was adjusted to 7.0 with diluted hydrochloric acid. The mixture was concentrated to about 20 mL and dialyzed (MWCO=10000) against deionized water for 7 days. Finally, a white powder was obtained via lyophilization. Methacrylation of PCD was performed via the ring-opening reaction of the epoxy group of GMA with hydroxyl groups of PCD using DMAP as a catalyst, based on protocols previously described with some minor modifications.43 Briefly, PCD (3.5 g) and DMAP (0.1 g) was dissolved in 20 mL of dried DMF. Afterwards, excess GMA (0.5 mL) was added to the mixture. After degassing with N2 for 30 min, the mixture was stirred at 45 °C for 24 h. Finally, the product was precipitated in excess acetone, filtrated and


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washed three times using a large amount of acetone and dried under vacuum. The chemical structures of PCD and MPCD were investigated with a 400 MHz NMR (AvanceIII, Bruker, Switzerland) by using TMS as an internal standard. Fourier transform infrared spectroscopy (FT-IR) measurements were recorded using a PerkinElmer Spectrum Two instrument.

2.4. Synthesis of Acid-labile Adamantane-modified Doxorubicin Prodrug AD-DOX AD-DOX was synthesized using the established procedure described elsewhere.44 Briefly, hydrazine monohydrate (10 mL, 98%) and 10 mL of anhydrous dichloromethane containing 0.5 g of 1-Adamantanecarbonyl chloride were introduced into a 50 mL round-bottom flask. The mixture was stirred for 12 hours under N2 at room temperature. After the reaction was completed, the mixture was dried by rotary evaporation. The residue was dissolved in dichloromethane (5 mL) and washed with deionized water (3 × 5 mL), and then the organic phase was dried overnight by MgSO4 followed by filtration. Finally, the filtrate was dried through rotary evaporation and the product was dried under vacuum for 24 h to gain 0.3 g of adamantine-1-carboxylic acid hydrazide. Afterwards, adamantine-1-carboxylic acid hydrazide (60 mg) was dissolved in 50 mL of anhydrous methanol, and then doxorubicin hydrochloride (100 mg) and trifluoroacetic acid (50 µL) were added. The reaction was allowed to proceed for 48 h at 50 °C in the dark. The resulting mixture was concentrated under reduced pressure followed by precipitated in excess ethyl acetate. The pure product was obtained after centrifugation at 4000 rpm for 15 min and dried under vacuum. 1H NMR spectra were recorded with a 400 MHz NMR (AvanceIII, Bruker, Switzerland). The ESI-MS spectrum of AD-DOX was recorded by electrospray ionization mass spectrometry (ESI-MS, LCQ DECA XP, Thremo, USA).

2.5. In Vitro Hydrogel Formation and Drug Loading Nanocomposite hydrogels were synthesized by copolymerizing MPCD and NIPAm with



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different feed compositions with GNRs incorporated using MPCD as a cross-linker and APS and AA redox system as the initiator. In brief, 150 mg total mass of MPCD and NIPAm were dissolved in 0.5 mL of GNR-PEG aqueous solution (200 µg/mL) to obtain a homogeneous solution. After the monomer dissolved in the solution, 50 µL of APS (1%, 2%, 3%, 5% w/w), and 50 µL of AA (1%, 2%, 3%, 5% w/w) were added to initiate polymerization. The feed compositions of the composite hydrogels in this study were shown in Table 1. The precursor mixture solutions were transferred to a 10 mm diameter plastic syringe and the polymerization were conducted at room temperature (25 °C) and physiological temperature (37 °C), respectively. The gelation time was monitored by the vial inverting method described elsewhere.45 The resulting cylindrical hydrogels with diameter of 10 mm were gently pushed out of the syringe and cut into discs with thickness of 3 mm.

To prepare DOX-loaded nanocomposite hydrogels, 10 mg AD-DOX was added to 5 mL of MPCD aqueous solution (30 mg/mL) under ultrasonic treatment. The mixture was stirred for 2 days at 37 °C, then transferred into a dialysis bag (MWCO = 3000) and dialyzed for 24 h against deionized water. Finally, red MPCD/AD-DOX inclusion complex powder was obtained via lyophilization. Then, the DOX-loaded hydrogels were fabricated following the same procedure as described in the preparation of blank nanocomposite hydrogels.


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Table 1. The feeding compositions of poly(NIPAm-co-MPCD)/GNRs nanocomposite hydrogels. Sample NCD-1 NCD-2 NCD-3 NCD-4 NCD-5 NCD-6 NCD-7 NCD-8 NCD-9

NIPAm:MPCD (mass ratio, mg/mg) 1:3 1:2 1:1 2:1 3:1 1:1 1:1 1:1 1:1

GNR-PEG (µg) 100 100 100 100 100 100 100 100 0

APS (w/w) 5% 5% 5% 5% 5% 3% 2% 1% 2%

AA (w/w) 5% 5% 5% 5% 5% 3% 2% 1% 2%

H2O (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

2.6. Characterization of Nanocomposite Hydrogels 2.6.1. Thermo-responsive Swelling Behavior of the Nanocomposite Hydrogels The research of thermo-responsive swelling behavior of nanocomposite hydrogel was carried out in buffer solutions of different temperatures from 25 to 55 °C, which included the prospective extent of the lower critical solution temperature (LCST) of the hydrogel samples. The lyophilized hydrogels were immersed and reached swollen state in phosphate buffer saline (PBS) at pH 7.4 for 48 h. Thereafter, the swollen samples were taken out and wiped using humid filter paper to remove excess water on the hydrogel surface followed by weighed. All tests were conducted in triplicate. The equilibrium swelling ratio (ESR) was determined according to the following equation: ESR (%) = (Ws-Wd)/Wd × 100% where Ws and Wd are the weight of equilibrium swollen and dried hydrogels, respectively.

