Biocompatible Iodine-Starch-Alginate Hydrogel for Tumor

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Biocompatible Iodine-Starch-Alginate Hydrogel for Tumor Photothermal Therapy Haoyu Wang, Limei Jiang, Huanhuan Wu, Weiya Zheng, Di Kan, Ran Cheng, Juanjuan Yan, Chunshui Yu, and Shao-Kai Sun ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00280 • Publication Date (Web): 25 May 2019 Downloaded from http://pubs.acs.org on May 27, 2019

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ACS Biomaterials Science & Engineering

Biocompatible Iodine-Starch-Alginate Hydrogel for Tumor Photothermal Therapy Haoyu Wang,‡ Limei Jiang,§ Huanhuan Wu,‡ Weiya Zheng,† Di Kan,† Ran Cheng,† Juanjuan Yan,‖ Chunshui Yu,*,‡ and Shao-Kai Sun*,† †School

of Medical Imaging, Tianjin Medical University, No.1, Guangdong Rong, Hexi District,

Tianjin 300203, China ‡Department

of Radiology, Tianjin Key Laboratory of Functional Imaging, No.154, Anshan

Road, Heping District, Tianjin Medical University General Hospital, Tianjin 300052, China §Department

of Radiology, Tianjin First Central Hospital, No.24, Kangfu Road, Nankai District,

Tianjin 300192, China ‖School

of Medical Laboratory, Tianjin Medical University, No.1, Guangdong Rong, Hexi

District, Tianjin 300203, China *E-mail: [email protected] (S.-K. Sun) *E-mail: [email protected] (C. Yu) KEYWORDS: Iodine-starch-ALG hydrogel, Photothermal therapy, Biocompatibility

ABSTRACT: Photothermal therapy (PTT) with the advantages of high efficiency and minimal invasiveness is a promising technique for tumor therapy, but clinical application of PTT agents has been stifled by the great safety concerns. Herein, a deep blue iodine-starch-alginate (ALG)

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hydrogel is elegantly fabricated based on the classic and simple “iodine-starch test” for in vivo tumor PTT in a facile and mild way. The iodine-starch-ALG hydrogel composed of clinically used agents is fabricated by dispersing blue iodine-starch complex into alginate-Ca2+ hydrogel, which guarantees the good chemical stability of iodine-starch complex via separating them from surrounding reductive environment. The iodine-starch-ALG hydrogel possesses favorable biocompatibility derived from the biosafe and degradable components, and good photothermal heating ability based on iodine-starch chromophore. The proposed iodine-starch-ALG hydrogel is successfully applied in tumor PTT in vitro and in vivo for the first time. This work lays down a novel way for the development of high-performance and biocompatible biomaterials via teaching old drugs new tricks.

INTRODUCTION Photothermal therapy (PTT) is emerging as a high-efficient strategy for tumor therapy, which converts light energy into heat to ablate tumors.1-4 PTT agents are the key to high-efficient PTT by remarkably improving photothermal conversion efficiency. Ideal PTT agents should own strong near-infrared (NIR) light absorption, high photothermal conversion efficiency and outstanding biocompatibility.5,6 Generally, PTT agents can be divided into inorganic and organic agents. The inorganic PTT agents, such as metal nanomaterials (e.g. Au nanomaterials,7,8 Pd nanosheets,9 and Bi nanostructures10,11), metal oxide (e.g. W18O4912 and MoOx13), transitional metal dichalcogenides (e.g. CuS,14,15 MoS2,16 WS2,17 Bi2S3,18 and Bi2Se319,20), carbon-based nanomaterials (e.g. carbon nanotubes21 and graphene22,23), black phosphorus,24,25 and MXenes,26 have desirable NIR absorption and favorable photothermal conversion efficiency and photothermal stability. However, many inorganic nanomaterials are difficult to be metabolized and suffer from great concerns regarding their long-term toxicity due to the non-biodegradable


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components, thus restricting their clinical implementations.27 The organic PTT agents, such as organic dyes,28 porphyrins,29 prussian blue nanostructures,30 polydopamine nanoparticles,31 polyaniline nanomaterials,32 make a great progress in the aspect of biocompatibility of PTT agents, but most of them are still facing the great challenges of complicated synthesis processes and ambiguous biosafety.33 Therefore, it is highly desired to develop simply synthetic and biocompatible PTT agents for potential clinical transformation of PTT. The iodine starch test is a widely used and classic method for iodometric titration in chemical analysis and starch determination in food industry, textile industry and biological field.34 This simple science phenomenon was firstly discovered in 1814 and many efforts have been devoted to investigate the chemical structure of iodine-starch complex.35-38 The aqueous solubility of iodine can be highly improved with iodide ions due to the formation of polyiodides (In⁻, mostly I3⁻).39 These polyiodides can interact with diverse natural polymers (starch, albumin, glycogen, chitosan, and so on) and form versatile complexes with special function (chromophore formation, polymer morphology changes, biological activity or electrical conductivity enhancement).40 Starch as the main reserve polysaccharide of green plants is the principal food of many animals including human, which consists of linear and helical amylose and branched amylopectin.41 The iodine-starch complex consists of dark blue iodine-amylose complex with maximum absorption at 650 nm and reddish-purple iodine-amylopectin complex with maximum absorption at 540 nm. The characteristic deep blue color of iodine-starch complex is mainly derived from the iodine-amylose complex.34,40 Structurally, amylose is organized as left-handed helices, and each turn of the helix contains of six 1, 4-anhydroglucose units. The polyiodides are linearly arranged in the inner cavity of the helices to form iodine-amylose complex.35-38 The


