In Situ Fabrication of Intelligent Photothermal Indocyanine Green

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Biological and Medical Applications of Materials and Interfaces

In Situ Fabrication of Intelligent Photothermal Indocyanine Green-Alginate Hydrogel for Localized Tumor Ablation Haiyan Pan, Cai Zhang, Tingting Wang, Jiaxi Chen, and Shao-Kai Sun ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16517 • Publication Date (Web): 25 Dec 2018 Downloaded from http://pubs.acs.org on December 26, 2018

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In Situ Fabrication of Intelligent Photothermal Indocyanine Green-Alginate Hydrogel for Localized Tumor Ablation Haiyan Pan,‡ Cai Zhang,§ Tingting Wang,† Jiaxi Chen,† and Shao-Kai Sun,*,† †School

of Medical Imaging, Tianjin Medical University, Tianjin 300203, China

‡Department

of Radiology, Tianjin Medical University General Hospital, Tianjin

300052, China. §College

of Chemistry, Research Center for Analytical Sciences, State Key

Laboratory of Medicinal Chemical Biology (Nankai University), Tianjin Key Laboratory of Molecular Recognition and Biosensing, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, 94 Weijin Road, Tianjin 300071, China KEYWORDS: ICG, alginate, hydrogel, photothermal therapy, tumor

ABSTRACT:

Simplifying

synthesis

and

administration

process,

improving

photothermal agents’ accumulation in tumors, and ensuring excellent biocompatibility and biodegradability are the key to promoting the clinical application of photo-thermal therapy. However, current photothermal agents have great difficulties in meeting the requirements of clinic drugs from synthesis to administration. Herein, 1 ACS Paragon Plus Environment

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we reported in situ formation of a Ca2+/Mg2+ stimuli-responsive ICG-alginate hydrogel in vivo for localized tumor photothermal therapy. ICG-alginate hydrogel can form by the simple introduction of Ca2+/Mg2+ into ICG-alginate solution in vitro, and the widely distributed divalent cations in organization in vivo enabled the in situ fabrication of ICG-alginate hydrogel without the leakage of any agents by simple injection of ICG-alginate solution into the body of mice. The as-prepared ICG-alginate hydrogel not only owns good photothermal therapy efficacy and excellent biocompatibility, but also exhibits strong ICG fixation ability, greatly benefiting the high photothermal agents’ accumulation and minimizing the potential side effects induced by the diffusion of ICG to surrounding tissues. The in situ fabricated ICG-alginate hydrogel was applied in high-efficient PTT in vivo without obvious side effects successfully. Besides, the precursor of the hydrogel, ICG and alginate, can be stored in a stable solid form, and only simple mixing and noninvasive injection are needed to achieve PTT in vivo. The proposed in situ gelation strategy using biocompatible components lays down a simple and mild way for the fabrication of high-performance PTT agents with the superiors in the aspects of synthesis, storage, transportation and clinic administration.

1. INTRODUCTION Photothermal therapy (PTT), which converts near-infrared (NIR) light energy to heat based on PTT agents, is an attractively noninvasive approach with the advantages of 2 ACS Paragon Plus Environment

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high selectivity, powerful tumor ablation, and minimal damage with normal tissues.1,2 Current PTT agents mainly include nanostructures,1,2 hydrogels3-19 and organic dyes20-22 with strong NIR absorption. Up to now, various nanoparticles, such as gold nanostructures,23,24 nanocarbons,25-28 transition metal sulfide/oxide nanoparticles29-33 and organic nanoparticles,34-39 hydrogel containing various PTT agents3-13, and others (phosphorene,40 antimonene,41-43 borophene44 and MXene45), have been widely applied

for

PTT.

