In Situ Fabrication of Intelligent Photothermal Indocyanine Green−Alginate Hydrogel for Localized Tumor AblationHaiyan 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 (NankaiUniversity), Tianjin Key Laboratory of Molecular Recognition and Biosensing, and Collaborative Innovation Center of ChemicalScience and Engineering (Tianjin), Nankai University, 94 Weijin Road, Tianjin 300071, China

*S Supporting Information

ABSTRACT: Simplifying synthesis and administration process, improvingphotothermal agents’ accumulation in tumors, and ensuring excellentbiocompatibility and biodegradability are keys to promoting the clinicalapplication of photothermal therapy. However, current photothermal agentshave great difficulties in meeting the requirements of clinic drugs from synthesisto administration. Herein, we reported the in situ formation of a Ca2+/Mg2+

stimuli-responsive ICG−alginate hydrogel in vivo for localized tumorphotothermal therapy. An ICG−alginate hydrogel can form by the simpleintroduction of Ca2+/Mg2+ into ICG−alginate solution in vitro, and the widelydistributed divalent cations in organization in vivo enabled the in situfabrication of the ICG−alginate hydrogel without the leakage of any agents bysimple injection of ICG−alginate solution into the body of mice. The as-prepared ICG−alginate hydrogel not only owns good photothermal therapyefficacy and excellent biocompatibility but also exhibits strong ICG fixationability, greatly benefiting the high photothermal agents’ accumulation and minimizing the potential side effects induced by thediffusion of ICG to surrounding tissues. The in situ-fabricated ICG−alginate hydrogel was applied successfully in highly efficientPTT in vivo without obvious side effects. Besides, the precursor of the hydrogel, ICG and alginate, can be stored in a stable solidform, and only simple mixing and noninvasive injection are needed to achieve PTT in vivo. The proposed in situ gelationstrategy using biocompatible components lays down a simple and mild way for the fabrication of high-performance PTT agentswith the superiors in the aspects of synthesis, storage, transportation, and clinic administration.

KEYWORDS: ICG, alginate, hydrogel, photothermal therapy, tumor

1. INTRODUCTION

Photothermal therapy (PTT), which converts near-infrared(NIR) light energy to heat based on PTT agents, is anattractively noninvasive approach with the advantages of highselectivity, powerful tumor ablation, and minimal damage tonormal tissues.1,2 Current PTT agents mainly includenanostructures,1,2 hydrogels,3−18 and organic dyes19−21 withstrong NIR absorption. Up to now, various nanoparticles, suchas gold nanostructures,22,23 nanocarbons,24−27 transition metalsulfide/oxide nanoparticles,28−32 and organic nanopar-ticles,33−38 hydrogels containing various PTT agents,3−13 andothers (phosphorene,39 antimonene,40−42 borophene,43 andMXene44), have been widely applied for PTT. Despite the hightherapeutic efficacy, their inherent nonbiodegradable compo-nents and potential long-term toxicity of the nanoparticleshindered further clinical translation.1,2 Organic dyes withdefinite structure and physicochemical properties are anothertype of PPT agents; however, the great safety concerns make

most organic dyes difficult in clinic applications, apart fromindocyanine green (ICG).19−21

ICG with intense NIR-absorbing and fluorescence proper-ties is an ideal theranostic agent approved by the FDA becauseof its low toxicity. ICG is widely used in clinic for fluorescenceimaging, cardiac output analysis, plasma volume, and liverfunction assessment. Recently, ICG and ICG-loaded nano-structures have been explored for PTT in vivo.45−50 However,most intravenously injected ICG-based PTT agents suffer fromlimited ICG accumulation in tumors, and potential side effectsresulted from unspecific distribution in various or-gans,19−21,45−50 although intratumorally injected ICG-basedagents undergo the obstacle of the strong pressure in a solidtumor, leading to leakage of ICG along the path of the needleand limiting the injected dose of agents. Ideally, theranostic

Received: September 21, 2018Accepted: December 25, 2018Published: December 25, 2018

Research Article

www.acsami.orgCite This: ACS Appl. Mater. Interfaces 2019, 11, 2782−2789

© 2018 American Chemical Society 2782 DOI: 10.1021/acsami.8b16517ACS Appl. Mater. Interfaces 2019, 11, 2782−2789

