Injectable and Near-Infrared-Responsive Hydrogels Encapsulating

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Injectable and NIR-Responsive Hydrogels Encapsulating Dopamine-Stabilized Gold Nanorods with Long Photothermal Activity Controlled for Tumor Therapy Jinfeng Zeng, Dongjian Shi, Yanglin Gu, Tatsuo Kaneko, Li Zhang, Hongji Zhang, Daisaku Kaneko, and Mingqing Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00600 • Publication Date (Web): 07 Aug 2019 Downloaded from pubs.acs.org on August 8, 2019

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Injectable and NIR-Responsive Hydrogels Encapsulating Dopamine-Stabilized Gold Nanorods with Long Photothermal Activity Controlled for Tumor Therapy

Jinfeng Zenga, Dongjian Shia*, Yanglin Gub, Tatsuo Kanekoc, Li Zhanga, Hongji Zhanga, Daisaku Kaneko a, Mingqing Chena* a Key

Laboratory of Synthetic and Biological Colloids, Ministry of Education, School

of Chemical and Material Engineering, Jiangnan University, Wuxi, China b

The Affiliated Wuxi No.2 People’s Hospital of Nanjing Medical University, Wuxi,

China c

Graduate School of Advanced Science and Technology, Japan Advanced Institute of

Science and Technology, Ishikawa, 923-1292, Japan Corresponding author: Dr. Dongjian Shi, [email protected], Prof. Mingqing Chen, [email protected]

Keywords Gold Nanorods; Photothermal Therapy; Injectable Hydrogel; Polydopamine 1

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Abstract Gold nanorods (AuNRs) are confirmed to have excellently and repeatedly photothermal property under near-infrared (NIR) light irradiation above 780 nm. However, AuNRs easily leaked out from local pathological tissues and circulated in the body, reducing photothermal therapy (PTT) efficacy. By complexing AuNRs with a scaffold via interactions, AuNRs might be dispersed in the scaffold and fixed in the tumor site. Thus, based on mussel-mimetic adhesion concept, AuNRs were designed to be coated with polydopamine (PDA), and then the prepared polydopamine-coated AuNRs (AuNR-PDA) were incorporated into a thermo-sensitive injectable hydrogel composed of β-glycerophosphate-bound chitosan (CGP) and dopamine-modified alginate (Alg-DA) efficiently. Thanks to the strong interactions between PDA and polymers, AuNR-PDA could be immobilized stably and evenly into the obtained CGP/Alg-DA/AuNRs composite hydrogel, which can avoid over-heating locally or leaking out. Sol-gel transition temperature of the composite hydrogel was adjusted to the body temperature at around 37 C to be conveniently injectable in vivo. With NIR irradiation at 808 nm of wavelength, the composite hydrogel was locally heated quickly to over 50 oC depending on controlling the irradiation powers and times. Moreover, in vitro cytotoxicity test of the composite hydrogel showed good biocompatibility to normal cells, but obvious suppression to tumor cells’ growth under multiple photothermal therapy. Furthermore, in vivo antitumor test demonstrated the obvious suppression to tumor growth of the CGP/Alg-DA/AuNRs composite hydrogel under 2


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multiple PTTs. Therefore, the injectable CGP/Alg-DA/AuNRs hydrogel could be a promising candidate for the long-term repeated photothermal treatment of tumor.

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Introduction Chemotherapy, as a main tumor therapy, might induce a risk of cumulating side effects to normal tissues and patients’ immune system, because that the aggressive and resistant of tumor requiring drug injection repeatedly.1,2 Due to free of invasiveness and side effects, photothermal therapy (PTT) has become mostly useful method for tumor therapy. PTT commonly utilizes photothermal agents to strongly absorb near-infrared (NIR) light and translate it into hyperthermia for the ablation of adjacent tumor cells.3,4 Photothermal agents used in PTT mainly include gold nanoparticles5-7, graphene oxide nanosheets8, platinum nanoparticles9, organic compounds10,11 such as porphyrins and indocyanine green (ICG)12,13, and carbon nanomaterials14. Gold nanorods (AuNRs) are one of the most popular photothermal agents for PTT due to their excellent physicochemical

properties,

including

easy

surface

functionalization,

good

biocompatibility and remarkable light-heat transition ability.3,15 They have excellently and repeatedly photothermal property under NIR light irradiation above 780 nm which could penetrate biotic tissues.16-19 However, AuNRs are found to easily aggregate and leak out of the tumor site.20, 21 This phenomenon reduces the PTT efficacy, and then induces increment of drug dosages and injection times to keep PTT efficacy, which bring pains to the patients. Therefore, an additional carrier for encapsulating AuNRs is necessary to achieve high long-term stability and PTT efficacy of AuNRs under NIR irradiation. Hydrogels have been confirmed to have the capability to load drugs and then 4