Despite

the

high

therapeutic

efficacy,

their

inherent

non-biodegradable components and potential long-term toxicity of the nanoparticles hindered further clinical translation.1,2 Organic dyes with definite structure and physicochemical properties are another type of PPT agents, however, the great safety concerns make most organic dyes difficult in clinic applications apart from indocyanine green (ICG).20-22 ICG with intense NIR-absorbing and fluorescence properties is an ideal theranostic agent approved by FDA due to its low toxicity. ICG is widely used in clinic for fluorescent imaging, cardiac output analysis, plasma volume and liver function assessment. Recently, ICG and ICG-loaded nanostructures have been explored for PTT in vivo.46-51 However, most intravenously injected ICG-based PTT agents suffer from limited ICG accumulation in tumors, and potential side effects resulted from unspecific distribution in various organs.20-22,46-51 While intratumorally injected ICG-based agents undergo the obstacle of the strong pressure in solid tumor, leading to leakage of ICG along the path of the needle and limiting the injected dose of agents. Ideally, theranostic PTT agents should be guided by following criteria: 1) 3 ACS Paragon Plus Environment

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ease of preparation, storage, transportation and administration; 2) high accumulation capability in tumors; 3) excellent biocompatibility and biodegradability; 4) cost and time effectiveness. Therefore, it is of great significance to develop novel strategies to fabricate an ideal PTT agent using ICG as the only FDA-approved dye. Sodium alginate, a kind of polysaccharide carbohydrate extracted from brown seaweed, is widely applied in clinic and food industry with their inherent biodegradability and biocompatibility.52,53 Sodium alginate consists guluronic (G) and mannuronic (M) acid regions, and divalent cations such as Ca2+/Mg2+ prefer to bind the G-blocks in the linear copolymer to form the structure of “egg-box”, thereby obtaining a hydrogel in a ultrasimple way.54,55 Alginate hydrogels with superior biocompatibility and biodegradability are widely used in medical treatment due to the structural similarity in extracellular matrixes of the living tissues,56-68 such as the carrier of drugs,62,66 cell transplantation57,63,67 and tissue engineering56,59,65,68 compared to other hydrogels.3-13,61,69,70 The body fluid circulation is a process of dynamic balance and the ions in the body are constantly updated but relatively constant,

which

provides

great

opportunities

for

in

situ

formation

of

Ca2+/Mg2+-responsive alginate hydrogel in vivo.18 Thus, the integration of ICG and alginate hydrogel with unique advantages makes it possible to build a localized ICG depot for improving ICG accumulation in tumors and reducing the side effects induced by the leakage of ICG. Herein, we show in situ fabrication of photothermal ICG-alginate hydrogel for localized tumor ablation in an ultrafacile way. The precursor of ICG-alginate hydrogel 4 ACS Paragon Plus Environment

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was synthesized by simply mixing ICG and alginate at room temperature. The introduction of Ca2+/Mg2+ enables the transformation of ICG-alginate from solution to hydrogel easily, and ICG distributes in the hydrogel homogeneously. In vivo gelation study confirmed ICG-alginate hydrogel could generate quickly as soon as the ICG-alginate solution was injected into the body of mice. The in situ fabrication process of hydrogel can avoid the leakage of ICG along the path of the needle, improve the accumulation of ICG in tumors and minimize the diffusion of ICG to surrounding tissues. The ICG-alginate solution exhibits intense NIR absorption and fluorescence as same as free ICG, and the formed ICG-alginate hydrogel own excellent photothermal heating ability. In vitro and in vivo toxicity assessment demonstrated the favorable biocompatibility of the hydrogel due to the inherent biosafety of ICG and alginate. The in situ fabricated ICG-alginate hydrogel was applied in localized PTT in vitro and in vivo successfully, and the fluorescence of ICG enables monitoring the distribution of ICG accurately. Besides, ICG and alginate can be stored as a stable solid form for a long time, and only the simple mixing in water and noninvasive injection are needed to perform for high-efficient localized PPT of tumors, greatly benefiting the preparation, storage, transportation and potential clinic usage. Scheme 1. (A) Schematic diagram of transformation from alginate solution to hydrogel. (B) Stimuli-responsive photothermal ICG-alginate hydrogel for localized tumor ablation.

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2. RESULTS AND DISCUSSION 2.1. Synthesis of ICG-Alginate Hydrogel in Vitro. The viscosity of ICG-alginate hydrogels highly depends on the concentrations of alginate and divalent cations. Considering the constant concentration of Ca2+/Mg2+ in vivo, various concentrations of alginate solutions were employed to tune the viscosity of hydrogel (Figure S1). High viscosity benefits the loading of ICG in ICG-alginate hydrogel, but is adverse to syringeability. Therefore, 20 mg/mL alginate as the optimum concentration was chosen to ensure the high ICG loading efficiency and good syringeability. The ICG-alginate hydrogel was synthesized via a simple mixing method. Briefly, 35 mg alginate and 7.5 mg ICG were mixed in 1.75 mL H2O and stirred for 30 min. The ICG-alginate solution is homogeneous and stable, exhibiting excellent syringeability compared to hydrogels. In order to access the gelation properties in vitro, the well-dispersed ICG-alginate solution (60 μL, 4.28 mg mL-1 of ICG, 20 mg mL-1 of alginate) was injected into 8 mL of Ca2+/Mg2+ solution in a meter glass. The 6 ACS Paragon Plus Environment