2.6.2. Rheological Experiments The rheological analysis of hydrogels was conducted with Kinexus Pro rheometer



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(Malvern Instruments Ltd., Worcestershire, UK). The measurement was performed by using a parallel plate geometry (PU25 SR 2426 SS, 25 mm diameter). The gap between the plates was 1 mm. Cylindrical hydrogel (0.5 mL) was put on the bottom plate, and the measurement was carried out at 37 ± 0.1 °C. Storage modulus (G’) and loss modulus (G’’) of hydrogels were monitored over frequency ranging from 0.1 to 10 Hz at a constant strain of 0.5% by oscillatory frequency sweep tests.

2.6.3. Morphology of the Hydrogels The morphologies of hydrogels were examined via field emission scanning electron microscopy (SEM, JSM-6330F, JEOL, Japan). The hydrogels incorporated with or without GNR-PEG, NCD-7 and NCD-9, after reaching swelling equilibrium in PBS at pH 7.4, were rapidly frozen by liquid nitrogen followed by lyophilized for 2 days. The lyophilized samples were fractured carefully and coated with gold before observation under SEM.

2.7. NIR Light-Responsive Property Assay The NIR light-responsive assay in vitro was carried out according to a method reported previously.8,

12

An infrared diode laser (Changchun New Industries Optoelectronics

Technology, Changchun, China) with a wavelength of 785 nm was used for the NIR light-responsive assay. The irradiation area of the laser at 785 nm was a circle spot about 1 cm in diameter. To investigate the temperature and volume change of the hydrogels responding to NIR light, the swollen hydrogel discs with thickness of 3 mm were exposed to NIR light laser with power densities of 0.6 W/cm2 and 1 W/cm2 in air at an initial temperature of 25 °C. The surface temperature change of the hydrogel discs was monitored in real time by an infrared thermometer (HT-866, HCJYET, Guangzhou, China). Meanwhile, the dimension change of the hydrogel discs was monitored using a digital camera.


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To investigate the thermal reversibility of nanocomposite hydrogels, the swollen hydrogel discs with thickness of 3 mm were exposed under NIR light (0.6 W/cm2) in air at 25 °C for 5 min (LASER ON) and reswelled in deionized water at 25 °C for 6 h (LASER OFF) repeatedly. The dimension change of the hydrogel discs was monitored using a digital camera. The dynamic volume change ratios were determined by the equation Vt/V0 = (dt/d0)3, in which dt was the diameter of hydrogel at time t while d0 was the diameter of the hydrogel at beginning.

2.8. In Vitro Drug Release Test DOX-loaded nanocomposite hydrogel samples (0.5 mL) were immersed in 20 mL of PBS at three different pH values, 5.5, 6.5 and 7.4, respectively. Then the samples were incubated in a thermostatic rotary shaker (ZWY-200D, Zhicheng, Shanghai, China) at a shaking speed of 150 rpm at 37 °C. At predetermined time intervals, 3 ml of medium were taken out with 3 mL of fresh dissolution medium added. The released drug amount in the PBS was calculated through measuring the UV absorbance of the solutions at 490 nm by a UV-vis spectroscopy (UV2600, Techcomp, Shanghai, China). All tests were conducted in triplicate.

To demonstrate NIR light-triggered drug release behavior, DOX-loaded nanocomposite hydrogel samples were immersed in 20 mL of PBS solution (pH 6.5) and exposed to a NIR diode laser at 785 nm, 1 W/cm2 for 10 min, which was defined as LASER ON stage and then incubated without NIR laser irradiation for 24 h at 37 °C, which was defined as LASER OFF stage. This process was repeated for 6 cycles. At certain time intervals, the release medium was collected with an equal volume of fresh medium added to analyze the cumulative amount of DOX released from the hydrogels.



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2.9. In Vitro Cytotoxicity Assay Cell viabilities were determined using MTT assay and Live/Dead assay with MCF-7 cells and HeLa cells. To investigate the hyperthermia effect of nanocomposite hydrogel on the viabilities of cancer cells, cells were plated at a density of 5×104 cells/well in 12-well plates and cultured in a 5% humidified CO2 incubator for 24 h at 37 °C. Then, Transwell inserts with nanocomposite hydrogel discs with thickness of 3 mm were placed in the wells containing 1 ml of media and an additional 500 µL was added to the insert. Cells incubated with nanocomposite hydrogel discs were exposed to NIR laser irradiation (785 nm, 1 W/cm2) for 0, 3, 5, 10, 15 min. To investigate the safety of the NIR irradiation, cells grown in absence of hydrogel discs were exposed to NIR laser irradiation (785 nm, 1 W/cm2) for 15 min. The laser spot size was a circle spot about 1 cm in diameter. Then the cells were cultured for 24 h at 37 °C. Thereafter, 150 µL of MTT solution was added to each well and the cells were incubated for another 4 h at 37 °C. After the media was carefully removed, the formazan crystals generated by live cells were dissolved in dimethyl sulfoxide. The absorbance at 490 nm was determined by a microplate reader (ELX800, Bio-Tek, USA). The relative cell viability (%) was calculated by contrasting the absorbance of treated wells with that of control wells.

For the determination of synergistic effect of chemo-photothermal therapy in vitro, the cells were treated with the blank nanocomposite hydrogels and DOX-loaded nanocomposite hydrogels with or without NIR laser irradiation. Briefly, cells incubated with nanocomposite hydrogel discs with thickness of 3 mm were or weren’t exposed to NIR laser irradiation (785 nm, 1 W/cm2) for 10 min. Next, the cells were cultured for another 24 h at 37 °C. Finally, the cell viabilities were determined using previous procedures. For Live/Dead assay, the medium was replaced with 1 mL of PBS after 24 h incubation. Then, each well was treated with FDA solution (staining live cells, green) with a final concentration of 100 µg/mL and PI solution (staining dead cells, red) with a


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final concentration of 40 µg/mL followed by 10 min incubation at 37 °C in the dark. Finally, each well was washed with PBS three times and pictures were taken by a fluorescence microscopy (DMIL LED, Leica, Germany).

