In vivo investigation

This animal experiment was approved by the ethical committee for animal care of National Taiwan University and was performed following the national regulations' guidelines.

Mice (C57BL/6) were purchased from BioLASCO Co. (Taipei, Taiwan).

2.2.3.1 Subcutaneous tumor implant model of mice

Subcutaneous glioma implants model was adopted to monitor tumor volume serially according to published studies [70-72]. Six-week-old male C57BL/6 mice were used and ALTS1C1 glioma cells with luciferase reporter gene [73, 74] were injected subcutaneously

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in their right thigh [70, 71]. When the tumor diameter grew up to 3 to 4 mm and adequate for intratumoral drug injection, mice were categorized into different treatment groups.

2.2.3.2 Intratumoral dye injection

Blue colorant (20 μL) containing methylene blue (Sigma-Aldrich, Missouri, USA) was mixed with oxi-HA/ADH hydrogel (80 μL) and injected into the subcutaneous tumor via a syringe with a 28-gauge needle. The mice were sacrificed 1 day and 3 days after intratumoral dye injection respectively, followed by tumor excision and gross section to evaluate the distribution of dye loaded by hydrogel [49].

2.2.3.3 Irradiation setting

The mice were anesthetized by intramuscular injection (1 µl/g) of a mixture with Zoletil 50 (Virbac Laboratory, France) and 2% Xylazine (Bayer, Germany) in a 2:1 ratio [75, 76].

The radiation was delivered with 20 Gy (10 Gy daily fraction for 2 fractions, at a dosage rate of 0.5 Gy/min) adjusted according to the published data and tumor growth condition [48, 49, 51] by a cobalt-60 unit (V-9, PICKER, USA). The distance from the radiation source to the skin of right thigh was 80 cm. A customized harness was used to immobilize the mice with only the right hind leg was exposed and the remainder of the body was shielded with five times the half-value thickness of lead [77].

2.2.3.4 Different treatment combinations of carboplatin and irradiation

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The total carboplatin dose was classified as low-dose (60 µg, 3 µg/g) and high-dose (300 µg, 15 µg/g) calculated by mouse body weight and adjusted according to the published studies [56, 78] as well as the feasible drug loading volume by mouse tumor size to explore the adequate carboplatin dose combined with RT. In order to compare the treatment effect of hydrogel carboplatin with aqueous carboplatin, the dose of hydrogel carboplatin was injected in a single dose, while the equal amount of aqueous carboplatin was divided into 3 doses and injected in 3 consecutive days. Hydrogel carboplatin or aqueous carboplatin was injected into the subcutaneous tumor by a syringe with a 28-gauge needle. The mice were categorized into the following groups according to their treatments: including shame (S), hydrogel (H), aqueous carboplatin (AC), hydrogel carboplatin (HC), RT (R), RT with hydrogel (HRT), RT with aqueous carboplatin (ACR), and RT with hydrogel carboplatin (HCR).

The purpose of the first-stage in vivo experiment was to investigate the synergistic effect of low-dose hydrogel carboplatin or aqueous carboplatin with RT. Mice were randomized into 5 treatment groups, including S, R, HRT, ACR, and HCR groups. The purpose of the second-stage in vivo experiment was to investigate the synergistic effects of high-dose hydrogel carboplatin or aqueous carboplatin with RT. Mice were randomized into 7 treatment groups, including S, H, AC, HC, R, ACR, and HCR groups.

Figure 8A and Figure 8B illustrated the cell number, dosage of hydrogel carboplatin and aqueous carboplatin, radiation dose, and schedules of drug delivery for first and second in vivo experiments, respectively [59]. The protocol of the second-stage experiment was modified according to the results of the first-stage experiment. The primary endpoint of the in vivo investigation was the tumor growth control effect according to different treatments.

The secondary endpoints included survival and treatment-related local and systemic

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toxicities. The survival time was calculated from the day of tumor cell implant to the day of mice death or being sacrificed due to tumor dimension ≥ 20 mm or skin ulcer development.

The treatment effects were compared among different treatment groups, especially between the single injection of hydrogel carboplatin and multiple injection of aqueous carboplatin.

2.2.3.5 Tumor growth evaluation by gross volume measurement and bioluminescence imaging (BLI)

The length and width of tumors were measured by an electronic caliper and the tumor volume was estimated by the formula: volume = 1/2 × length × width² [79]. Tumor volume was evaluated every 2 to 5 days according to the tumor growth condition. BLI was used for sequential tumor growth monitor [80]. Mice were anesthetized by isoflurance gas (8.6 mg/kg, respectively) and D-luciferin (0.2 mL) was injected intraperitoneally. BLI was acquired by spectrometer, In Vivo Image System (IVIS) SPECTRUM (exposure time: 30 sec; binning:

medium; f/stop: 1) with Living Image® software (PerkinElmer), 10 minutes after D-luciferin injection with exposure for 30 seconds. BLI was evaluated every 5 to 8 days according to the tumor growth condition and facility availability [81]. The intensity of bioluminescence signal (expressed in radiance) from ALTS1C1-luc tumors and the maximum value was quantified by Living Image® software to estimate the tumor burdens [55].

2.2.3.6 Treatment effect evaluation by tumor gross and slice

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The gross tumors of different treatment combinations were dissected after mice being sacrificed, and slice with hematoxylin and eosin (H&E) stain was used to evaluate the histologic tumor cell proliferation and death.

2.2.3.7 Toxicity evaluation by blood analysis and skin survey

Systemic toxicity of carboplatin was evaluated by the blood biochemistry study (BCS), including liver and kidney functions, and complete blood count (CBC), which were sampled 6 days after intratumoral injection of high-dose aqueous carboplatin [82]. The BCS and CBC were analyzed by ProCyte Dx® hematology analyzer (IDEXX, Maine, USA) and Cobas c111 analyzer (Roche, New Jersey, USA) respectively. Local toxicity of RT, such as skin ulcer, usually developing within 2 weeks after RT, was monitored sequentially up to 3 weeks after treatment.

2.2.4 Statistical analysis

Data analysis and statistical tests were performed using SPSS. OS was calculated on the basis of the date of tumor plant to the date of death or being sacrificed. Survival was calculated using the Kaplan-Meier product-limit method. Differences in survival were compared between the groups by using the log-rank test. Biocompatibility investigation of oxi-HA/ADH and tumor volume differences on a specific day among different treatment groups were analyzed by using one-way analysis of variance (ANOVA) or t-test according to the number of groups. All tests were two-sided and results with p < 0.05 were considered statistically significant.

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