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absorption of iodine-starch complex ranges from ultraviolet to NIR region, and thus showing great potential for PTT. In addition, iodine is a vital element for body and mostly accumulates in the thyroid gland. The main function of iodine as a constituent of the thyroid hormones is to maintain proper functioning of the vertebrate endocrine system.42 Iodine deficiency can lead to severe abnormalities, such as thyroid function abnormalities, endemic goiter, cretinism, and mental deficiency.43 Lugol’s solution (LS) composed of potassium iodide (KI, 10%) and iodine (I2, 5%) is frequently used in clinic for the treatment of iodine deficiency. Besides, LS is widely applied in water disinfectant, starch detection, histologic preparations, dental procedures and diagnosis of cervical cell alterations. Especially, LS has been applied as a standard pre-operative treatment in thyroid surgery since 1920s to control hyperthyroidism by oral administration of the mixture of LS and starch contained food.44,45 Therefore, the iodine-starch complex produced by mixing exhibits appealing biocompatibility. Herein, we show the fabrication of a biocompatible iodine-starch-ALG hydrogel for PTT based on the classic and simple “iodine starch test” in a facile and mild way (Scheme 1). The iodine-starch complex was synthesized by simply mixing starch and LS, and further encapsulated into an alginate (ALG)-Ca2+ hydrogel to generate iodine-starch-ALG hydrogel. ALG is an FDA-approved polysaccharide, and ALG-Ca2+ hydrogel is widely used in various biomedical applications, such as wound healing, drug delivery and cell transplantation.46 The ALG-Ca2+ hydrogel plays a vital role in improving the chemical stability of iodine-starch complex by slowing down the interaction between iodine and surrounding reductive molecules. As all the components of iodine-starch-ALG hydrogel are frequently used in clinic, the hydrogel exhibits excellent biosafety. The proposed iodine-starch-ALG hydrogel has favorable


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photothermal conversion capability, and in vitro and in vivo PTT demonstrated its powerful tumor suppressing effect. More importantly, the iodine-starch-ALG hydrogel can be degraded in physiological environment, avoiding the potential long-term biosafety. To the best of our knowledge, it is the first time that iodine-starch complex is employed for tumor PTT. Scheme 1. Schematic Illustration of the Synthesis of Iodine-starch-ALG hydrogel for Tumor Photothermal Therapy

EXPERIMENTAL SECTION Chemicals. All the reagents used were of analytical pure. Potassium iodide (KI), iodine (I2), starch, calcium chloride anhydrous (CaCl2, 96%), sodium alginate (200 ± 20 mpa.s, ALG), 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dinitrosalicylic acid (DNS) were purchased from Aladdin Chemistry Corporation (Shanghai, China). 2′,7′dichlorofluorescin diacetate (DCFH-DA) was obtained from Beijing Solarbio Science &


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Technology Co., Ltd. (Beijing, China). The deionized water used throughout the experiments was provided by Wahaha Group Co., Ltd. (Hangzhou, China). Synthesis of Iodine-Starch. The iodine-starch was prepared by a facile and mild reaction. LS was prepared by adding solid I2 (120 mg) into KI aqueous solution (10 mg mL-1, 24 mL) with stirring and ultrasonic treatment. Soluble starch was firstly dissolved in deionized water (20 mg mL-1) by heating with an oil bath at 95 °C for 30 min, and then cooled to 37 °C. For the synthesis of iodine-starch, LS (24 mL) was rapidly introduced into starch solution (12 mL) with a feeding mass ratio of 1.25 for iodine to starch. After continuously stirring for 60 min, the reaction product was precipitated by ethanol (40 mL) and centrifuged at 9500 rpm for 5 min. The precipitate was purified by water (4 mL) and ethanol (16 mL) at 9500 rpm for 5 min twice, and the deep blue iodine-starch complex was obtained after vacuum drying. To obtain iodine-starch complex with strong absorption, the reaction conditions were optimized by monitoring reaction kinetics (5, 10, 20, 30, 60, 90 and 120 min) and tuning the feeding mass ratio of iodide to starch (0.313, 0.625, 0.938, 1.25, 1.563 and 1.875). Synthesis of Iodine-starch-ALG hydrogel. The iodine-starch-ALG hydrogel was prepared via ionic cross-linking of alginate and Ca2+. Briefly, various masses of iodine-starch powder were dissolved in CaCl2 (1 mg mL-1) solution, and then ALG solution (10 mg mL-1) with same volume was rapidly added under stirring for 10 min. The formed iodine-starch-ALG hydrogel was used for further studies. ALG-Ca2+ hydrogel was prepared by the mixing of CaCl2 (1 mg mL-1) solution and ALG solution (10 mg mL-1) for 10 min. Characterization. The UV-vis-NIR absorption spectra were recorded by a UV-3600 spectrophotometer with QS-grade quartz cuvettes at room temperature (Shimadzu, Japan). The Fourier transform infrared (FT-IR) (500–3500 cm-1) spectra were obtained on a Nicolet IS10