2.10. In Vivo Gel Formation and Biocompatibility Kunming mice (6-8 weeks old) were obtained from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China). All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (Guangzhou, China). For assessing the feasibility of in vivo hydrogel formation, 200 µL of the sterilized precursor solution was injected subcutaneously into the back of Kunming mice by a 1 cc hypodermic syringe with a 26-gauge needle. The mice were euthanized 10 min after implantation. Subsequently, the appearance of in-situ forming gel was monitored using a digital camera. For histological evaluation, mice were sacrificed 21 days after implantation. The surrounding tissues of the formed hydrogels were surgically excised and processed histologically using hematoxylin-eosin (H&E) stains to evaluate the in vivo biocompatibility of the hydrogels in mice.

2.11. In Vivo Drug Accumulation and Antitumor Activity Tumor-bearing mice were prepared by implanting 100 µL of S180 cell suspension (2.0×107 cells/mL) at the right hind hip in male Kunming mice (around 20g body weight). About 7-9 days after inoculation, the subcutaneous mouse tumor model was well built. When the tumor sizes reached about 150 mm3, mice were given an intratumoral injection with free TPZnPc solutions as the control group and TPZnPc-loaded hydrogels with or without NIR laser irradiation (785 nm, 1 W/cm2) as experimental groups, with the same dosage of TPZnPc (200 µL, 10 mg/kg). In the case of TPZnPc-loaded hydrogels with laser irradiation groups, the NIR laser irradiation (785 nm, 1 W/cm2) was applied on the



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tumor regions for 10 min at 6h, 2d, 5d, 11d and 18d post-injection. Near-infrared fluorescence imaging (NIRF) experiment was performed at 0h, 6h, 1d, 3d, 7d, 14d and 21d post-injection using a Berthold In Vivo Imaging System (IVIS, excitation wavelength = 630 nm, emission wavelength = 680 nm) (NightOWL LB983, Berthold, Germany).

The mice were used for in vivo antitumor test when the tumor volume reached nearly 150~200 mm3. The mice were then randomly divided into six groups (n=5). Each group received one of the following treatments: (1) intratumoral injection of saline; (2) intratumoral injection of the blank nanocomposite hydrogels (Gel only); (3) intratumoral injection of the free DOX solution (Free DOX); (4) intratumoral injection of blank nanocomposite hydrogels + NIR (laser irradiation at day 2, 4, 6) (Gel+NIR); (5) intratumoral injection of DOX-loaded hydrogels (Gel/DOX) and (6) intratumoral injection of DOX-loaded hydrogels + NIR (laser irradiation at day 2, 4, 6) (Gel/DOX+NIR). The dose of DOX administrated was 20 mg/kg per animal, and the injection volume for each intratumoral administration was 200 µL. In the case of NIR treatment group, mice were anesthetized and then tumor areas were exposed to the NIR laser (785 nm, 1 W/cm2) for 10 min. The tumor volumes and body weights were measured at desired time intervals for an additional 21 days. The tumor volumes of mice were measured using a vernier caliper and computed as follows: V = (length) × (width)2/2. All the mice were sacrificed at day 21. The tumors in various groups were excised, fixed and sliced for H&E staining for histopathological assay.

2.12. Statistical Analysis All the data were presented as mean ± standard deviation (SD), unless stated otherwise. To determine a statistically significant difference between groups, either Student’s two-tailed t test or a one-way analysis of variance (ANOVA) was applied to analyze the data. A p-value less than 0.05 was considered to be statistically significant. All the


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statistical analyses were performed by SPSS for Windows (version 13.0, IBM Corporation, USA).

3. Results and Discussion 3.1. Preparation and Characterization of GNRs GNRs were prepared by a seed-mediated method described in the literature with slight modification.39-40 The synthesized GNRs were characterized by the TEM image and UV-vis absorption spectrum analysis. TEM image revealed that GNRs were well dispersed in water and showed good uniformity. The average length and width of the prepared GNRs with an aspect ratio of around 4 were 55 ± 5 nm and 14 ± 2 nm, respectively (Figure 1A). The GNRs exhibited a longitudinal surface plasmon resonance band at around 800 nm in the NIR region (Figure 1B). Then, to eliminate the cytotoxicity of CTAB and improve the biocompatibility and stability, the GNRs were conjugated with mPEG-SH through a ligand exchange reaction using a previously described method.7, 46 The positive zeta potential of the CTAB-coated GNRs (+40 mV) was changed to neutral (-3 mV) after surface functionalization (Figure S1), demonstrating an adequate exchange of the cationic CTAB by mPEG-SH. The UV-vis absorption spectrum exhibited that GNR-PEG had a similar absorption to original GNR-CTAB with transverse and longitudinal surface plasmon resonance bands at 520 and 800 nm, respectively. Furthermore, there was no aggregation of GNR-PEG occurring in PBS while CTAB-coated GNRs aggregated seriously (Figure S2), suggesting the enhanced stabilities of particles by surface functionalization. The photothermal conversion experiments showed that GNRs can rapidly and efficiently convert NIR light into thermal energy as highly localized heat sources when irradiated with a laser (Figure 1C), indicating that it had great potential for cancer hyperthermia therapy and NIR light-triggered drug release.



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Figure 1. (A) Representative TEM image of GNR-PEG. Scale bar: 50 nm. (B) UV-vis-NIR absorption spectrum of GNR-CTAB, GNR-PEG in water or PBS. (C) Temperature variations in the different concentrations of GNR-PEG aqueous solution with NIR laser irradiation (785 nm, 1 W/cm2).