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release them in a fixed site with high drug concentration at the pathological tissues over a long period of time.22,23 In order to make the hydrogels reach the pathological tissues accurately, thermo-sensitive hydrogels with a lower critical solution temperature (LCST) are the most extensive carriers because they can be injected into body in a liquid state, and then undergo gelation at a physiological temperature to form a crosslinked hydrogel.24 Specially, fabrication of thermo-responsive hydrogels with “off-on” fashion could allow the control of the cancer drugs release in highly spatial and temporal resolutions for even several weeks of continuous drug release, which could enhance bioavailability and reduce side effects.25,26 Accordingly, if AuNRs were encapsulated into the thermo-sensitive hydrogels, AuNRs could reach the tumor site easily and then be immobilized for repeated PTTs. However, most of the reported thermo-sensitive hydrogels are undegradable, fail to be used in the clinical bioapplications. Chitosan (CS) as a major component with good biocompatibility has been widely used in drug delivery carriers and scaffolds for cell therapy.5,27,28 When CS was mixed with β-glycerophosphate (β-GP), the CS/β-GP (CGP) composite showed interesting temperature-dependent gelation behavior under physiological condition29-31, i.e. the injectable property. However, this property is easily changed or even disappeared by mixing a third-component.32 Selecting an appropriate component to keep the temperature-dependent gelation property is very important to achieve the injectable gel with high stability of AuNRs. In our previous work, we had prepared the stable Au nanoparticles or Au nanorods 5



by coating dopamine6,31, which was inspired by the strong interactions inducing well adhesive property of mussel. Herein, with mussel-mimic design, we employ dopamine to modify AuNRs and a functioned anionic polysaccharide derivative, dopamine modified alginate (Alg-DA) to composite with CGP for fabrication of an injectable hydrogel, whose gelation behaviors could be controlled based on the molecular interaction. As a result, composite hydrogels with biodegradability could deliver AuNRs accurately to the pathological tissues by injection and work as PTT materials stably for a long time under repeated NIR laser irradiation.

Scheme 1. Illustration for preparation of (a) AuNRs-PDA and (b) CGP/AlgDA/AuNRs thermo-sensitive hydrogel.

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2 Experimental Section 2.1 Materials Chitosan (CS) with viscosity of 100~200 mPa·s was purchased from Macklin and used without further purification. Alginate (Alg, 200 ± 20 mPa·s), dopamine hydrochloride

(DA),

ascorbic

acid,

and

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride (EDC) were purchased from Aladdin. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O), SH-PEG-CH3 (Mw = 2000) and sodium β-glycerophosphate (β-GP) were purchased from Alfa Aesar. Hexadecyl trimethyl ammonium bromide (CTAB) and sodium borohydride (NaBH4) were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Tetramethyl azo salt (MTT) was purchased from Amersco. Human hepatocellular carcinoma cell (HepG2) was obtained from Jindou Biomedical Company (Shanghai China). All the materials were used as received. 2.2 Synthesis and modification of gold nanorods (AuNRs) AuNRs were prepared in two steps using seed-mediated growth as reported (the detail procedure was shown in supporting information).33 AuNRs was then modified with SH-PEG-CH3 (2 mg/mL, Mw = 2000) in aqueous solution for 16 h at room temperature. AuNRs-PEG was obtained by centrifuged three times at 7500 r/min for 10 min. AuNRs-PEG was further coated with polydopamine (PDA) by dispersing AuNRsPEG into 5 mL DA buffer solutions at room temperature for 30 min. Polydopamine coated AuNRs (AuNRs-PDA) were washed twice with deionized water. Morphologies 7



and sizes of AuNRs and their composites after modification were examined using a JEM-2100 Transmission electron microscope (TEM) operating at an acceleration voltage of 200 KV (Japan) and dynamic laser light scattering (DLS, ALV/DLS/SLS5022F, Germany). Optical properties of AuNRs and their composites were evaluated using UV-Vis spectroscopy (UV-1100, Beijing). Zeta potentials were performed using Zeta potential and nano particle size analyzer (Zeta PALS, USA). 2.3 Preparation of the composite hydrogel Firstly, chitosan/dopamine modified alginate hydrogel (CGP/Alg-DA) was prepared as following. CS was firstly dissolved in 1.0 % (v/v) lactic acid solution under vigorous stirring to prepare clear solution. After 3.0 mL CS solution was chilled to 4 oC for 30 min, 0.5 mL ice-cold β-GP (60 wt％) solution was added dropwise to the CS solution until pH to 7.2 and further stirred for 30 min to prepare the CS-β-GP compound (abbreviated as CGP). The aqueous solutions of DA-modified Alg (Alg-DA) with various DA contents34 (shown in Supporting Information) were respectively added into CGP solutions with a given ratio (CGP: Alg-DA=3:1, w/w) under vigorous stirring for 30 min. Then, the mixed solutions with various weight ratios were injected into a cylindrical mold using syringes and subsequently incubated in a water bath at 37 oC to form gels. CGP/Alg hydrogels with various weight ratios of CGP and Alg (5:0, 4:1, 3:1, 2:1 and 1:1) were also prepared according to the above procedure. Then, AuNRs doped chitosan/dopamine-modified alginate composite hydrogel (CGP/Alg-DA/AuNRs) was also prepared, similar to the above procedure. A mixture 8