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spectrometer using KBr pellets (Nicolet, USA). Field emission scanning electron microscopy (FE-SEM) images and element mapping images of freeze-drying iodine-starch-ALG hydrogel were acquired on a JSM-7800F microscopy at an accelerating voltage of 15 kV. The content of I element in iodine-starch complex was measured by sodium thiosulfate titration and absorption measurement. Standard KI solutions were prepared with I⁻ concentrations from 5 ppm to 50 ppm, and typical UV absorptions at 226 nm were recorded to obtain a linear calibration curve. Iodine-starch complex was treated with excess sodium thiosulfate to entirely convert polyiodides into iodide ions. The absorbance of iodide in iodine-starch was collected and calculated based on the calibration curve of I⁻. Stability Assessment. Iodine-starch solution (0.5 mL, 4 mg mL-1), iodine-starch-ALG hydrogel (0.5 mL, 4 mg mL-1 iodine-starch) and LS (0.5 mL) were incubated with various main biomolecule solutions present in organisms (0.5 mL) including 2 mg mL-1 glucose, DNA, glycine, glutamic acid, lecithin, glutathione and cysteine and 80 mg mL-1 BSA. Then photographs of these mixtures were acquired by a canon 60D camera and the UV-vis-NIR absorption spectra were recorded after appropriate dilution. Besides, the in vitro stability of iodine-starch-ALG hydrogel was assessed. Iodine-starch-ALG hydrogel (0.5 mL, 2 mg mL-1 iodine-starch) was treated with 0.5 mL water, PBS and normal saline, and incubated at 37 °C. The photographs of the above mixtures were taken at different time points. Biodegradability Assessment of Iodine-starch Complex in Vitro. For in vitro biodegradability assessment, iodine-starch solution and iodine-starch-ALG hydrogel were incubated with or without α-amylase under 37 °C for 6 h respectively. The iodine test was performed on the faded mixtures to determine the starch. Besides, another group of mixtures were investigated by DNS method to determine the generation of reducing sugar.


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Photothermal Performance Measurement. To assess the photothermal efficacy of iodinestarch-ALG hydrogel, 1 mL deionized water, ALG-Ca2+ hydrogel and iodine-starch-ALG hydrogels containing different concentrations of iodine-starch (0.5, 1, 2 mg mL-1) were added in a quartz cell (1-cm path length) and irradiated with an 808 nm NIR laser at a density of 2 W cm-2 for 10 min, respectively. A thermocouple probe was putted into colloid solution to monitor the temperature change and an infrared camera (FLIR E75) was used to record the real-time infrared thermal images at the same time. Cell Cultures and Tumor Models. 4T1 cells (mouse breast cancer cells) and 3T3 cells (mouse fibroblast cells) were cultured in DMEM culture medium supplemented with 10% fetal calf serum in an incubator at 37 °C under a humidified atmosphere with 5% CO2. To establish 4T1 tumor in situ in the Balb/c mice (6 weeks), 4T1 cells (5 × 106) in 50 µL PBS was subcutaneously injected into the right back of each mouse. All the animal experiments were approved by the Tianjin Medical University institutional ethical committee and carried out according to Tianjin Medical University guidelines for animal research. In Vitro Cytotoxicity. 4T1 cells and 3T3 cells were seeded in a 96-well plate with a density of 104 per well and cultured for 24 h, respectively. Then culture medium was replaced with fresh culture medium containing iodine-starch solution or iodine-starch-ALG hydrogel (40 µL, 0, 2, 4, 10, 15, 20 and 25 mg mL-1 iodine-starch). Five replicates were set for each sample. After 24 h incubation, the culture medium was removed, and cells were washed with PBS and treated with MTT (10 µL, 5 mg/mL) dispersing into fresh culture medium for 4 h. Subsequently, DMSO (120 µL) was added to dissolve the formed formazan crystals after discarding the culture medium. The absorbance (490 nm) of each well was recorded by a Synergy HTX multi-mode microplate


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reader. The cell viability was calculated according to the following formula equation 1: (A is the average absorbance) Cell viability (%) = (𝐴𝑠𝑎𝑚𝑝𝑙𝑒 ― 𝐴𝑏𝑙𝑎𝑛𝑘) (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ― 𝐴𝑏𝑙𝑎𝑛𝑘) × 100 %