3.2. Synthesis and Characterization of MPCD and AD-DOX PCD was synthesized by cross-linking β-CD with EPI in the presence of toluene in an alkaline solution. Then, it was methacrylated via the ring-opening reaction of the epoxy group of GMA with hydroxyl groups of PCD using DMAP as a catalyst. The 1H NMR spectra of the PCD and MPCD macromer measured in D2O were shown in Figure 2. The 1

H NMR spectra showed that most bands of β-CD at 3.4-4.2 ppm were broadened in the

spectrum of PCD due to the cross-linking reaction of β-CD. Upon the 1H NMR spectra from the integration ratio of signals in the extent of 5.0-5.2 and 3.3-4.2 ppm, the β-CD content in PCD was determined as around 70%. The typical 1H NMR peaks ascribed to the protons of double bond (CH2=C) and methyl group (-CH3) of GMA were observed at 6.18 ppm, 5.76 ppm and 1.97 ppm, respectively, indicating the successful methacrylation of PCD. As shown in Figure S3, the FTIR spectrum of PCD demonstrated that the stretching vibrations of -OH, -CH2- and -C-O-C- are around 3380, 2930 and 1040 cm−1, respectively, which were the representative peaks of β-CD. Moreover, the absorption bands of stretching vibration of C-O-C at 1020~1100 cm-1 were broadened in the spectrum of PCD due to the cross-linking reaction of β-CD. As shown in the spectrum of MPCD, the peak of ester group at 1720 cm−1 confirmed the successful methacrylation of PCD again.


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Compared with pristine β-CD, the PCD was much more dissoluble in water and proved to be more effective as a potential host component for drugs due to its stability, high solubilization and biocompatibility.47 While cyclodextrin cavities were capable to load anticancer drugs, their inclusion capacities were quite restricted. The complex stability constant for β-CD with DOX was reported to be 188 M-1, which was consistent with an encapsulation efficiency of around 7.5%.48 As one of the most popular host-guest pairs, β-CD and adamantane has been traditionally applied in molecular self-assembly. There is firm interaction between β-CD and adamantane with an binding constant around 1×105 M-1 in water.49 Due to the strong interaction between β-CD and the adamantyl group, an acid-labile adamantane-modified doxorubicin prodrug AD-DOX was synthesized as the guest molecule to improve encapsulation efficiency of DOX. Adamantane was conjugated with anticancer drug doxorubicin through a well-known acid-cleavable hydrazone bond. As shown in Figure S4 and S5, the 1H NMR and ESI-MS spectra demonstrated the AD-DOX was successfully prepared.

Figure 2. 1H NMR spectra of β-CD, PCD and MPCD in D2O. Red rectangles indicate



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methylene protons and methyl protons on the methacrylate group.

3.3. In Vitro Hydrogel Formation and Characterization 3.3.1. Thermo-responsive Swelling Behavior of the Nanocomposite Hydrogels PNIPAm, which is a commonly investigated thermal-responsive polymer for biomedical applications, undergoes a phase transition in pure water from hydrophilic, fully hydrated polymer chains to hydrophobic-collapsed chains as the temperature is changed around its LCST.50 The LCST of pure PNIPAm is around 32 °C, which is lower than the physiological temperature (37 °C). Therefore, for in vivo applications, the LCST should be tuned to a pertinent range between 37 °C, which represented a characteristic physiological temperature and 42 °C, which indicated a hyperthermia temperature.

Fortunately, the LCST of PNIPAm could be readily adjusted by copolymerizing with a second monomer to control the hydrophilic/hydrophobic balance of the polymer. Copolymerizing with a hydrophilic monomer generally resulted in an increased LCST while hydrophobic monomer caused a decreased LCST.12, 51 Here, for increase the LCST, NIPAm was copolymerized with the hydrophilic MPCD macromer to obtain poly(NIPAm-co-MPCD)/GNRs nanocomposite hydrogels. The feed compositions of the composite hydrogels in this study were shown in Table 1.

The LCSTs of hydrogels were determined by investigating thermo-responsive swelling behavior of these hydrogels. The LCST of the hydrogel was determined as the temperature at which the swelling ratio of a hydrogel decreased most dramatically. As shown in Figure 3A, the poly(NIPAm-co-MPCD) hydrogels had higher LCSTs (above 32 °C) on account of copolymerization with the hydrophilic monomer MPCD, and the LCST of the hydrogel increased gradually with the increase of the percentage of MPCD. Moreover, the obstruction of PNIPAm chain segments by MPCD macromer caused broadening of the phase transition of the polymers. It can be seen that the temperature


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response of hydrogels decreased gradually with the increase of the percentage of MPCD. The fading of thermosensitivity of the poly(NIPAm-co-MPCD) hydrogel was ascribed to the incorporation of co-monomer MPCD. The introduction of hydrophilic monomers MPCD would interrupt the hydrophilic/hydrophobic balance and increase the hydrophilicity of the hydrogel, resulting in an increased LCST. Meanwhile, the thermosensitivity of the PNIPAm hydrogel was attributed to the continuous isopropylamide sequence with a specific hydrophilic/hydrophobic balance. With the introduction of hydrophilic moiety, the proportion of PNIPAm chain segments in hydrogel reduced and the hydrophobic interactions between isopropyl groups would be much weaker at an equal temperature, causing a weaker thermosensitivity of the PNIPAm hydrogel.52 Thus, the hydrogel with a monomer mass ratio of 1:1 was chosen for further research. This material with a LCST around 40 °C, would be in a highly swollen state at 37 °C, but a highly collapsed state at 40-45 °C, exhibiting great potential to be used as a depot for drug delivery system.

3.3.2. Gelation Time Determination In this study, nanocomposite hydrogels were synthesized by copolymerizing MPCD and NIPAm using APS and AA redox system as the initiator. As an oxidant, APS is widely applied with a reductant N, N, N’, N’- tetramethylethylenediamine (TEMED) to trigger polymerization in room temperature. However, both of them are toxic to a certain extent.53 The toxicity of initiator and their outgrowths raise considerable obstruction for further biomedical applications. Therefore, we chose APS and AA as a safe redox initiator system to reduce the toxicity of initiator system.54

The effect of initiator concentration on hydrogel formation was investigated. The gelation time of hydrogels was determined using the inverted tube test. As shown in Figure 3B, as expected, with the increase of initiator concentration or/and temperature, the gelation



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time gradually decreased. When the concentration of initiators was 1%, the gelation time of hydrogel was about 22 min at 25 °C and about 14 min at 37 °C. When the concentrations of initiators increased to 2%, 3% and 5%, the gelation time were about 13 min, 8 min, 5 min at 25 °C and 6.5 min, 3.5 min, 1.5 min at 37 °C, respectively. The injectable hydrogels should maintain liquid state within sufficient time at room temperature to enable injection and gel quickly in human physiological environment. When the concentration of initiator was 2%, the hydrogel can keep in liquid state for about 12 min at room temperature and gel in around 6 min at physiological temperature in vitro. Therefore, considering the harmful effects of initiator and proper gelation time in vivo, NCD-7 formed with an initiator concentration of 2% are acceptable for practical application.