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of AuNRs-PDA and Alg-DA was prepared and then added into the CGP solution. The CGP/Alg-DA/AuNRs composite hydrogel could be prepared after incubating the mixture at 37 oC. Depending on the DA contents, the obtained composite hydrogels were abbreviated as CGP/Alg-DA1/AuNRs, CGP/Alg-DA3/AuNRs, and CGP/AlgDA5/AuNRs. Microstructural and EDS element mappings of the composite hydrogels were analyzed by scan electric microscope (SEM, S-4800, Japan). 2.4 Gelation time of hydrogels The gelation times of CGP/Alg-DA and CGP/Alg-DA/AuNRs composite hydrogels were determined by a test tube-inverting method.35 Briefly, the mixture solution of CGP, Alg-DA and AuNRs-PDA was injected into glass vial. With incubation at 37 oC, the glass vial was inclined or inverted every minute to observe the fluidity of the samples. The gelation point was recorded once the sample stopped flowing, which was referred to gelation time. All samples were repeated three times under the similar environmental condition. 2.5 Photothermal property of hydrogel Photothermal effect of the hydrogel was evaluated using a NIR laser (MDL-Ⅲ808-2.0 W/cm2, Changchun). For this purpose, cylindrical hydrogel (1.0×1.0 cm2×cm) was irradiated with 808 nm of NIR laser at different setting powers (1.0, 1.5 and 2.0 W/cm2) for 6 min. Temperature of the hydrogel after laser irradiation was recorded by infrared imager (Fluke Ti400, USA). For comparison, the temperature of the pure H2O and PBS buffer solution (pH 7.4) without the hydrogels were also measured with laser 9



irradiation. Moreover, the cylindrical hydrogel was repeatedly irradiated with 808 nm laser (1.5 W/cm2, 6 min) for 10 times to analyze the long and repeated efficiency of photothermal conversion. 2.6 Cytotoxicity of hydrogel in vitro Cytotoxicity of the composite hydrogel was evaluated using methylthiazolyl diphenyl tetrazolium bromide (MTT) assays. Firstly, mouse fibroblast cells (L929 cells) as a model normal cells were cultured in DMEM medium containing 10 % FBS and 1 % penicillin/streptomycin with a density of 5×104 cells/mL in a standard humidified 5 % CO2 at 37 oC. After 24 h of incubation, the culture medium was replaced with extract liquids of the hydrogels. Followed by another 24 h of incubation, 20 μL MTT (2 mg/mL) was added into each well. After incubation for 4 h in dark, 150 μL DMSO was added into the incubators and the plate was shaken for 10 min to dissolve all of the formed formazan crystal. Absorbance was then measured with a microplate reader (Infinite M200Pro) at a test wavelength of 570 nm (reference: 630 nm). Cell viability was calculated as following: OD (experiment ― blank)

Cell viability=

OD (control ― blank)

× 100 %

Then, effects of photothermal therapy of the composite hydrogel based on AuNRs on tumor cells in vitro were demonstrated with HepG2 cells as model cancer cells. HepG2 cells with a density of 5×104 cells/mL were incubated in a 96-well plate in a standard humidified 5 % CO2 incubator at 37 oC for 24 h, according to the above method. The CGP/Alg and CGP/Alg-DA/AuNRs composite hydrogels were fabricated firstly. 10


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After soaked in 75 % ethanol and washed for three times with PBS, the hydrogels were placed into in culture inserts. After incubation, the cells were washed with PBS, replenished with fresh culture medium, and treated with NIR laser irradiation at different power settings (1.0, 1.5 and 2.0 W/cm2) for 6 min and repeated for various times. After laser irradiation, the cells were further cultivated for 24 h to evaluate cell viability using the MTT assay. Moreover, in order to detect the sustained anti-tumor property of the CGP/Alg-DA/AuNRs composite hydrogel under repeated NIR irradiations in vitro, HepG2 cells with a density of 1.25×104 cells/mL were incubated in the CGP/Alg-DA/AuNRs hydrogel. In 24 h interval, the cells were treated with repeatedly NIR laser irradiation at 1.5 W/cm2 for 6 min in each irradiation. After continued cultivated for 24 h, cell viability was evaluated using the MTT assay. 2.7 In Vivo Anti-tumor Anti-tumor experiments of nude mice were commissioned to Shanghai Kelton Biotechnology Co., Ltd. All animal surgical experiments were approved by the Animal Care and Ethics Committees of the Shanghai Institute of Science and Technology. The tumor-bearing mouse was established by injecting subcutaneously 100 μL HepG2 cells suspension (5.0×106 cells/mL) into the right flank region of the Balb/c nude mice with an average weight of about 19 g. When the tumor sizes reached to an average size of 100 mm3, the mice were randomly divided into four groups with five mice in each group. Each group received one of the following treatments: I. The first group was injected with saline (200 μL) as the control group. II. The second group was irradiated with NIR 11