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concentrations of Ca2+ and Mg2+ were fixed to be 1.8 mmol L-1 and 1.5 mmol L-1 to mimic the extracellular microenvironment in the living tissue.18 The ICG-alginate hydrogel were immediately formed while an ICG-alginate aqueous solution was injected into the Ca2+/Mg2+ solution (Figure 1). We further investigated the ICG loading capacity of ICG-alginate hydrogel, which could reach as high as 10 mg/mL ICG in the hydrogel (Figure S2). The formed uniform hydrogel enables encapsulating ICG and overcoming the burst release of ICG efficiently (Figure S3).

Figure 1. Photos of ICG and ICG-alginate fluid after injected into Ca2+ and Mg2+ solution. 2.2. Characterization of ICG-alginate Solution. The UV-vis-NIR absorption and fluorescence of ICG-alginate solution were similar to ICG solution, indicating negletable interaction between ICG and alginate (Figure 2A-D). The fluorescence spectra of ICG-alginate solution showed a maximum emission at 813 nm, benefiting in vivo fluorescence imaging with high tissue penetration depth. The strong NIR absorption peaked at 808 nm linearly increases with the concentrations of ICG in ICG-alginate solution with a fixed alginate concentration, which demonstrated the high photothermal potential with an 808 nm laser. The UV-vis-NIR absorption and 7 ACS Paragon Plus Environment

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fluorescence spectra of ICG-alginate hydrogel indicated the formed hydrogel also exhibited a strong NIR absorption and an intense fluorescent emission due to effective encapsulation of ICG in the hydrogel (Figure S4). SEM images showed the morphology of lyophilized ICG-alginate hydrogel (Figure S5).

Figure 2. (A) and (B) The fluorescence spectra of different concentrations of ICG and ICG-alginate solution. (C) and (D) The UV-vis-NIR absorption spectra of ICG and ICG-alginate solution. 2.3. Photothermal Performance of ICG-alginate Hydrogel. In order to evaluate the photothermal performance of the ICG-alginate hydrogel, ICG-alginate gel prepared by adding different volumes of ICG-alginate solution to Ca2+/Mg2+ solution were irradiated with an 808 nm laser for 7 mins at a power density of 0.9 W cm-2. Figure 3 revealed the increased temperature of ICG-alginate hydrogel varied from 12.4 C to 21.4 C with the volumes of ICG-alginate solution changed from 10 μL to 100 μL. In contrast, the temperature of Ca2+/Mg2+ solution pure water was only raised 8 ACS Paragon Plus Environment

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5.8 C. Besides, IR thermal photos were taken to monitor the temperature change of the solution during the irradiation process, further demonstrating the favorable photothermal performance of the ICG-alginate hydrogel (Figure 3). We found 660 nm or 980 nm laser irradiation also can lead to photothermal heating effect of ICG-alginate hydrogel. However, 660 nm laser irradiation suffers from lower photothermal heating efficacy compared to 808 nm laser irradiation and 980 nm laser illumination results in a strong undesired photothermal heating of pure water due to the intense absorption of water at 980 nm.71 Therefore, 808 nm laser irradiation is the most suitable light source for photothermal heating of ICG-alginate hydrogel (Figure S6 and Figure S7).

Figure 3. (A) IR thermal images of the ICG-alginate hydrogel with different volume, pure alginate hydrogel and water under NIR laser irradiation (0.9 W cm-2, 7 min). (B) Photothermal heating curves of the ICG-alginate hydrogel with different volume, pure alginate hydrogel and water with 808 nm laser irradiation (0.9 W cm-2, 7 min).