3.3.3. Rheological Characterization It is a significant indicator to achieve knowledge about the viscoelastic properties of hydrogels using rheological characterization. The optical photo showed the color changes of the hydrogels in the absence (NCD-9) and presence (NCD-7) of GNRs, evolving from transparent to red (Figure 3C). As shown in Figure S3, a broad peak at 3200-3500 cm−1 assigned to N-H stretching and those peaks at 1650 cm−1 and 1540 cm−1 corresponding to C=O stretching and N-H stretching of the amide group from PNIPAm component could be observed while most absorption bands of β-CD also appeared in both spectra of NCD-7 and NCD-9. These results provided direct evidence that nanocomposite hydrogel were successfully fabricated and the existence of GNRs had negligible impact on the FTIR spectra. Then, the storage modulus (G’) and loss modulus (G’’) of the two hydrogel samples were analyzed. As shown in Figure 3D, for both the tested samples, the value of G’ was much higher than the value of their corresponding G’’ and exhibited no dependence on frequency, suggesting the high elasticity of hydrogels. It should be noted that the final G' moduli of NCD-7 was slightly higher than that of the NCD-9, which


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meant the incorporation of GNRs could improve the mechanical strength of the hydrogel to some extent. Similar phenomena were observed in other nanocomposite hydrogel systems.1, 6-7, 9 Therefore, GNRs were expected to serve as reinforcing fillers and thus induced enhanced mechanical properties of hydrogel.55

3.3.4. Interior Morphology of the Hydrogels The morphologies of the hydrogels incorporated with or without GNRs assessed by SEM were showed in Figure 3E and 3F. Both the lyophilized hydrogels revealed highly homogeneous porous structure. The highly interconnected pores of the hydrogels were regular in shapes. The GNRs embedded into hydrogel were too small to be observed in the SEM image mainly due to the large scale bar, indicating that there was no micro-agglomeration

of

GNRs

in

nanocomposite

hydrogel.

Furthermore,

the

nanocomposite hydrogel NCD-7 with a smaller average pore size was denser than NCD-9. This was likely because the abundant hydrophilic PEG chains on GNRs allowed the formation of hydrogen bond interaction with cyclodextrin and increased the cross-linking density, thus leading to a denser network with smaller pore sizes, which was corresponding to the results of rheological experiments.56-57

This microporous morphology of hydrogel due to the high water content can play an important role for controlled and sustained drug release by obvious swelling-shrinking transition of hydrogel. With plenty interconnected pores in the hydrogel networks, the obvious shrinking process could force large amount of water to run out of the hydrogel while the entrapped drug in the hydrogel networks was squeezed out with water for on demand release.



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Figure 3. Characterization of nanocomposite hydrogels. (A) Swelling ratios of the nanocomposite hydrogels formed with different feed compositions (NIPAm:MPCD). (B) The gelation time of nanocomposite hydrogels with various initiator concentrations at 25 °C and 37 °C. (C) Photograph of the hydrogels incorporated with (NCD-7) or without GNRs (NCD-9). (D) Frequency dependency of the storage modulus (G’) and loss modulus (G’’) of NCD-7 and NCD-9 and SEM images of NCD-7 (E) and NCD-9 (F). Scale bar: 100 µm.

3.4. NIR Light-Responsive Property Assay As we all know, GNRs exhibited strong absorption in the NIR region and could convert it into heat efficiently. Therefore, the nanocomposite hydrogels combining GNRs and thermo-responsive PNIPAm networks exhibited excellent NIR light-responsive properties. The volume changes of the hydrogels incorporated with or without GNRs under the equal NIR light irradiation were shown in Figure 4A. Under NIR laser irradiation with a power density of 0.6 W/cm2 for 3 min, NCD-7 shrank dramatically and had a significant volume decrease. The volume of the hydrogel after irradiation was much smaller than that of before irradiation. The weight of the hydrogel also decreased by 86%. However, NCD-9 displayed no obvious volume change under the equal NIR light irradiation for 5 min. The GNRs in the hydrogels absorbed the NIR light and converted it into heat efficiently, which caused the local temperature increase of the nanocomposite hydrogels. When the


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temperature increased to above the LCST (40 °C) of hydrogel, the nanocomposite hydrogels shrank significantly to extrude the water in the polymeric networks, resulting in a significant volume decrease while the hydrogel without GNRs had no obvious volume change after equal NIR light irradiation due to the temperature below the LCST.

For assessing the photothermal conversion efficiency of GNRs embedded into the hydrogel, the hydrogels were exposed to a 785 nm NIR laser at power densities of 0.6 W/cm2 and 1 W/cm2. As shown in Figure 4B, there were no significant temperature changes occurring for NCD-9 exposed to NIR laser irradiation (0.6 W/cm2, 5 min). However, once the hydrogels incorporated with GNRs (NCD-7) were exposed under NIR laser (0.6 W/cm2), their surface temperatures can increase significantly and rapidly. NIR laser irradiation at power density of 0.6 W/cm2 and 1 W/cm2 can increase the surface temperatures of the hydrogels to about 47.5 °C and 58.2 °C in 5 min, respectively. These results indicated that no aggregation of GNRs occurred with high stability in hydrogels.