laser in addition to 200 μL saline injection. III. In the third group, the CPG/AlgDA/AuNRs hydrogels were injected around the tumor. IV. The mice were injected with the CPG/Alg-DA/AuNRs hydrogels around the tumor and then were irradiated with NIR laser. In the case of the groups with laser irradiation, the NIR laser at 808 nm, 1.5 W/cm2 was applied to irradiate the tumor regions for each 3 min at 1, 3, 6, 9, 12, 15, 18, 21 days. Simultaneously, the temperature at tumor site of four group of mice were also recorded at 1, 3, 6, 9, 12, 15, 18, 21 days postinjection using an infrared imager. The tumor sizes and body weights were measured at planned time intervals within 21 days. The length and width of the tumors were measured using a Vernier caliper and calculated as follows36: V = (length) × (width)2/2 All of the mice were sacrificed after treatment for 21 days. The tumors in the various groups were excised, fixed, and sliced with H&E staining for the histopathological assay.

3 Results and discussion AuNRs are well-known to have an adjustable longitudinal localized surface plasmon resonance (LSPR) peak at NIR region.37 Because the attenuation of NIR by blood and soft tissue is relatively low38, AuNRs are supposed to be an attractive candidate for PTT under NIR irradiation. In order to encapsulate AuNRs in hydrogel 12


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matrix with high stability, we designed to mussel-mimetic DA: (i) a thin layer of PDA was coated on the surface of AuNRs; (ii) dopamine-modified alginate (Alg-DA) was synthesized. Alg-DA can interact with CGP via electrostatic interactions to control gelation behavior, as well as with PDA-coated AuNRs to make an efficient encapsulation, due to the interactions among the catechol groups in DA. 3.1 Synthesis, modification and characterizations of AuNRs AuNRs with an appropriate length-diameter ratio were synthesized via seedmediated growth procedures by controlling amount of AgNO3 and ascorbic acid. TEM images (Figure 1a) showed that average length and width of the obtained AuNRs were around 60 nm and 16 nm, respectively, resulting in an aspect ratio of AuNRs at ∼4.0. AuNRs with this aspect ratio showed the plasma resonance wavelength at around 830 nm (shown in Figure 1c), which was well satisfied with NIR light irradiation for PTT. Since the catechol groups in DA have strong interactions with metals and polymers via chelating, hydrogen-bond, and covalent interactions39, DA could easily coat on the AuNRs surface to form PDA layer. The formed PDA has hydroxyl (-OH) and secondary amine (-NH) groups, which endow the PDA coated materials with high water dispersibility.40 Thus, DA was employed to self-polymerize and coat on the AuNRs in Tris-HCl buffer solution for improving the uniform and stable dispersion of AuNRs in hydrogel. However, the prepared AuNRs are unstable and inclined to aggregate in alkaline conditions during self-polymerization of DA. To solve this problem, thiolterminated polyethylene glycol (PEG-SH) ligand was firstly introduced to graft on the 13



AuNRs surface via Au-S binding (Scheme 1a), which was verified by X-ray photoelectric spectroscopy (XPS) (Figure S1) and Raman spectra (Figure S2). The obtained PEG grafted AuNRs (AuNRs-PEG) showed the similar size with AuNRs, but better dispersibility than that of AuNRs. Then, PDA could be coated onto the AuNRsPEG surface to form the uniform and stable AuNRs-PDA nanorods, as shown in Figure 1a. By modulating the concentrations of DA solutions at 0.1, 0.25 and 0.5 mg/mL during the self-polymerization reaction, the sizes of the obtained AuNRs-PDA (abbreviated as AuNRs-PDA0.1, AuNRs-PDA0.25, and AuNRs-PDA0.5) increased with the increment of DA concentrations, and the shell thicknesses of PDA could be calculated to 6.0, 8.3 and 8.5 nm from TEM images (Figure 1a), respectively. The sizes and their distributions of AuNRs before and after PDA modification were also characterized by DLS (Figure S3), which were well in accordance with the TEM results. PDA coated AuNRs could also be easily ascertained by the surface charge of Zeta potential value. As shown in Figure 1b, zeta potential of AuNRs-PEG changed to a nearly neutral charge (8.9±0.6 mV), compared with the positively charged AuNRs (21.3±2.1 mV). While the Zeta potential of AuNRs-PDA gradually decreased from positive value to -10.0±1.2 mV, -10.9±0.7 mV and -15.9±0.6 mV with increasing DA contents. These were ascribed to multiple effects of the catechol groups on PDA shells and -OH groups of PEG exposed outside from the shells.38 Moreover, UV-vis spectra were also carried to confirm the modification of AuNRs, which were shown in Figure 1c. Characteristic absorption of AuNRs was at 800 nm, attributed to the resonant propagation of the surface plasmon along the longitudinal axis. The absorption of 14


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AuNRs-PEG was almost similar to that of AuNRs, indicating no influence of PEG on the physical and optical properties of AuNRs. After PDA depositing, the LSPR peak of AuNRs was obviously red-shifted and became broadening, because of strong adsorption of PDA on the AuNRs surfaces inducing the larger refractive index of PDA.38 These results also confirmed the successful preparation and formation of the stable AuNRs-PDA nanorods. Since AuNRs-PDA0.25 showed more appropriate thickness of the PDA layers and better dispersibility, AuNRs-PDA0.25 as an example was mainly used in the following research.