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2.4. Cytotoxicity of ICG-alginate Hydrogel. The cytotoxicity of ICG-alginate hydrogel was evaluated by a standard MTT assay with 4T1 cells. The cells were treated with ICG-alginate solution or preformation ICG-alginate hydrogel containing different concentrations of ICG. Figure 4A indicated the cell viability kept above 85% even when the concentration of ICG reached to 1 mg mL-1 in ICG-alginate solution, and the cell viability kept above 82% even when the concentration of ICG reached to 500 mg L-1 in preformation ICG-alginate hydrogel (Figure S8), demonstrating the excellent biocompatibility of in situ formation and preformation of ICG-alginate hydrogel and their great potential for biological applications. 2.5. Photothermal Therapy in Vitro. We then investigate the PTT capability of ICG-alginate hydrogel in vitro. 4T1 cells treated with ICG-alginate gel containing 200 mg L-1 of ICG were irradiated with an 808 nm laser at power densities of 0.3 W cm-2 or 1 W cm-2 for 5 min. The viabilities of 4T1 cells treated with the hydrogel and laser irradiation are only 5.20 % and 4.84 % at the laser power densities of 0.3 W cm-2 and 1 W cm-2, respectively (Figure 4B). While the cells without any treatments and treated with the hydrogel or laser irradiation alone all showed a neglectable cell death. Moreover, calcein-AM/PI dual staining was performed to show the cell viability directly. The fluorescent images indicated few live cells can be seen after the combined treatments of ICG-alginate hydrogel and laser irradiation (Figure 4C). In contrast, majority of cells were alive with other treatments. These results indicated that ICG-alginate hydrogel possessed excellent photothermal therapy ability.

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Figure 4. (A) Relative cell viabilities of 4T1 cells treated with different concentrations of ICG-alginate hydrogel. (B) Relative cell viabilities after treated with ICG-alginate hydrogel and laser irradiation at the power density of 0.3 W cm-2 or 1 W cm-2. (C)

4T1 cells with different treatments were obained with an inverted

luminescence microscope, and live and dead cells were stained to be green and red respectively with calcein-AM/PI. 2.6. In Situ Fabrication of ICG-alginate Hydrogel in Vivo. We reasonably assumed that the injection of ICG-alginate solution into the living organism will lead to the formation of ICG-alginate hydrogel due to the presence of appropriate concentration of Ca2+/Mg2+ in biological fluids. Like many PTT agents solution, ICG 11 ACS Paragon Plus Environment

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solution leaked along the path of the needle from the pinhole because of the enormous pressure in solid tumor when free ICG was injected directly into tumors. However, there was not any leakage of solution from the injection sites after the administration of ICG-alginate solution due to the in situ formation of ICG-alginate hydrogel in vivo (Figure 5A). To evaluate the feasibility of in situ fabrication of ICG-alginate hydrogel in vivo, four Kunming mice were subcutaneously injected with 60 μL ICG-alginate solution (ICG 4.28 mg mL-1, alginate 20 mg mL-1) was injected on the one side and 60 μL ICG solution (4.28 mg mL-1) on the other side as the control group. Moreover, the state of injected solutions was monitored by ex vivo imaging at different time points. Figure 5B indicated the formation of ICG-alginate hydrogel, and green color of ICG distributed into the hydrogel, avoiding the self-aggregation of ICG and favoring the uniform heating triggered by laser illumination. Both ICG solution and ICG-alginate hydrogel was gradually metabolized along with the time, while the metabolism rate of ICG-alginate hydrogel was obviously slower than the bare ICG solution. These results demonstrated ICG-Alginate hydrogel can be easily in situ formed by the injection of ICG-Alginate hydrogel without any leakage, proving a stable ICG depot for PTT in vivo.

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Figure 5. (A) The pictures of 4T1 bearing mice were recorded after injecting 60 µL of ICG solution or ICG-alginate solution for 0.5 h. (ICG 4.28 mg mL-1, alginate 20 mg mL-1). (B) Photos showing the metabolism of ICG fluid and ICG-alginate hydrogel in vivo. (C) The dynamic fluorescence imaging of ICG-alginate hydrogel and ICG solution in vivo. 2.7. Fluorescence Imaging of ICG-alginate Hydrogel in Vivo. To investigate the ICG fixation capability of ICG-alginate hydrogel, fluorescent imaging of mice was performed after the administration of ICG and ICG- alginate solution (Figure 5C). Two groups of Kunming mice were subcutaneously administrated with 60 μL of ICG-alginate solution (ICG 4.28 mg mL-1, alginate 20 mg mL-1) and 60 μL of ICG 13 ACS Paragon Plus Environment