(1)

Reactive oxygen species (ROS) generation analysis: 3T3 cells were seeded in a 96-well plate and incubated with iodine-starch solution or iodine-starch-ALG hydrogel (40 µL, 1, 10 and 15 mg mL-1 iodine-starch) for 4 h. Then culture medium was replaced with fresh culture medium containing DCFH-DA and incubated for 30 min. DCFH-DA can traverse cell member and react with ROS to form DCFH with green fluorescence. Then, the cells were washed with PBS for three times, and the fluorescence images were obtained by an inverted fluorescence microscope. In Vitro PTT Study. 4T1 cells were seeded in a 96-well plate with a density of 104 per well and incubated for 24 h. The culture medium was replaced with fresh PBS containing iodinestarch-ALG hydrogel (40 µL, 0, 10, 15 and 20 mg mL-1 iodine-starch), and then cells were illuminated with or without an 808 nm laser (1.5 or 2 W cm-2) for 5 min. After irradiation, the medium was replaced with fresh DMEM culture medium and cells were incubated for another 1 h. Thereafter, the cell viability was evaluated by MTT assay. For in vitro PTT assessment intuitively, the post-irradiation cells were co-stained with calcein-AM (2 mM) and PI (5 mM) for 15 min, and then carefully rinsed twice with PBS. The fluorescence images were obtained by an inverted fluorescence microscope. In Vivo PTT and Toxicity. The chemical stability of iodine-starch-ALG hydrogel was assessed by subcutaneous injection of iodine-starch-ALG hydrogel and iodine-starch solution (100 µL, 50 mg mL-1 iodine-starch) on the back of mice in vivo, respectively. The mice were sacrificed and the skin in the injection site was peeled at different time points (0.5 h, 1 h, 4 h, 6 h and 7 day) to assess the chemical stability. For in vivo biodegradability assessment, iodine-starch


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solution and iodine-starch-ALG hydrogel (100 µL, 50 mg mL-1 iodine-starch) were subcutaneously injected on the back of Kunming mice respectively. The mice were sacrificed and the skin at the injection site was peeled at different time points (6 h, 12 h, 1 day and 7 day). Subsequently, iodine test was performed on the collected skin to determine the starch. For the in vivo PTT study, when the tumor volume was around 100 mm3, the 4T1 tumorsbearing mice were randomly divided into 4 groups (n = 5 for each group): (i) control group without any treatments; (ii) ALG-Ca2+ hydrogel + laser irradiation; (iii) iodine-starch-ALG hydrogel alone; (iv) iodine-starch-ALG hydrogel + laser irradiation. The mice were anaesthetized and intratumor injected with ALG-Ca2+ hydrogel (100 µL) or iodine-starch-ALG hydrogel (100 µL, 50 mg mL-1 iodine-starch), and then the tumors were irradiated by an 808 nm laser (1 W cm-2) for 5 min. The infrared thermal images were collected by an infrared camera (FLIR E75) to monitor the temperature of tumors during laser irradiation. After treatment, the tumors of mice were monitored by taken photos every other day, and the tumor sizes were measured by a caliper and calculated according to the formula equation 2: volume (𝑉) = (tumor length × tumor width2)/2

(2)

The relative tumor volume was calculated as V/V0 (V0 is the initial tumor volume before treatment). The weights of mice were recorded and behaviors of mice were also monitored. After 14 days of monitoring, the mice were euthanized, followed by the collection of tumors and major organs (heart, liver, spleen, lung and kidney). H&E analysis was carried out to assess the histopathological damage of major organs in each group. For the blood biochemistry analysis and histopathological study of tissues at injection sites, different groups of Kunming mice were subcutaneously injected with or without iodine-starchALG hydrogel (100 µL, 50 mg mL-1 iodine-starch) (n = 3). At 1, 7 and 15 day after injection, the