Figure 1. (a) TEM images, (b) Zeta potential values, and (c) UV-vis spectra of AuNRs, AuNRs-PEG and AuNRs-PDA with PDA compositions. 3.2 Preparation and characterization of CGP/Alg-DA/AuNRs hydrogel I. Gelation behaviors

15



To increase the intermolecular interactions and the hydrogel strength, Alg-DA was designed to conjugate with CGP to form a composite hydrogel via electrostatic interactions. Firstly, DA was introduced into the Alg chain using EDC/NHS as catalysts34, which was illustrated in the supporting information in Figure S3. Alg-DA with various DA contents of 9.0%, 27.9% and 49.8% were obtained by controlling the molar ratios of DA to alginate at 0.1, 0.25, 0.5 mg/mL, and were abbreviated as AlgDA1, Alg-DA3, Alg-DA5. After mixing the solutions of CGP, Alg-DA and AuNRsPDA at 4 oC, a uniform and flowing solution was obtained. Then, the mixture changed into semisolid composite hydrogels at 37 oC for a few minutes, as shown in the inset figure in Figure 2a, suggesting the injectable property of the formed hydrogel. The CGP was reported to have the thermal sensitivity31, which led to the injectable property. Moreover, the gelation of the complex was partly induced by the formation of the crosslinking bonds among the catechol groups in Alg-DA and AuNRs-PDA in the presence of oxide. For biomedical application, gelation temperature and time in vivo must be appropriate and clear. The gelation time was detected by analyzing changes of storage (G’) and loss (G’’) modulus with the increase of temperature (Figure 2a). When temperature was less than 35 oC, G’ value was lower than G’’, showing a characteristic of liquid-state. At 36.5 oC, a crossover point formed in the G’ and G’’ curves, indicating transition of hydrogel from liquid-like to elastic gel-like. Thus, the gelation temperature was recorded as 36.5 oC. Then, fixing the gelation temperature at 36.5 oC, the gelation time was determined 16


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by the test tube-inverting method. For the pure CGP hydrogel, its gelation time was highest, around 40 min (Figure S6). After adding Alg into CGP, the gelation time of CGP/Alg tended to decrease to below 30 min with the increment of the Alg contents, due to the hydrogen-bond interaction and electrostatic interaction between CS and Alg. Taking into account the appropriate gelation time and mechanical stability in vivo, the weight ratio of CGP: Alg at 3: 1 and the gelation time of 26 min were chosen for the further experiments. When DA was introduced into Alg, CGP/Alg-DA1 took about 29 min to form gel, a slightly longer than the CGP/Alg hydrogel (Figure 2b), due to few DA groups destroyed the interactions between CS and Alg. Further increasing the DA composition, the gelation time then gradually decreased, indicating the interactions of the hydrogel became stronger again. With high DA composition, the interactions among the polymers became strong enough to form more crosslinking points, resulting in quick gelation. Interestingly, in the presence of AuNRs, the gelation times of the CGP/Alg-DA/AuNRs hydrogel with different DA contents were 19 ± 1.1, 12 ± 0.7 and 8 ± 0.5 min, respectively, much quicker than the CGP/Alg-DA hydrogels. This phenomenon might be attributed to the strong interactions such as hydrogen-bonds and covalent-bonds among DA groups in both Alg-DA and AuNRs-PDA. Accordingly, the gelation time of hydrogel largely depended on the intermolecular interactions. Moreover, the DA group was one of main factors to adjust the hydrogel with an appropriate gelation time for not only easily operating but also satisfying the requirement of rapid prototyping in vivo.