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solution (4.28 mg mL-1) respectively. Fluorescent images indicated free ICG was distributed throughout the body quickly due to the continuous body fluid circulation. In contrast, the ICG in ICG-alginate hydrogel penetrated to bloodstream and surrounding tissues slowly due to strong fixation capability of the hydrogel. The high ICG accumulation capacity of the in situ formed hydrogel shows great potential in improving the PTT efficacy and minimizing the side effects induced by the fast leakage of PTT agents. 2.8. Toxicity of ICG-alginate Hydrogel in Vivo. Before the in vivo PTT application, the in vivo toxicity of ICG-alginate was evaluated via body weight change monitoring and histopathological analysis (Figure 6A). The Kunming mice were injected ICG-alginate solution (ICG 4.28 mg mL-1, alginate 20 mg mL-1) or 60 μL of PBS as control or subcutaneously, and the mice treated with the hydrogel showed a similar increase tendency in weight in comparison with the control group (Figure 6B). Hematoxylin-eosin staining analysis of the main organs (liver, heart, spleen, kidney, and lung) revealed there were no obvious histopathological lesions for mice in both experimental and control groups. In physiological condition, ion replacement and the elution of the cross-linking calcium cation results in osmotic swelling of Ca-alginate hydrogel, leading to increased pore size and destabilization and rupture of the hydrogel. Alginate is also a biodegradable polymer as the backbone of alginate can be degraded via hydrolysis of the glycosidic bonds. Therefore, the proposed hydrogel can be biodegraded in vivo base on these mechanisms, ensuring

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the biosafety of the hydrogel.52-55 The in vivo toxicity investigation demonstrated the excellent biocompatibility of ICG-alginate hydrogel in vivo.

Figure 6. (A) Histopathological images of the major organs in mice treated with ICG-alginate solution (60 μL) at different time points. (B) Weight changes of the mice after injection. (C) IR thermal images of the mice before and after the treatments of PBS or ICG-alginate solution (60 μL) and laser irradiation (0.7 W cm-2 for 15 min, n=3). (D) Tumor volume changes of mice after treatment. (E) The photos of mice at different time points before and after treated with PBS or ICG-alginate solution (60 μL) plus laser irradiation (0.7 W cm-2). 2.9. Photothermal Therapy in Vivo Using in Situ Fabricated ICG-alginate Hydrogel. Inspired by the excellent performance of photothermal ability and biocompatibility, the ICG-alginate hydrogel was employed in in vivo PTT. The 4T1 15 ACS Paragon Plus Environment

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tumor-bearing mice were injected 60 μL of PBS or ICG-alginate (ICG: 0, 0.1, 1 and 4.28 mg mL-1, alginate: 20 mg mL-1) solution intratumorally, followed by the 808 nm laser irradiation at a power density of 0.7 W cm-2 for 15 min. The temperature of tumors was monitored by an IR thermal camera during the irradiation process. In order to achieve obvious PTT efficacy and minimize the administration dose, ICG-alginate solution containing 4.28 mg mL-1 ICG was chosen as the optimum PTT agent for tumor ablation (Figure S9 and Figure S10). Figure 6C indicated the temperature of tumors the mice injected ICG-alginate solution (ICG: 4.28 mg mL-1, alginate: 20 mg mL-1) exhibited a remarkable increase, while the mice in the control group only have a much slower temperature. Furthermore, the sizes of tumors were measured with a vernier caliper every day after various treatments (Figure 6D). In addition, the photos of tumors were taken every day to observe the tumor change visually in a week. Figure 6E showed the tumors of mice treated with PBS and laser irradiation kept growing all the time, while the tumors of mice treated with ICG-alginate solution almost disappeared in a week. These results obviously demonstrated that ICG-alginate hydrogel with strong ICG fixation capability can serve as an excellent photothermal agent with outstanding biocompatibility. 3. CONCLUSION In summary, we proposed an in situ gelation strategy to synthesis ICG-alginate hydrogel in vivo for PTT, uniting the advantages of ultrasimple synthesis and administration procedures, excellent biocompatibility, avoiding the leakage from injection sites, improving the accumulation in tumors, minimized side effects induced 16 ACS Paragon Plus Environment