In the case of cancer photothermal therapy, it is highly desirable to use a repeatable biomaterial that is able to repeated heating upon laser exposure.57 Reversibility of NIR light-responsive property of the composite hydrogels was investigated by repeatedly exposing the nanocomposite hydrogel under NIR laser in air and reswelling it in deionized water at 25 °C. Once exposed under NIR light in air, NCD-7 exhibited a rapid shrinking response behavior. When immersed in water at 25 °C without NIR light irradiation, the hydrogel slowly returned to its original equilibrium swollen state through reswelling process in water within 6 h (Figure 4C). The shrinking-swelling transition proceeded repeatedly with the NIR light irradiation-reswell cycles. The results showed that the NIR light-responsive behaviors of the nanocomposite hydrogels were reversible.



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Figure 4. NIR light-responsive characteristics of nanocomposite hydrogels. (A) Photographs of the cycles of NIR light-responsive volume change of NCD-7 and NCD-9 exposed under the same NIR light irradiation (785 nm, 0.6 W/cm2) for different time periods and the recovery to original expansive state after reswelled in deionized water at 25 °C for 6 h. (B) The temperature change of NCD-7 and NCD-9 after NIR exposure (785 nm, 0.6 or 1 W/cm2) for 5 min. (C) Volume changes of NCD-7 as a function of NIR laser ON-OFF cycles.

3.5. In Vitro Drug Release Test The significant difference in pH values between cancerous environment (pH 6.5) and


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normal tissue environment (pH 7.4) has provided a theoretical foundation for the design of pH-responsive tumor targeted drug delivery vehicles. In this study, the DOX-loaded nanocomposite hydrogels were prepared by copolymerizing MPCD/AD-DOX inclusion complex and NIPAm with GNRs incorporated. There was no significant difference in gelation time, swelling behavior and NIR-light responsive property between the blank hydrogel and DOX-loaded hydrogel (Figure S6). Adamantane was conjugated with DOX through a hydrazone bond which was stable at physiological pH while labile at acidic pH. To investigate in vitro sustained and controlled drug release behavior, the prepared DOX-loaded nanocomposite hydrogel were immersed in buffer solution of pH 5.5, 6.5 and 7.4 at 37 °C for 30 days. The release profiles of DOX from the nanocomposite hydrogel under different conditions were shown in Figure 5A. We can see that the hydrogel was capable to markedly reduce the burst release effect and maintained the sustained release of DOX. The pH of the buffer solution has a significant effect on drug release. Only less than 23% DOX was released after 30 days from the hydrogels in PBS of pH 7.4. However, the DOX was released much faster under acidic conditions of pH 5.5, 6.5 compared to pH 7.4. It was seen that the cumulative amount of DOX released was about 66% and 45% after 30 days of incubation in PBS of pH 5.5 and 6.5, respectively. The release of DOX from the nanocomposite hydrogels also could be readily accelerated via NIR laser irradiation (Figure 5B). The amount of DOX released from the hydrogel increased significantly when the hydrogel was exposed to the NIR laser for only 10 min, which was much higher than that of without irradiation for 24 h, clearly verifying a NIR-triggered release behavior of hydrogel based on NIR exposure.

It should be noted that the slow and long term DOX release was primarily attributed to the formation of MPCD/AD-DOX inclusion complex. With the strong affinity between β-CD and the adamantyl group, the prodrug can form inclusion complex with β-CD polymer chains efficiently by both hydrophobic and host-guest interactions. Thus, drug



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release from this hydrogel system was mainly determined by two steps: first, free DOX was released from MPCD/AD-DOX inclusion complex slowly, which was controlled by the cleavage of acid-labile hydrazone bond in weak acidic conditions; then, free DOX remained in the 3D networks of the hydrogel was slowly released into the medium through passive diffusion. Upon NIR light exposure, GNRs introduced into hydrogel networks could generate localized heat by excellent photothermal conversion ability. When the temperature increased to above the LCST of hydrogel, the hydrogel underwent a prominent shrinking process, which could squeeze the water along with entrapped drug in the networks out of hydrogel in an on demand way. These two distinct and related release behaviors determined the entire DOX release profile and the corresponding curative effect. At lower pH value, DOX was released faster and more completely. These results indicated the superiority of injectable nanocomposite hydrogel, which could release DOX in an acidic tumor tissue environment and avoid undesirable drug accumulation in normal tissues to diminish side effects. Furthermore, nanocomposite hydrogel with excellent NIR light-responsive properties was able to release drug in an on demand way with the aid of NIR irradiation, which can satisfy the requirements for clinical drug delivery with tunable release kinetics.


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Figure 5. (A) In vitro DOX release profiles of DOX-loaded nanocomposite hydrogels in PBS at pH 5.5, 6.5 and 7.4. (B) Cumulative DOX release (µg) from nanocomposite hydrogel in response to NIR laser irradiation and without laser irradiation. Six cycles of ON (10 min NIR laser, 1 W/cm2) and OFF (24 h, 37 °C) were evaluated.

3.6. In Vitro Cytotoxicity Assay The in vitro cell cytotoxicity of this nanocomposite hydrogel was studied using a



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Transwell inserts device in Figure 6A. The localized photothermal effects of nanocomposite hydrogel on MCF-7 and HeLa cells were assessed respectively following exposure to the NIR laser (785 nm, 1 W/cm2) for 0, 3, 5, 10 and 15 min. As shown in Figure 6B, for cells grown in absence of hydrogel discs, there was no significant difference between the cells treated with or without laser irradiation for 15 min, which proved the safety of the NIR irradiation. No significant cytotoxicity was observed when the cancer cells were incubated with hydrogels for 24 h without laser irradiation. Furthermore, the cytocompatibility of the eluants of nanocomposite hydrogel at longer time points (24 h, 48 h, 72 h, 96 h and 120 h) was measured. The result showed that more than 95% of cells are viable, even treated with the eluants of nanocomposite hydrogel at 120 h (Figure S7). These results demonstrated that the hydrogel had a favorable biocompatibility. On the contrary, with the increase of NIR irradiation time, the cells viabilities of MCF-7 dramatically decreased from 80.3% at 5 min to 42.8% and 26.7% at 10 min and 15 min, respectively. Similar results were observed in HeLa cells (Figure S8A). These results demonstrated that nanocomposite hydrogel can rapidly and efficiently convert NIR light into thermal energy as highly localized heat sources to kill cancer cells directly, indicating that it had a great potential for cancer hyperthermia therapy.