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Figure 2. Gelation behaviors of CGP/Alg-DA solution. (a) Temperature dependence of dynamic moduli, G’ and G’’, in the presence of AuNRs-PDA. Cross-over of G’ and G” refers to sol-gel transition point. (b) Gelation times of the hydrogels with various DA contents in the absence and presence of AuNRs-PDA. II. Stability and distribution of AuNRs in CGP/Alg-DA/AuNRs hydrogel To assure high and long-time PTT efficacy, AuNRs should keep stable in hydrogel. Thus, the stability of AuNRs in hydrogel was detected by immersing the hydrogel in PBS buffer solution (pH 7.4) at 37 C for 1 and 15 days. Results from UV-vis spectrum of the PBS solution showed no special absorbance of AuNRs (Figure S7), indicating no AuNRs leaked from CGP/Alg-DA/AuNRs composite hydrogel even after 15 days. Digital photographs showed that the CGP/Alg-DA/AuNRs hydrogel kept their shapes well even after 15 days of immersing (Figure 3a). Additionally, distribution of AuNRs in the hydrogel has significantly influenced on the PTT efficacy. Therefore, the EDS element mapping was used to observe the distribution of AuNRs in the CGP/AlgDA/AuNRs hydrogel. From Figure 3b, Au element was distributed homogeneously 18


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without aggregation in the hydrogel (CGP/Alg-DA5/AuNRs as an example), which can avoid the localized overheating efficiently and keep the scaffold’s stability. Compression strength and biodegradation behaviors of the hydrogels also indicated

the no leakage of AuNRs (Figure S8). These results suggested that AuNRs could be stable in the CGP/Alg-DA/AuNRs hydrogels, which mainly contributed to the strong interactions of DA and AuNRs-PDA.40

(a)

(b)

Figure 3. (a) Photographs of CGP/Alg-DA/AuNRs hydrogels after 15 days of immersing in PBS buffer solution (pH 7.4) and (b) EDS element mapping of Au in CGP/Alg-DA/AuNRs hydrogel.

3.3 Photothermal effects of CGP/Alg-DA/AuNRs hydrogel It is well known that AuNRs show strong absorption in NIR region and then translate the energy to heat, exhibiting excellent photothermal conversion efficiency.36 Then, the photothermal conversion efficiency of CGP/Alg-DA/AuNRs composite 19


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hydrogel containing the same amounts of AuNRs was detected using NIR laser irradiation (808 nm) with different setting powers (1.0, 1.5 and 2.0 W/cm2) for 6 min. Figure 4 showed the thermographic images and temperature changes of the hydrogels with various irradiation times. In Figure 4a, PBS buffer solution showed negligible temperature change after NIR laser irradiation for 6 min. While the temperature of the CGP/Alg-DA/AuNRs hydrogel increased significantly and rapidly. Moreover, the higher setting power could lead to a rapider heating efficiency for the samples. The detailed temperature changes with irradiation time were showed in Figure 4b. When the CGP/Alg-DA/AuNRs hydrogel was exposed under NIR laser at lowest power (1.0 W/cm2), the temperature raised to 52 oC after 6 min. With higher power at 1.5 W/cm2 and 2.0 W/cm2, the temperature elevated to around 60 oC and 77 oC, respectively. Since the temperature above 50 oC is sufficient to kill cancer cells41, the NIR laser power setting at 1.5 W/cm2 was used in the following research. (a)

t=0Ss t=0

t=30 s t=60 t=360Ss t=30 S t=60 sS t=180 t=180 Ss t=360

(b)

1.0W/cm W/cm22 1.0

100

CGP/Alg-DA/AuNRs 2.0 W/cm2 PBS 2.0 W/cm2 CGP/Alg-DA/AuNRs 1.5 W/cm2 PBS 1.5 W/cm2 CGP/Alg-DA/AuNRs 1.0 W/cm2 PBS 1.0 W/cm2

90 80

Temperature (℃ )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.5 1.5W/cm W/cm22

2.0 2.0W/cm W/cm22

70 60 50 40 30 20

Figure

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Photothermal

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Thermographic images of hydrogel irradiated at 808 nm for 6 min with different 20


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powers. Upper images: PBS buffer solution (pH 7.4), lower images: CGP/AlgDA/AuNRs hydrogel. (b) Temperature elevation curves of hydrogels under different laser powers with irradiations at 808 nm up to 360 seconds. In order to kill tumor cells completely, the AuNRs based composite materials are generally required to be immobilized in the tumor site for long time and kept repeated PTT efficacy under laser irradiation. Thus, the repeatability of photothermal conversion capability of CGP/Alg-DA/AuNRs hydrogel was investigated via 10 times of repeatedly exposing the composite hydrogel to NIR laser (1.5 W/cm2, 6 min). The temperature of the CGP/Alg-DA/AuNRs composite hydrogel could always increase to 55~60 oC after each NIR laser irradiation, and the temperature increment could maintain at least 10 times with negligible decrement after multiple laser irradiations (Figure 5b). These results demonstrated that the multiple PTTs based on one-dose injection of CGP/Alg-DA/AuNRs hydrogel would be promising for inhibiting tumor growth efficiently in vivo.