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PTT agents should be guided by the following criteria: (1)ease of preparation, storage, transportation, and administra-tion; (2) high accumulation capability in tumors; (3) excellentbiocompatibility and biodegradability; (4) cost and timeeffectiveness. Therefore, it is of great significance to developnovel strategies to fabricate an ideal PTT agent using ICG asthe only FDA-approved dye.Sodium alginate, a kind of polysaccharide carbohydrate

extracted from brown seaweed, is widely applied in clinic andfood industry with their inherent biodegradability andbiocompatibility.51,52 Sodium alginate consists of guluronic(G) and mannuronic (M) acid regions, and divalent cationssuch as Ca2+/Mg2+ prefer to bind the G-blocks in the linearcopolymer to form the structure of “egg box”, therebyobtaining a hydrogel in an ultrasimple way.53,54 Alginatehydrogels with superior biocompatibility and biodegradabilityare widely used in medical treatment because of their structuralsimilarity with extracellular matrixes of the living tissues,55−67

such as the carrier of drugs,61,65 cell transplantation,56,62,66 andtissue engineering55,58,64,67 compared to other hydro-gels.3−13,60,68,69 The body fluid circulation is a process ofdynamic balance, and the ions in the body are constantlyupdated but relatively constant, which provides greatopportunities for in situ formation of Ca2+/Mg2+-responsivealginate hydrogel in vivo.17 Thus, the integration of ICG andalginate hydrogel with unique advantages makes it possible tobuild a localized ICG depot for improving ICG accumulationin tumors and reducing the side effects induced by the leakageof ICG.Herein, we show the in situ fabrication of a photothermal

ICG−alginate hydrogel for localized tumor ablation in anultrafacile way (Scheme 1). The precursor of the ICG−alginatehydrogel was synthesized by simply mixing ICG and alginate atroom temperature. The introduction of Ca2+/Mg2+ enables the

transformation of ICG−alginate from solution to hydrogeleasily, and ICG distributes in the hydrogel homogeneously. Invivo gelation study confirmed that the ICG−alginate hydrogelcould generate quickly as soon as the ICG−alginate solutionwas injected into the body of mice. The in situ fabricationprocess of the hydrogel can avoid the leakage of ICG along thepath of the needle, improve the accumulation of ICG intumors, and minimize the diffusion of ICG to surroundingtissues. The ICG−alginate solution exhibits intense NIRabsorption and fluorescence as same as free ICG, and theformed ICG−alginate hydrogel owns an excellent photo-thermal heating ability. In vitro and in vivo toxicity assessmentdemonstrated the favorable biocompatibility of the hydrogeldue to the inherent biosafety of ICG and alginate. The in situ-fabricated ICG−alginate hydrogel was applied in localizedPTT in vitro and in vivo successfully, and the fluorescence ofICG enables the monitoring of the distribution of ICGaccurately. Besides, ICG and alginate can be stored as a stablesolid form for a long time, and only the simple mixing in waterand noninvasive injection are needed to perform high-efficientlocalized PPT of tumors, greatly benefiting the preparation,storage, transportation, and potential clinic usage.

2. RESULTS AND DISCUSSION

2.1. Synthesis of ICG−Alginate Hydrogel in Vitro. Theviscosity of ICG−alginate hydrogel highly depends on theconcentrations of alginate and divalent cations. Consideringthe constant concentration of Ca2+/Mg2+ in vivo, variousconcentrations of alginate solutions were employed to tune theviscosity of the hydrogel (Figure S1). High viscosity benefitsthe loading of ICG in the ICG−alginate hydrogel but isadverse to syringeability. Therefore, 20 mg/mL of alginate asthe optimum concentration was chosen to ensure the high ICGloading efficiency and good syringeability. The ICG−alginate

Scheme 1. (A) Schematic Diagram of Transformation from Alginate solution to Hydrogel. (B) Stimuli-ResponsivePhotothermal ICG−Alginate Hydrogel for Localized Tumor Ablation

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hydrogel was synthesized via a simple mixing method. Briefly,35 mg of alginate and 7.5 mg of ICG were mixed in 1.75 mL ofH2O and stirred for 30 min. The ICG−alginate solution ishomogeneous and stable, exhibiting excellent syringeabilitycompared to hydrogels. To access the gelation properties invitro, the well-dispersed ICG−alginate solution (60 μL; 4.28mg mL−1 of ICG and 20 mg mL−1 of alginate) was injectedinto 8 mL of Ca2+/Mg2+ solution in a meter glass. Theconcentrations of Ca2+ and Mg2+ were fixed to be 1.8 and 1.5mM to mimic the extracellular microenvironment in the livingtissue.17 The ICG−alginate hydrogel immediately formedwhile an ICG−alginate aqueous solution was being injectedinto the Ca2+/Mg2+ solution (Figure 1). We further

investigated the ICG loading capacity of the ICG−alginatehydrogel, which could reach as high as 10 mg/mL of ICG inthe hydrogel (Figure S2). The formed uniform hydrogelenables encapsulation of ICG and overcomes the burst releaseof ICG efficiently (Figure S3).2.2. Characterization of ICG−Alginate Solution. The