To investigate the combinational effect of chemo-photothermal therapy, different drug formulations were carried out for treatment with MCF-7 cells and HeLa cells followed by the MTT assay and Live/Dead assay. As shown in Figure 6C, Figure S8B, the cell viability of NIR and Gel only groups had no obviously decrease, indicating that the NIR laser and nanocomposite hydrogels had no obvious cytotoxicity towards MCF-7 cells and HeLa cells. In addition, the cell viabilities in irradiated groups (Gel+NIR) were much lower than those of the cells in non-irradiated groups (Gel only), verifying the photothermal effect of nanocomposite hydrogels. However, only a mild cytotoxicity was


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observed in Gel/DOX group owing to the sustained release of DOX from hydrogel. DOX was minimally released from the nanocomposite hydrogel in the absence of NIR irradiation while the hydrogel discs were incubated with media for 24 h. The dual-therapy group (Gel/DOX+NIR) exhibited a remarkable combinational effect of chemotherapy and photothermal therapy and excellent theraputic efficiency as compared to those of the monotherapy groups (chemotherapy or photothermal therapy). The result of Live/Dead assay also confirmed the combinational effect of chemo-photothermal therapy (Figure 6D). In conclusion, in this study, NIR laser irradiation not only induced heat for cancer therapy but also could accelerate the release of DOX from hydrogel leading to an enhanced chemotherapy. The nanocomposite hydrogel with NIR laser irradiation could realize chemo-photothermal synergistic therapy.

Figure 6. (A) Schematic diagram of the device used to study the in vitro cytotoxicity of the nanocomposite hydrogel. (B) Cell viability of MCF-7 cancer cells incubated with the blank nanocomposite hydrogel exposed to NIR laser irradiation (785 nm, 1 W/cm2) for 0,



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3, 5, 10, 15 min. Cells grown in absence of hydrogel discs were exposed to NIR laser irradiation (785 nm, 1 W/cm2) for 15 min. Cell viability (C) and cell Live/Dead staining (D) of MCF-7 cancer cells incubated with the blank nanocomposite hydrogel, DOX-loaded nanocomposite hydrogel with or without NIR laser irradiation (785 nm, 1 W/cm2, 10 min). Scale bar: 100 µm. Data are presented as means ± SD (n = 3, **P < 0.01, ***P < 0.001).

3.7. In Vivo Gel Formation and Biocompatibility For assessing the feasibility and biocompatibility of in vivo hydrogel formation, the precursor solution was injected subcutaneously into the back of Kunming mice. Figure 7A showed the representative macroscopic images of the implanted hydrogels after being injected subcutaneously into the dorsal side of mice for 10 min. The gross appearance of hydrogel was pink due to the color of GNRs embedded into hydrogel networks, somewhat cohesive and adhered to tissue planes (Figure 7B). The efficiency of the sol-gel transition in vivo was around 94.7% by measuring the net weight of the lyophilized hydrogel. Furthermore, the shape of the hydrogel remained stable for more than 20 days (Figure 7C), indicating that the implanted hydrogel had a potential for long term applications in vivo. In order to investigate initial biocompatibility of the hydrogels, the inflammatory response of hydrogels was evaluated at day 21 using H&E staining. No obvious infections or inflammatory reactions were observed in the subcutaneous tissues (Figure 7D) and skins (Figure 7E and F) of the injected sites at day 21, even if longer evaluation should be performed to confirm whether the implant would have further effects. Therefore, this injectable nanocomposite hydrogel was formed immediately after injection, which laying the foundation as a local and sustained long term drug delivery carrier, and might be a promising material for in vivo applications.


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Figure 7. In vivo nanocomposite hydrogel formation and tissue biocompatibility. (A) Photograph of the implanted hydrogel 10 min after subcutaneous injection. The gel region is denoted by the white dashed line in (B). (C) Gross appearance of hydrogel implant in mice after 21 days. H&E staining of the subcutaneous tissues (D) and skins (E, F) surrounding the injection site after 21 days of implantation.

3.8. In Vivo Drug Accumulation and Antitumor Activity To investigate the sustained/controlled release and local retention of drugs loaded in the nanocomposite hydrogels after intratumoral injection, a near infrared fluorescent dye, TPZnPc, was used as a fluorescence probe. After injection, optical fluorescence imaging was obtained by using a Berthold In Vivo Imaging System while NIR laser irradiations were performed at the indicated time points (Figure 8A). As shown in Figure 8B, in free TPZnPc treated group, the fluorescence intensity at the tumor site decayed quickly and nearly vanished at 24 h post injection. By contrast, the TPZnPc-loaded hydrogels showed persistent retention of TPZnPc within a much longer time period. The fluorescence signals of experimental groups were able to maintain for more than 21 days. Furthermore, TPZnPc-loaded hydrogels with NIR laser irradiation groups showed fast fluorescence attenuation in 21 days, which was much quicker than TPZnPc-loaded hydrogels without NIR laser irradiation groups. These results confirmed a NIR-triggered release behavior of



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hydrogel and NIR irradiation can efficiently accelerate drug release in vivo in an on demand way. Moreover, various drugs which were also suitable for loading into such a unique and versatile hydrogel networks can realize such a durable retention and sustained release manner after intratumoral injection. Thus, the nanocomposite hydrogel had a promise to be used as a long term drug delivery platform for cancer therapy in vivo.

Figure 8. In vivo evaluation of the retention and accumulation of drug in nanocomposite hydrogel by NIRF imaging. (A) Schedules of gel injection, NIR irradiation (785 nm, 1 W/cm2, 10 min) and IVIS. (B) Representative in vivo fluorescence imaging of tumor-bearing mice intratumoral injected with free TPZnPc solution, TPZnPc-loaded hydrogels and TPZnPc-loaded hydrogels with NIR laser irradiation at different time points over 21 days.