Figure 5. Temperatures of CGP/Alg-DA/AuNRs hydrogel after multiple laser irradiations (808 nm, 1.50 W/cm2, 6 min). (a) Thermographic images and (b) temperatures as a result of repeating irradiations for 360 seconds. 21



3.4 In vitro cytotoxicity I. Biocompatibility to the normal cells Cytotoxicity of the hydrogel is an important considered factor for photothermal cancer therapy. In order to verify the non-cytotoxicity of the composite hydrogel, the biocompatibility of hydrogel was estimated using L929 cells as the model normal cells via MTT assay method. In Figure 6a, the cell viabilities of L929 were 98.1%, 90.1%, 93.7%, and 96.1% after incubated in the CGP/Alg, CGP/Alg-DA1/AuNRs, CGP/AlgDA3/AuNRs, and CGP/Alg-DA5/AuNRs composite hydrogel for 24 h at 37 oC, respectively. Accordingly, all the hydrogels have good biocompatibility to the normal cells. Morphologies of cells were also observed with fluorescence microscopy after incubation with CGP/Alg-DA/AuNRs hydrogel extract liquid for 24 h at 37 oC (Figure 6b). The cells exhibited good growth with a fibrous structure. Thus, these composite hydrogels with good biocompatibility would not affect growth and proliferation of the normal cells and could be a potential candidate in the biomedical fields.

Figure 6. In vitro cytotoxicity test. (a) Cell viability and (b) fluorescence images of L929 cells incubated with CGP/Alg-DA/AuNRs composite hydrogel extract liquid for 22


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24 h at 37 oC. The insert scale bars are 50 μm. II. Photothermal therapy on tumor cells in vitro For assessing the photothermal ablation of the CGP/Alg-DA/AuNRs hydrogel on tumor cells, the hydrogels accompanied with HepG2 cells (as the model tumor cells) constructs were exposed to NIR laser irradiation at various powers, and the cell viabilities were then analyzed by MTT method, as shown in Figure 7. In the case of the hydrogel without NIR irradiation, the HepG2 cells could grow well (Figure 7a). As expected, after exposed to NIR laser, the cell viability decreased with the increment of laser power. Adjusting the irradiation power at 1.0 W/cm2, the HepG2 cell viability was still high, above 40% after 6 min of irradiation, suggesting lower PTT efficacy toward HepG2 cells. However, with higher irradiation power, most of the tumor cells were killed by the hydrogels (Figure 7a). As mentioned in Figure 5, the temperature of the hydrogel could increase up even to 59.7 oC with high power, in which cells happened to necrocytosis easily.42 These results further confirmed that AuNRs could absorb energy sustainably, and then translated it into high heat energy to kill tumor cells. Meanwhile, using CGP/Alg-DA5/AuNRs as an example, inhibitory effect of composite hydrogel on tumor cells under multiple NIR irradiation was also evaluated. As shown in Figure 7b, the CGP/Alg-DA and CGP/Alg-DA/AuNRs composite hydrogel showed no effect on HepG2 cells without laser irradiation. With laser irradiation, the CGP/Alg-DA hydrogel showed high cell viabilities of HepG2 cells, even after three times of laser irradiation, meaning no PTT property of CGP and Alg23



DA polymers. Whereas, the cell viabilities declined sharply after cultured with the CGP/Alg-DA/AuNRs hydrogel under NIR irradiation. Repeated irradiation of the hydrogels for 2 and 3 times, the cell viabilities further decreased to 15.2 % and 9.0 %, respectively. Thus, HepG2 cells could be damaged more severely with more NIR irradiation times. All these results demonstrated that the CGP/Alg-DA/AuNRs composite hydrogels had better PTT efficacy and could be promising in tumor control. Moreover, the tumor cell necrosis efficiency could be controlled by the higher irradiation power, longer NIR irradiation time and repeated NIR irradiation times.

Figure 7. Cell viability of HepG2 tumor cells incubated with the CGP/Alg-DA/AuNRs hydrogel for 24 h at 37 oC (a) after exposed to laser irradiation at 808 nm with different powers and (b) after various times of laser irradiation (808 nm, 1.5 W/cm2) for each 6 min. 3.5 In vivo anti-tumor activity In vivo anti-tumor efficacy of the CGP/Alg-DA/AuNRs hydrogel with photothermal therapy was also investigated by injecting the hydrogel into the xenograft 24


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HepG2 tumor model. As shown in Figure 8a, the temperature of the tumor site in saline group (as a control) had no change. Then, under NIR irradiation for 3 min (in the in vivo experiments, irradiation time was shorten to 3 min to decrease some unforeseeable injuries, induced by high temperature), the temperature of the tumor site in the saline group rose from 33.5 oC to 38.0 oC, without significant change treated with NIR irradiation. The injectable composite hydrogel without NIR irradiation also showed negligible change. With the help of CGP/Alg-DA/AuNRs hydrogel under NIR irradiation in the tumor site for 3 min, the temperature rose significantly to 62.3 oC. Further repeatedly irradiating the tumor site, the temperature could maintain at 55~65 oC

for each NIR irradiation (Figure 8b). These results demonstrated that the CGP/Alg-