UV−Vis−NIR absorption and fluorescence of the ICG−alginate solution were similar to that of the ICG solution,indicating neglectable interaction between ICG and alginate(Figure 2A−D). The fluorescence spectra of the ICG−alginate

solution showed a maximum emission at 813 nm, benefiting invivo fluorescence imaging with high tissue penetration depth.The strong NIR absorption peak at 808 nm linearly increasedwith the concentrations of ICG in the ICG−alginate solutionwith a fixed alginate concentration, which demonstrated thehigh photothermal potential with an 808 nm laser. The UV−Vis−NIR absorption and fluorescence spectra of the ICG−

alginate hydrogel indicated that the formed hydrogel alsoexhibited a strong NIR absorption and an intense fluorescenceemission due to effective encapsulation of ICG in the hydrogel(Figure S4). SEM images showed the morphology of alyophilized ICG−alginate hydrogel (Figure S5).

2.3. Photothermal Performance of ICG−AlginateHydrogel. To evaluate the photothermal performance of theICG−alginate hydrogel, an ICG−alginate gel prepared byadding different volumes of ICG−alginate solution to Ca2+/Mg2+ solution was irradiated with an 808 nm laser for 7 min ata power density of 0.9 W cm−2. Figure 3 revealed that the

increased temperature of the ICG−alginate hydrogel variedfrom 12.4 to 21.4 °C with the volumes of the ICG−alginatesolution changed from 10 to 100 μL. In contrast, thetemperature of Ca2+/Mg2+ solution pure water was only raisedto 5.8 °C. Besides, IR thermal photos were taken to monitorthe temperature change of the solution during the irradiationprocess, further demonstrating the favorable photothermalperformance of the ICG−alginate hydrogel (Figure 3). Wefound that 660 or 980 nm laser irradiation could also lead to aphotothermal heating effect of the ICG−alginate hydrogel.However, 660 nm laser irradiation suffers from lowerphotothermal heating efficacy compared to 808 nm laserirradiation, and 980 nm laser illumination results in a strongundesired photothermal heating of pure water due to theintense absorption of water at 980 nm.70 Therefore, 808 nmlaser irradiation is the most suitable light source forphotothermal heating of the ICG−alginate hydrogel (FiguresS6 and S7).

2.4. Cytotoxicity of ICG−Alginate Hydrogel. Thecytotoxicity of the ICG−alginate hydrogel was evaluated by astandard MTT assay with 4T1 cells. The cells were treatedwith ICG−alginate solution or preformation ICG−alginatehydrogel containing different concentrations of ICG. Figure 4Aindicated that the cell viability remained above 85% even whenthe concentration of ICG reached 1 mg mL−1 in ICG−alginatesolution, and the cell viability remained above 82% even whenthe concentration of ICG reached to 500 mg L−1 in thepreformation ICG−alginate hydrogel (Figure S8), demonstrat-ing the excellent biocompatibility of in situ formation andpreformation of the ICG−alginate hydrogel and its greatpotential for biological applications.

Figure 1. Photos of ICG and ICG−alginate (ALG-ICG) fluid afterbeing injected into the Ca2+ and Mg2+ solution.

Figure 2. (A, B) Fluorescence spectra of different concentrations ofICG and ICG−alginate solution. (C, D) UV−Vis−NIR absorptionspectra of ICG and ICG−alginate solution.

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

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2.5. Photothermal Therapy in Vitro. We theninvestigated the PTT capability of the ICG−alginate hydrogelin vitro. 4T1 cells treated with ICG−alginate gel containing200 mg L−1 of ICG were irradiated with an 808 nm laser at apower density of 0.3 or 1 W cm−2 for 5 min. The viabilities of4T1 cells treated with the hydrogel and laser irradiation wereonly 5.20% and 4.84% at the laser power densities of 0.3 and 1W cm−2, respectively (Figure 4B), whereas the cells withoutany treatments and treated with the hydrogel or laserirradiation alone all showed a neglectable cell death. Moreover,calcein-AM/PI dual staining was performed to show the cellviability directly. The fluorescence images indicated that fewlive cells could be seen after the combined treatments of theICG−alginate hydrogel and laser irradiation (Figure 4C). Incontrast, a majority of cells were alive with other treatments.These results indicated that the ICG−alginate hydrogelpossessed an excellent photothermal therapy ability.2.6. In Situ Fabrication of ICG−Alginate Hydrogel in