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blood samples of mice were collected by eyeball extirpating and centrifugated at 3000 rpm for 10 min. The supernatant serum was collected and stored at -80 ºC for blood biochemistry analysis, including liver function indicators: alanine aminotransferase (ALT), aspartate aminotransferase (ASL), albumin (ALB), alkaline phosphatase (ALP) and typical biomarkers of kidney function: serum creatinine (CREA), urea nitrogen (BUN) and uric acid (UA). Besides, the skin tissues at the injection site of the sacrificed mice were harvested and fixed with 4% paraformaldehyde. The H&E analysis of skin tissues were performed to evaluate the in vivo toxicity and local inflammation induced by iodine-starch-ALG hydrogel. RESULTS AND DISCUSSION Synthesis and Characterization of Iodine-Starch Complex and Iodine-starch-ALG hydrogel. The iodine-starch complex was synthesized via a simple one-pot reaction. Soluble starch is slight soluble in cold water but is irreversibly dissolved in hot water in a starch gelatinization process. LS was prepared by dissolve I2 into KI solution via the formation of polyiodides (mostly I3⁻, confirmed by two typical absorption peaks at 287 nm and 355 nm) (Figure 1a).47 The colorless starch solution immediately turned into dark blue after the addition of LS due to the formation of iodine-starch complex. The absorption spectrum revealed that the iodine-starch mixture exhibited a broad absorption band from ultraviolet to NIR region centered at 585 nm (Figure 1a). To obtain iodine-starch complex with strong absorption, the reaction conditions were optimized by monitoring reaction kinetics and tuning the feeding mass ratio of iodide to starch. The absorbance of iodine-starch mixture at 808 nm slowly increased along with the reaction time and reached to a plateau at 60 min (Figure S1). Besides, the absorbance intensity at 808 nm increased noticeably with the mass ratio of iodine to starch, and the increase tendency became slower when the ratio is larger than 1.25 (Figure S2). Thus 60 min and the ratio


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of 1.25 for iodine to starch were chosen as the optimal reaction time and amounts of agents. The content of I element in iodine-starch was measured to be 8.5% by sodium thiosulfate titration and absorption measurement of I⁻ at 226 nm. The FT-IR spectra demonstrated the presence of starch and iodine-starch complex (Figure S3). Typically, the four absorbance bands in the fingerprint region at 1156, 1083, 1023 and 937 cm-1 are attributed to C–O stretching in starch. The characteristic peak at 1640 cm-1 and 2926 cm-1 are attributed to the tightly bound water present in the starch and C–H stretches, respectively.48

Figure 1. (a) Absorption spectra of LS (84 µg mL-1 I element), starch solution (67 µg mL-1), and iodine-starch mixture (84 µg mL-1 I element, 67 µg mL-1). Inserts: 1, LS; 2, starch; 3, iodine starch mixture. (b) Photographs of ALG-Ca2+ hydrogel, iodine-starch solution (0.5 mg mL-1) and iodine-starch-ALG hydrogel (0.5 mg mL-1 iodine-starch) (from left to right). (c) Pattern of “TMU” formed by iodine-starch-ALG hydrogel (50 mg mL-1 iodine-starch). (d) SEM image and (e) element mapping images of freeze-dried iodine-starch-ALG hydrogel (50 mg mL-1 iodinestarch). (f) Absorption spectra of iodine-starch solution (0.5 mg mL-1) and iodine-starch-ALG hydrogel (0.5 mg mL-1 iodine-starch). (g) Absorption spectra of water and iodine-starch-ALG hydrogel with various iodine-starch concentrations (0, 0.25, 0.5, 0.75, 1, 1.5 and 2 mg mL-1).


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Considering the potential influence of reductive molecules in physiological conditions on the chemical stability of iodine-starch complex, ALG-Ca2+ hydrogel was employed to encapsulate iodine-starch complex and isolate the complex from the surrounding environments. Alginate is composed of β-D-mannuronate (M units) and α-L-glucuronate (G units), the G units in different alginate chains can interact with calcium ions to form network based on ionic covalent crosslinks.49 The iodine-starch-ALG hydrogel was simply synthesized by mixing ALG and iodine-starch/Ca2+ solutions (Figure 1b). The formed hydrogel displayed a good syringeability, and pattern of “TMU” can be readily produced by extruding the hydrogel from an injection syringe (Figure 1c). The scanning electron microscope images indicated feezed-drying iodinestarch-ALG hydrogel had a flake structure (Figure 1d). The element mapping analysis demonstrated O, I and Ca elements were well distributed in the hydrogel, indicating the homogeneous distribution of iodine-starch complex and ALG (Figure 1e). The absorption spectrum of ALG-Ca2+ hydrogel is similar to that of iodine-starch complex, which indicated the encapsulation of ALG-Ca2+ have neglectable influence on the structure of iodine-starch complex (Figure 1f). The obtained iodine-starch-ALG hydrogel showed a strong absorption at 808 nm, ensuring its great potential in the application of PTT (Figure 1g). Stability Assessment of Iodine-Starch Complex and Iodine-starch-ALG hydrogel. To evaluate the chemical stability, iodine-starch solution is incubated with various solutions containing the main biomolecules present in organisms (glucose, DNA, glycine, glutamic acid, lecithin, glutathione (GSH), bovine serum albumin (BSA) and cysteine). Figure S4a indicated that the introduction of glucose, DNA, glycine, glutamic acid or lecithin did not lead to obvious color change, while the dark blue color of iodine-starch solution disappeared quickly when GSH, BSA or cysteine were added. The absorption spectra of the mixtures of iodine-starch solution