DA/AuNRs composite hydrogel could efficiently immobilize AuNRs in the tumor site to ensure the repeated PTTs, attributing to the strong interactions between AuNRs and hydrogel induced high mechanical property. This stability of AuNRs led the composite hydrogel to effectively suppress the tumor growth for long time. In order to investigated effect of PTT on the tumor growth in vivo, the tumor was taken out from the mice after predetermined time, as shown in Figure 8c. Then, the tumor volume in each group of mouse was also calculated by measured its length and width. As shown in Figure 8d, the tumor of mice in saline group grew rapidly and unrestrainedly, and the average size in this group was approximately 48.1 times than the initial size after 21 days. Similar results were also observed in the saline group even with NIR irradiation and in the hydrogel group without NIR irradiation. Accordingly, 25



the tumor could not be inhibited or killed by saline or the hydrogel directly because of no PTT property in these groups. In the group treated with the CGP/Alg-DA/AuNRs hydrogel under NIR irradiation for 3 min in each period time, the tumor size became bigger very slowly, indicating the tumor growth was significantly suppressed after the repeated PTTs (Figure 8d). It was worth mentioning that the body weight of the mice maintained the normal growth characteristics without sharp reduction or increase during 21 days (Figure S9), suggesting that multiple PTTs were tolerated well without any side effects. Therefore, the CGP/Alg-DA/AuNRs hydrogel could efficiently suppress the tumor growth by multiple PTTs and achieve the “one-dose injection, multiple PTTs”, which could largely reduce the patient pain but increase the treatment efficacy. Although the tumor hadn’t been completely suppress growth or killed in vivo, the hydrogel already showed high PTT efficacy in absence of anti-tumor drugs. Encapsulating anti-tumor drugs into the hydrogel is now processing to wipe out the tumor cells.

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Figure 8. In vivo anti-tumor activity of composite hydrogel in HepG2-bearing Balb/c mice. Infrared thermal images of (a) mice with various treatments and (b) the mice injected CGP/Alg-DA/AuNRs hydrogel under multiple NIR irradiation. (c) Photograph of the excised tumors after 21 days (І. saline, Ⅱ. Saline with NIR, Ⅲ. CGP/AlgDA/AuNRs, and Ⅳ. CGP/Alg-DA/AuNRs with NIR) and (d) changes in relative tumor volume of HepG2-bearing Balb/c mice upon various treatments, calculated from c. Histopathological analyses of tumor tissues were carried out for further evaluating the anti-tumor efficacies of the CGP/Alg-DA/AuNRs hydrogel, as shown in Figure 9. In saline group and saline with NIR irradiation group, the tumor cells grew and proliferated well which show normal cellular morphologies. Slight inflammation was observed in the hydrogel without irradiation group, possibly due to the implant of foreign materials. Compared with above three groups, massive cell necrosis was 27



observed in the tumor sections when the mice were administered the composite hydrogel with NIR irradiation, indicating the high tumor suppression efficiency. All of the above data demonstrated that the CGP/Alg-DA/AuNRs hydrogel could be potentially applied as a long-acting implant for effective tumor treatment in vivo.

Figure 9. H&E stained tumor sections after treated by saline without (a) and with (b) NIR irradiation and CGP/Alg-DA/AuNRs without (c) and with (d) NIR irradiation.

Conclusion We developed an injectable photothermal hydrogel composed of hydrogel matrix of CGP, Alg-DA and AuNRs PDA. The hydrogels were injectable under the function of CGP and stable in the hydrogel state via the interactions of Alg-DA with both chitosan and AuNRs PDA. The sol-gel transition of the composite hydrogel occurred at around 37 oC, and the transition time could be controlled by adjusting the mass ratio of CGP and Alg-DA. The AuNRs were stably immobilized in the hydrogel without leakage for a long time. The composite hydrogel quickly and repeatedly showed the local heating behavior under cycling of NIR laser irradiation at 808 nm, which can be used for multiple PTTs. The cytotoxicity test in vitro showed that composite hydrogel possessed a good biocompatibility, but the viability to tumor cells showed an excellent 28


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killing efficiency to tumor cells. The in vivo anti-tumor test demonstrated that the composite hydrogel could suppress tumor growth effectively without any other side effects under multiple PTTs. Therefore, the injectable composite hydrogel has great potential for the stable and long-term function of repeatedly PTT, achieving the requirement of “one-dose injection, multiple PTTs”.

Acknowledgements This study was supported by the National Nature Science Foundation of China (No.21571084), the Natural Science Foundation of Jiangsu Province (Grants No BK20181349 and BK20150135), National First-Class Discipline Program of Light Industry Technology and Engineering (LIFE2018-19), and MOE & SAFEA for the 111 Project (B13025). We would like to thank Xiang Gao for providing SH-PEG-CH3.

Supporting Information. Characterizations of AuNRs-PDA, synthesis and characterization of dopamine-modified alginate (Alg-DA), and characterization of the hydrogels. This material is available free of charge via the Internet at http://pubs.acs.org.

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Injectable and Near-Infrared-Responsive Hydrogels Encapsulating

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