Vivo. We reasonably assume that the injection of ICG−alginate solution into the living organism will lead to theformation of an ICG−alginate hydrogel due to the presence ofappropriate concentration of Ca2+/Mg2+ in biological fluids.Like many PTT agent solutions, ICG solution leaked along thepath of the needle from the pinhole because of the enormouspressure in the solid tumor when free ICG was injecteddirectly into tumors. However, there was not any leakage ofsolution from the injection sites after the administration ofICG−alginate solution due to the in situ formation of an

ICG−alginate hydrogel in vivo (Figure 5A). To evaluate thefeasibility of in situ fabrication of the ICG−alginate hydrogel invivo, four Kunming mice were subcutaneously injected with 60μL of ICG−alginate solution (4.28 mg mL−1 of ICG and 20mg mL−1 of alginate) on one side and 60 μL of ICG solution(4.28 mg mL−1) on the other side as the control group.Moreover, the state of injected solutions was monitored by exvivo imaging at different time points. Figure 5B indicated theformation of ICG−alginate hydrogel, and the green color ofICG was distributed into the hydrogel, avoiding the self-aggregation of ICG and favoring the uniform heating triggeredby laser illumination. Both ICG solution and ICG−alginatehydrogel were gradually metabolized along with the time,whereas the metabolism rate of the ICG−alginate hydrogel wasobviously slower than the bare ICG solution. These resultsdemonstrated that the ICG−alginate hydrogel could be easilyin situ-formed by the injection of ICG−alginate hydrogelwithout any leakage, proving a stable ICG depot for PTT invivo.

2.7. Fluorescence Imaging of ICG−Alginate Hydrogelin Vivo. To investigate the ICG fixation capability of theICG−alginate hydrogel, fluorescence imaging of mice wasperformed after the administration of ICG and ICG−alginatesolution (Figure 5C). Two groups of Kunming mice weresubcutaneously administrated with 60 μL of ICG−alginatesolution (4.28 mg mL−1 of ICG and 20 mg mL−1 of alginate)and 60 μL of ICG solution (4.28 mg mL−1), respectively.Fluorescence images indicated that free ICG was distributed

Figure 4. (A) Relative cell viabilities of 4T1 cells treated with different concentrations of ICG−alginate hydrogel. (B) Relative cell viabilities afterbeing treated with ICG−alginate hydrogel and laser irradiation at the power density of 0.3 or 1 W cm−2. (C) 4T1 cells with different treatmentswere obtained with an inverted luminescence microscope, and live and dead cells were stained to be green and red, respectively, with calcein-AM/PI.

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throughout the body quickly due to the continuous body fluidcirculation. In contrast, the ICG in the ICG−alginate hydrogelpenetrated to the bloodstream and surrounding tissues slowlydue to the strong fixation capability of the hydrogel. The highICG accumulation capacity of the in situ-formed hydrogelshowed a great potential in improving the PTT efficacy andminimizing the side effects induced by the fast leakage of PTTagents.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 andhistopathological analysis (Figure 6A). The Kunming micewere injected subcutaneously with ICG−alginate solution(4.28 mg mL−1 of ICG and 20 mg mL−1 of alginate) or 60 μLof PBS as control, and the mice treated with the hydrogelshowed a similar increase tendency in weight in comparisonwith the control group (Figure 6B). Hematoxylin−eosinstaining analysis of the main organs (liver, heart, spleen,kidney, and lung) revealed that there were no obvioushistopathological lesions for mice in both experimental andcontrol groups. Under physiological condition, ion replace-ment and the elution of the cross-linking calcium cationresulted in osmotic swelling of Ca−alginate hydrogel, leadingto increased pore size and destabilization and rupture of thehydrogel. Alginate is also a biodegradable polymer because thebackbone of alginate can be degraded via hydrolysis of theglycosidic bonds. Therefore, the proposed hydrogel can bebiodegraded in vivo on the basis of these mechanisms, ensuringthe biosafety of the hydrogel.51−54 The in vivo toxicityinvestigation demonstrated the excellent biocompatibility ofthe ICG−alginate hydrogel in vivo.2.9. Photothermal Therapy in Vivo Using in Situ-

Fabricated ICG−Alginate Hydrogel. Inspired by theexcellent performance of photothermal ability and biocompat-ibility, the ICG−alginate hydrogel was employed in vivo PTT.