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and glucose, DNA, glycine or glutamic acid showed a neglectable change compared to iodinestarch solution (Figure S4b), further confirming that these biomolecules have little influence on the iodine-starch complex. However, the typical absorption of iodine-starch solution disappeared after the addition of GSH, BSA or cysteine due to the oxidation reaction between I2 and these reductive agents. As expected, lecithin with a weak reducibility resulted in a weak influence on absorption of iodine-starch solution. The poor chemical stability of I2 in iodine-starch complex was confirmed by the photos and absorption spectra of LS incubated with various biomolecule solutions (Figure S4c and S4d). To separate iodine-starch complex from surrounding environments, iodine-starch complex was further encapsulated into AlG-Ca2+ hydrogel to form iodine-starch-ALG hydrogel. Compared with iodine-starch solution, the iodine-starch-ALG hydrogel displayed a much better chemical stability after incubated with biomolecule solutions (Figure S5), which makes it possible for further biological applications. In the presence of water, normal saline (NS) and PBS, the iodine-starch-ALG hydrogel could retain the structural stability at 37 °C for about 4 h, and then gradually swelled (Figure S6). The appropriate chemical and structural stability greatly benefit the effective PTT. Besides, the iodine-starch solution and iodine-starch-ALG hydrogel can be biodegraded by α-amylase. Figure S7 indicated that the introduction of α-Amylase could lead to obvious color disappearance of iodine-starch complex in the iodine-starch solution and iodine-starch-ALG hydrogel due to the degradation of starch. The iodine test and DNS method on degraded mixtures further demonstrated the absence of starch and generation of reducing sugar. In Vitro Photothermal Assessment. To evaluate the photothermal heating ability, iodinestarch-ALG hydrogels containing different concentrations of iodine-starch complex were irradiated with an 808 nm laser (2 W cm-2) for 10 min. Figure 2a and 2b showed the temperature


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of iodine-starch-ALG hydrogel increased obviously under the laser illumination, and the enhanced temperature increased obviously in a concentration-dependent manner. The iodinestarch-ALG hydrogel (2 mg mL-1 iodine-starch) showed an obvious temperature increase by 29.6 °C under the laser irradiation for 10 min. In contrast, the temperature of water and ALG-Ca2+ hydrogel only raised by 5.3 °C and 6.5 °C under the same conditions, respectively. The heat conversion efficiency (η) of iodine-starch-ALG hydrogel at 808 nm was calculated to be 17.2% (Figure S8),50 demonstrating the iodine-starch-ALG hydrogel possesses a good photothermal heating ability.

Figure 2. (a) Infrared thermal images and (b) time-dependent temperature increase curves of 1 mL deionized water, ALG-Ca2+ hydrogel and iodine-starch-ALG hydrogels (0.5, 1, 2 mg mL-1 iodine-starch) under a 808 nm laser irradiation (2 W cm-2) for 10 min. (c) Relative viability of 4T1 cells after incubation with iodine-starch-ALG hydrogel and iodine-starch solution (40 µL, 0, 2, 4, 10, 15, 20 and 25 mg mL-1 iodine-starch) for 24 h. (d) Relative viability of 4T1 cells after


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treatment with iodine-starch-ALG hydrogel (40 µL, 0, 10, 15 and 20 mg mL-1 iodine-starch) and the 808 nm laser irradiation (1.5 W cm-2 or 2 W cm-2) for 5 min. (e) Fluorescence images of 4T1 cells co-stained with calcein-AM (live cells, green fluorescence) and PI (dead cells, red fluorescence) after various treatments. Cytotoxicity Assessment and in Vitro PTT. Prior to in vitro PTT, the cytotoxicity of iodinestarch-ALG hydrogel was evaluated. 4T1 cells were treated with iodine-starch solution and iodine-starch-ALG hydrogel (containing various concentrations of iodine-starch) for 24 h, and the relative cellular viability was determined by the MTT assay. The iodine-starch complex showed a relatively high cytotoxicity, and only 10% of cells were alive when the initial concentration of iodine-starch solution reached to 25 mg mL-1. In contrast, the viability of 4T1 cells was above 85% after the treatment of iodine-starch-ALG hydrogel containing as high as 25 mg mL-1 of iodine-starch (Figure 2c). The viability of 3T3 cells was similar to 4T1 cells under the same treatments (Figure S9). Besides, the ROS levels analysis of 3T3 cells was monitored after incubated with iodine-starch solution and iodine-starch-ALG hydrogel for 4 h (Figure S10). Iodine-starch solution could induce the production of ROS in 3T3 cells in a dose-dependent manner, while iodine-starch-ALG hydrogel led to less ROS in 3T3 cells due to the presence of ALG-Ca2+ hydrogel. These results indicate that the encapsulation of ALG-Ca2+ hydrogel provides a favorable protection effect to reduce the cytotoxicity of iodine-starch complex, facilitating its safe biological applications. The PTT in vitro was further investigated using the biocompatible iodine-starch-ALG hydrogel. 4T1 cells were treated with different combinations of iodine-starch-ALG hydrogel with different concentrations of iodine-starch complex and laser irradiation with various power densities, and then cellular viabilities were determined by the MTT assay. Figure 2d revealed