The in vivo antitumor efficacy of nanocomposite hydrogel with combination of


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chemo-photothermal therapy was investigated on the xenograft S180 tumor model. As shown in Figure 9A, mice treated with saline showed a rapid and unrestrained tumor growth, reaching an average tumor size of approximate 12.1 times of the initial tumor size at the end of 21 days. There was no statistical difference between saline and Gel only groups. The tumors in free DOX and Gel/DOX groups grew slowly, and both of groups exhibited significant difference compared with control groups. The treatment of free DOX could inhibit the tumor growth effectively in the initial stage due to the high DOX concentration, but thereafter the tumor grew normally to 10.9 times of the initial tumor size. The overall tumor growth inhibition of free DOX was not effective, which could be attributed to its high diffusion and quick elimination. Compared to free DOX, the enhanced therapeutic efficacy of Gel/DOX was mainly resulted from the prolonged drug retention and improved drug accumulation at tumor tissue, but the efficacy was still insufficient to stop the tumor growth. Meanwhile, owing to the excellent photothermal effect of nanocomposite hydrogel in the tumor site, the mice in Gel+NIR groups exhibited a moderate tumor ablation. The mice that treated with Gel/DOX and were exposed to the NIR laser (Gel/DOX+NIR) exhibited obviously suppressed and outstanding tumor growth with the aid of the NIR irradiation treatment, which demonstrated the capacity of nanocomposite hydrogel for chemo-photothermal synergistic therapy.

To verify the advantages of nanocomposite hydrogel in vivo, the body weights as an important indicator were real-time monitored. All treatment groups except free DOX group had the similar increasing tendency for the body weights of the mice during the experimental time periods in Figure 9B. Compared to that of control group, free DOX-treated mice exhibited a significant body weight loss which was ascribed to the serious toxicity and side effects, whereas the body weights of mice in Gel/DOX group and Gel/DOX+NIR group were similar to that of control group, suggesting that both of



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treatments were tolerated well with reduced side effects compared with free DOX.

The images of excised tumors and their average sizes further confirmed the combination of chemo-photothermal cancer therapy (Figure 9C, Figure S9). To further evaluate the antitumor efficacies of the above formulations, the histopathological analyses of tumor tissues were carried out. As shown in Figure 9D, in the cases of saline group and Gel only group, tumor cells had obvious and normal cellular morphology and large cell nuclear, indicating the vigorous proliferation of tumor cells. However, the tumors treated with various formulations exhibited various degrees of tumor cell necrosis with apparent morphological characteristics, such as cell nuclear shrinkage, fragmentation and nuclear condensation. Compared with other formulation-treated groups, the tumors of the Gel/DOX+NIR group exhibited the largest necrosis areas, which indicated the best tumor suppression efficiency in the Gel/DOX+NIR group. All of the above data demonstrated that this nanocomposite hydrogel could be potentially applied as a long-acting implant for effective cancer treatment in vivo.


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Figure 9. In vivo antitumor activity of different formulations in S180-bearing mice. (A) Changes in relative tumor volume and (B) Body weight of S180-xenografted Kunming mice upon various treatments. Data are presented as mean ± SD (n = 5, ***P < 0.001). (C) Photograph of the excised tumors after the mice were sacrificed on the 21st day. (D) H&E staining of the tumor sections in different formulation-treated groups of mice at the end of experiment. Scale bar: 200 µm.

4. Conclusions In

summary,

we

have

developed

an

injectable

and

NIR/pH-responsive

poly(NIPAm-co-MPCD)/GNRs nanocomposite hydrogel as a long-acting implant for local chemo-photothermal synergistic cancer therapy. The nanocomposite hydrogel showed tunable mechanical and swelling properties, gelation characteristics and excellent NIR-responsive property. A hydrophobic acid-labile doxorubicin prodrug AD-DOX was



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loaded into hydrogel efficiently by host-guest interaction. The introduction of GNRs was proven effective in improving mechanical properties of hydrogels. The nanocomposite hydrogel exhibited a highly and homogeneous three dimensional porous structure and a sustained drug release manner and could sustain the slow and steady release of DOX for more than one month. The pH-responsive release of DOX from the nanocomposite hydrogel was observed owing to the cleavage of acid-labile hydrazone bond between DOX and adamantyl group in acidic environment. NIR irradiation can accelerate the release of free DOX from the networks, which was controlled by the collapse of the hydrogel networks induced by photothermal effect of GNRs. The in vitro cytotoxicity test demonstrated the excellent biocompatibility and photothermal effect of hydrogel. In addition, the in-situ forming hydrogel showed promising tissue biocompatibility in mouse model study. The in vivo antitumor test demonstrated the capacity of nanocomposite hydrogel for chemo-photothermal synergistic therapy with reduced adverse effects owing to the prolonged drug retention in the tumor region and efficient photothermal effect. Therefore, this injectable and NIR/pH-responsive hydrogel might be a promising candidate as a long term drug delivery platform for chemo-photothermal synergistic cancer therapy.

ASSOCIATED CONTENT Supporting Information The zeta potentials of GNRs before and after surface modification measured by DLS; optical photos of GNR-CTAB and GNR-PEG incubated in 10, 20 mM PBS; FT-IR spectra of β-CD, PCD, MPCD, NIPAm, NCD-9 and NCD-7; the 1H NMR spectra of adamantine-1-carboxylic acid hydrazide, DOX and the prodrug AD-DOX; the ESI-MS profile of AD-DOX; the properties of DOX-loaded hydrogel; the cytocompatibility of the eluants of nanocomposite hydrogel; the MTT assay of nanocomposite hydrogel on HeLa cells; optical photos of the excised tumor and in-situ forming hydrogel.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We are grateful for financial support from the National Natural Science Foundation of China (grant number: 81673369/H3008).

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TOC


pH-Responsive Nanocomposite Hydrogel as Long

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