The 4T1 tumor-bearing mice were injected with 60 μL of PBSor ICG−alginate (0, 0.1, 1, and 4.28 mg mL−1 of ICG and 20mg mL−1 of alginate) solution intratumorally, followed by the808 nm laser irradiation at a power density of 0.7 W cm−2 for15 min. The temperature of tumors was monitored by an IRthermal camera during the irradiation process. To achieveobvious PTT efficacy and minimize the administration dose,ICG−alginate solution containing 4.28 mg mL−1 of ICG waschosen as the optimum PTT agent for tumor ablation (FiguresS9 and S10). Figure 6C indicated that the temperature oftumors in mice injected with ICG−alginate solution (4.28 mgmL−1 of ICG and 20 mg mL−1 of alginate) exhibited aremarkable increase, whereas the mice in the control grouponly had a much slower temperature. Furthermore, the sizes oftumors were measured with a vernier caliper every day aftervarious treatments (Figure 6D). In addition, the photos oftumors were taken every day to observe the tumor changevisually for 15 days. Figure 6E showed that the tumors of micetreated with PBS and laser irradiation kept growing all thetime, whereas the tumors of mice treated with ICG−alginatesolution almost disappeared in a week. These results obviouslydemonstrated that an ICG−alginate hydrogel with strong ICGfixation capability could serve as an excellent photothermalagent with outstanding biocompatibility.

3. CONCLUSIONSIn summary, we proposed an in situ gelation strategy tosynthesize an ICG−alginate hydrogel in vivo for PTT, unitingthe advantages of ultrasimple synthesis and administrationprocedures, excellent biocompatibility, avoidance of theleakage from injection sites, improvement of the accumulationin tumors, and minimizing of side effects induced by themetabolization from tumors. The ICG−alginate hydrogel can

Figure 5. (A) Pictures of 4T1-bearing mice were recorded afterinjecting 60 μL of ICG solution or ICG−alginate solution for 0.5 h(4.28 mg mL−1 of ICG and 20 mg mL−1 of alginate). (B) Photosshowing the metabolism of ICG fluid and ICG−alginate hydrogel invivo. (C) Dynamic fluorescence imaging of ICG−alginate hydrogeland ICG solution in vivo.

Figure 6. (A) Histopathological images of the major organs in micetreated with ICG−alginate solution (60 μL) at different time points.(B) Weight changes of the mice after injection. (C) IR thermalimages 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 15min, n = 3). (D) Tumor volume changes of mice after treatments. (E)Photos of mice at different time points before and after being treatedwith PBS or ICG−alginate solution (60 μL) plus laser irradiation (0.7W cm−2).

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be generated by adding Ca2+/Mg2+ into the ICG−alginatesolution in vitro or simply injecting the ICG−alginate solutioninto organisms without any leakage of any agents. The strongICG-fixed ability of the hydrogel was demonstrated in vitroand in vivo, greatly benefiting the high accumulation of ICG intumors and minimizing the side effects from the fastmetabolization. The toxicity studies in vitro and in vivoconfirmed that its favorable biocompatibility derived from thesafety of ICG and alginate. The in situ-fabricated ICG−alginate hydrogel with outstanding photothermal performancewas applied in tumor PTT in vitro and in vivo successfully.Moreover, the convenient storage of ICG and alginate in asolid form and simple synthesis and administration processmake the in situ-formed ICG−alginate hydrogel promising inPTT in vivo. We will investigate the clinic transformation ofthe ICG−alginate hydrogel with unique superiorities in thefuture. The in situ fabrication of the photothermal hydrogelusing biocompatible components lays down a promising wayto revolutionize PTT toward clinic transformation fromsynthesis of administration.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsami.8b16517.

Optimization of alginate in ICG−alginate hydrogel, ICGloading capacity of ICG−alginate hydrogel, release ofICG from ICG−alginate hydrogel in vitro, SEM oflyophilized ICG−alginate hydrogel, photothermal per-formance of ICG−alginate hydrogel irradiated by a 660nm laser, photothermal performance of ICG−alginatehydrogel irradiated by a 980 nm laser, cytotoxicity ofpreformation ICG−alginate hydrogel, optimization ofICG in ICG−alginate hydrogel (PDF)

■ AUTHOR INFORMATIONCorresponding Author*E-mail: shaokaisun@tmu.edu.cn.ORCIDShao-Kai Sun: 0000-0001-6136-9969NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the National Natural ScienceFoundation of China (grants 21435001 and 21874101) andthe Natural Science Foundation of Tianjin City (no.18JCYBJC20800).

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