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by the metabolized from tumors. The ICG-alginate hydrogel can be gen erated by adding Ca2+/Mg2+ into ICG-alginate solution in vitro or simply injecting ICG-alginate solution into organisms without any leakage of any agents. The strong ICG fixed ability of the hydrogel was demonstrated in vitro and in vivo, greatly benefiting the highly accumulating of ICG in tumors and minimizing the side effects from the fast metabolization. The toxicity studies in vitro and in vivo confirmed In vitro and in vivo its favorable biocompatibility derived from the safety of ICG and alginate. The in situ fabricated ICG-alginate hydrogel with outstanding photothermal performance was applied in tumor PTT in vitro and in vivo successfully. Moreover, the convenient storage of ICG and alginate in a solid form and simple synthesis and administration process make in situ formed ICG-alginate hydrogel promising in PTT in vivo. We will investigate the clinic transformation of ICG-alginate hydrogel with unique superiorities in future. The in situ fabrication of photothermal hydrogel using biocompatible components lays down a promising way for revolutionizing PTT towards clinic transformation from synthesis of administration. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.xxxxxxx. Optimization of alginate in ICG-alginate hydrogel; ICG loading capacity of ICG-alginate hydrogel; the release of ICG from ICG-alginate hydrogel in vitro; SEM 17 ACS Paragon Plus Environment

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of lyophilized ICG-alginate hydrogel; photothermal performance of ICG-alginate hydrogel irradiated by a 660 nm laser; photothermal performance of ICG-alginate hydrogel irradiated by a 980 nm laser; Cytotoxicity of preformation ICG-alginate hydrogel; optimization of ICG in ICG-alginate hydrogel. AUTHOR INFORMATION Corresponding Author *[email protected]. ORCID Shao-Kai Sun: 0000-0001-6136-9969 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grants 21435001 and 21874101) and the Natural Science Foundation of Tianjin City (No. 18JCYBJC20800). REFERENCES (1) Lim, E. K.; Kim, T.; Paik, S.; Haam, S.; Huh, Y. M.; Lee, K. Nanomaterials for Theranostics: Recent Advances and Future Challenges. Chem. Rev. 2015, 115, 327-394.

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(2) Zhang, P.; Hu, C.; Ran, W.; Meng, J.; Yin, Q.; Li, Y. Recent Progress in Light-Triggered Nanotheranostics for Cancer Treatment. Theranostics 2016, 6, 948-968. (3) Conde, J.; Oliva, N.; Zhang, Y.; Artzi, N. Local Triple-Combination Therapy Results in Tumour Regression and Prevents Recurrence in a Colon Cancer Model. Nat. Mater. 2016, 15, 1128-1138. (4) Hsiao, C.-W.; Chuang, E.-Y.; Chen, H.-L.; Wan, D.; Korupalli, C.; Liao, Z.-X.; Chiu, Y.-L.; Chia, W.-T.; Lin, K.-J.; Sung, H.-W. Photothermal Tumor Ablation in Mice with Repeated Therapy Sessions Ssing NIR-Absorbing Micellar Hydrogels formed in situ. Biomaterials 2015, 56, 26-35. (5) Jin, H.; Wan, C.; Zou, Z.; Zhao, G.; Zhang, L.; Geng, Y.; Chen, T.; Huang, A.; Jiang, F.; Feng, J.-P.; Lovell, J. F.; Chen, J.; Wu, G.; Yang, K. Tumor Ablation and Therapeutic Immunity Induction by an Injectable Peptide Hydrogel. ACS Nano 2018, 12, 3295-3310. (6) Jin, H.; Zhao, G.; Hu, J.; Ren, Q.; Yang, K.; Wan, C.; Huang, A.; Li, P.; Feng, J.-P.; Chen, J.; Zou, Z. Melittin-Containing Hybrid Peptide Hydrogels for Enhanced Photothermal Therapy of Glioblastoma. ACS Appl. Mater. Interfaces 2017, 9, 25755-25766. (7) Lei, Z.; Zhou, Y.; Wu, P. Simultaneous Exfoliation and Functionalization of MoSe2 Nanosheets to Prepare "Smart" Nanocomposite Hydrogels with Tunable Dual Stimuli-Responsive Behavior. Small 2016, 12, 3112-3118.

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