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that the viability of cells treated with iodine-starch-ALG hydrogel and laser irradiation decreased obviously with increased iodine-starch-ALG hydrogel dose or/and laser power densities. Nearly 95% of 4T1 cells were killed after the treatments of iodine-starch-ALG hydrogel (20 mg mL-1 iodine-starch) and laser irradiation at a power density of 2 W cm-1 for 5 min. On the contrary, the treatment of ALG-Ca2+ hydrogel, iodine-starch-ALG hydrogel, laser irradiation and laser irradiation plus ALG-Ca2+ hydrogel lead to neglectable cell death. To evaluate the PTT in vitro intuitively, the live and dead cells in each group were co-stained by calcein acetoxymethyl ester (calcein-AM) and propidium iodide (PI), respectively (Figure 2e). The fluorescent images further confirmed that only the combination of iodine-starch-ALG hydrogel and laser irradiation enabled the effective cancer cell ablation. In vitro cytotoxicity and PTT results ensure that iodine-starchALG hydrogel can serve as a biocompatible and efficient PTT agent. In Vivo PTT and Toxicity. The stability of iodine-starch-ALG hydrogel is important for efficient PTT in vivo. We compared the chemical stability of iodine-starch-ALG hydrogel and iodine-starch solution (100 µL, 50 mg mL-1 iodine-starch) after subcutaneous injection of them on the back of mice in vivo respectively. The mice were sacrificed and the skin in the injection site was peeled at different time points. Figure 3a indicated that dark blue iodine-starch solution was relatively stable within 1 h after the injection, but gradually faded, and became colorless at 4 h. This phenomenon is attributed to the oxidation-reduction reaction between I2 and the reductive molecules in the tissue, which is adverse to the stable PTT in vivo. In contrast, iodine-starchALG hydrogel exhibited much better chemical stability, and the stable dark blue color could be observed for at least 4 h. The dark blue color of iodine-starch-ALG hydrogel disappeared after 6 h due to the degradation of I2. The effective inhibition effect on color fading of iodine-starch after the encapsulation of ALG-Ca2+ hydrogel ensured the high-performance of iodine-starch-


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ALG hydrogel as a PTT agent. There was little residue of iodine-starch-ALG hydrogel present in the injection site at 7 day after injection, ensuring the long-term biocompatibility of iodinestarch-ALG hydrogel. Besides, the biodegradation of iodine-starch complex in iodine-starch solution and iodine-starch-ALG hydrogel in vivo was evaluated. The iodine test showed the recovery of dark blue color of the hydrogel at 6 h after the injection of iodine-starch-ALG hydrogel, and there was no color recovery occurred 12 h later. These results confirmed that iodine and starch in iodine-starch-ALG hydrogel have been degraded at 6 h and 12 h respectively after the in vivo injection. In contrast, the color of iodine-starch solution after injection faded more quickly, and failed to recover to dark blue color with the iodine test at 6 h, demonstrating the quick degradation of iodine-starch complex without the protection of ALG-Ca2+ hydrogel (Figure S11). The iodine-starch-ALG hydrogel with good chemical stability and biocompatibility was then applied in PTT in vivo. The 4T1 tumors-bearing mice were randomly divided into four groups (n = 5 for each group): (i) control group without any treatments; (ii) ALG-Ca2+ hydrogel + laser irradiation; (iii) iodine-starch-ALG hydrogel alone; (iv) iodine-starch-ALG hydrogel + laser irradiation. After intratumoral injection of ALG-Ca2+ hydrogel (100 µL) or iodine-starch-ALG hydrogel (100 µL, 50 mg mL-1 iodine-starch), the tumors were irradiated with an 808 nm laser (1 W cm-2) for 5 min. The infrared thermal images were taken by an infrared camera to monitor the temperature change of tumors during laser irradiation. Figure 3b and 3c revealed that the temperature of tumors in mice treated with iodine-starch-ALG hydrogel remarkably raised by 20.8 °C during initial 1 min-irradiation. The highest temperature could reach and keep as high as about 55 °C during the following irradiation, which is sufficient to induce tumor ablation. On the contrary, the tumor temperature of mice in the ALG-Ca2+ hydrogel treated group only displayed


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a small increase by 10.8 °C under the same irradiation condition. These results demonstrate the good photothermal heating ability of iodine-starch-ALG hydrogel in vivo.

Figure 3. (a) Photographs of mice and peeled skin after subcutaneous injection with iodinestarch solution and iodine-starch-ALG hydrogel (100 µL, 50 mg mL-1 iodine-starch) at the back of mice at different time points. (b) Infrared thermal images and (c) time-dependent temperature change curves of tumors in mice (n = 5) during irradiation with an 808 nm laser (1 W cm-2) for 5 min after intratumoral injection with ALG-Ca2+ hydrogel (100 µL) or iodine-starch-ALG hydrogel (100 µL, 50 mg mL-1 iodine starch). After photothermal treatment, the tumor volumes in various groups were monitored every other day (Figure 4a, 4b and 4c). The tumors in mice treated with iodine-starch-ALG hydrogel were scared and completely destroyed at 2 day after the laser irradiation, and there was no tumor recurrence observed in 14 days. While the tumors of mice treated with ALG-Ca2+ hydrogel plus laser irradiation or iodine-starch-ALG hydrogel alone kept growth quickly without obvious


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tumor suppressing effect compared to the control group, and the tumors in these groups after 14 days were about 7-fold bigger than initial ones. These results demonstrated the excellent PTT capability of iodine-starch-ALG hydrogel in vivo. Besides, there were no abnormal behaviors for the mice in all groups, and no obvious differences in the body weight increasing were observed between groups (Figure 4d). The histopathological study via hematoxylin-eosin (H&E) staining of major organs (heart, liver, spleen, lung and kidney) indicated there were no obvious difference in histopathological damage between the experimental and control groups (Figure 4e). Besides, the blood biochemistry analysis and histopathological study of tissues at injection sites were carried out on Kunming mice after subcutaneously injected with iodine-starch-ALG hydrogel at different time points (Figure S12). Compared with control group, there was no significant difference in liver or kidney function indicators in iodine-starch-ALG hydrogel group at all time points. H&E staining analysis was performed to evaluate the inflammation of skin tissues at the injection sites at different time points. Figure S13 indicated that there were mild-tomoderate inflammatory cells infiltration in the skin tissues at 1 day and 7 day after injection, and few residual inflammatory cells remained at 15 day. Moreover, H&E analysis revealed the absence of necrosis in skin tissue in all conditions (Figure S13). The in vivo PTT study and toxicity assessment demonstrate the developed iodine-starch-ALG hydrogel is an excellent PTT agent with high PTT efficacy and appealing biocompatibility in vivo.


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Figure 4. The 4T1 tumors-bearing mice were divided into four groups (n = 5 for each group): (i) control group without any treatment; (ii) ALG-Ca2+ hydrogel + laser irradiation; (iii) iodinestarch-ALG hydrogel alone; (iv) iodine-starch-ALG hydrogel + laser irradiation. (a) Photographs and (b) growth curves of tumors in mice after various treatments. (c) Photographs of excised tumors and (e) H&E-stained images of major organs of 4T1 tumors-bearing mice at 14 day after


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various treatments (The scale bar is 100 µm for all panels). (d) The body weight change curves of mice in each group post treatment (n = 5) CONCLUSIONS In summary, the classic and simple “iodine-starch test” was employed to construct iodine-starchALG hydrogel as a novel PTT agent via a facile and mild way. The ALG-Ca2+ hydrogel plays a vital role in improving the chemical stability of iodine-starch by slowing down the interaction between iodine and surrounding reductive molecules. The formed iodine-starch-ALG hydrogel possesses good photothermal conversion capability and low cytotoxicity and in vivo toxicity. In vitro and in vivo PTT studies demonstrate the powerful tumor suppressing effect of iodinestarch-ALG hydrogel. This work opens up a new way for the development of high-performance and biocompatible biomaterials via teaching old drugs new tricks, greatly benefiting the clinical transformation of biomaterials. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.xxxxxxx. Photothermal conversion efficiency calculation, absorbance intensity curve of iodine-starch under various reaction conditions, FT-IR spectra of starch and iodine-starch complex, photographs and absorption spectra of iodine-starch solution, LS or iodine-starch-ALG hydrogel incubated with various biomolecules, biodegradability assessment in vitro and in vivo, photothermal effect of iodine-starch-ALG hydrogel, viability of 3T3 cells, ROS generation test, blood biochemistry analysis, H&E analysis of skin tissues (PDF).


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [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 Natural Science Foundation of Tianjin City (No. 18JCYBJC20800). ABBREVIATION PTT, photothermal therapy; NIR, near-infrared; LS, Lugol’s solution; ALG, alginate; GSH, glutathione; BSA, bovine serum albumin; DCHF-DA, 2′,7′-dichlorofluorescin diacetate; MTT, thiazolyl blue tetrazolium bromide; DNS, dinitrosalicylic acid; DMSO, dimethyl sulfoxide; ROS, reactive oxygen species REFERENCES (1) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z., Functional nanomaterials for phototherapies of cancer. Chem. Rev. 2014, 114, 10869-10939, DOI: 10.1021/cr400532z.


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For Table of Contents Use Only Biocompatible Iodine-Starch-Alginate hydrogel for Tumor Photothermal Therapy Haoyu Wang,‡ Limei Jiang,§ Huanhuan Wu,‡ Weiya Zheng,† Di Kan,† Ran Cheng,† Juanjuan Yan,‖ Chunshui Yu,*,‡ and Shao-Kai Sun*,†


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Biocompatible Iodine-Starch-Alginate Hydrogel for Tumor

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