Drug-Loaded Polymer-Coated Graphitic Carbon Nanocages for Highly

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Biological and Medical Applications of Materials and Interfaces

Drug-Loaded Polymer-Coated Graphitic Carbon Nanocages for Highly Efficient in Vivo Near-Infrared Laser-Induced Synergistic Therapy through Enhancing Initial Temperature Wenhao Li, Pomchol Han, Yang Chen, Yuliang Guo, Dan Li, Ying Wu, Yan Yue, and Maoquan Chu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11748 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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Drug-Loaded Polymer-Coated Graphitic Carbon Nanocages for Highly Efficient in Vivo Near-Infrared Laser-Induced Synergistic Therapy through Enhancing Initial Temperature

Wenhao Li†, #, Pomchol Han†, #, Yang Chen†,‡,§, Yuliang Guo†, Dan Li†, Ying Wu†, Yan Yue†, Maoquan Chu†,* †

Biomedical Multidisciplinary Innovation Research Institute and Research Center for Translational

Medicine at Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, P. R. China. ‡

§

Institute of Biophysics, Chinese Academy of Science, Beijing 100101, P. R. China. University of Chinese Academy of Sciences, Beijing 100049, P. R. China.

ABSTRACT: Graphitic carbon nanocages (GCNCs) have unique geometric structures and physical properties, which have been extensively investigated for various applications. However, no reports focusing on using GCNCs and polymer-coated GCNCs for solid tumor ablation induced by near-infrared laser irradiation under enhanced initial body temperature, or on the biosafety of GCNCs in vivo, have been published. Here we developed chitosan (CS)-coated GCNCs and show that both GCNCs and GCNCs/CS in mouse tumors can rapidly convert an 808-nm laser light energy into heat, which efficiently kill nasopharyngeal arcinoma cells and inhibit tumor growth. The tumors are further damaged by the phototoxicity of GCNCs/CS after loading with 5-fluorouracil (5FU). Tumors are no longer detected after 6 days of 5FU-GCNCs/CS treatment under irradiation, which is due to the synergistic effect of the

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photothermal response of GCNCs and the chemotherapy of 5FU. None of the tumors reappeared during the following 12 days of no irradiation. Interestingly, increasing the initial body temperature of the mice significantly improved the photothermal effect of GCNCs in vivo, and the synergistic effect of PTT and chemotherapy; and thus accelerated the shrinking of tumors. To the best of our knowledge, this is the first study to improve the photothermal ablation of GCNCs and synergetic photothermal-chemotherapy of drug-loaded GCNCs through enhancing the initial body temperature. As the results show that GCNCs, GCNCs/CS and 5FU-GCNCs/CS are safe in mice after intratumoral injection both with and without laser irradiation, our technique may have great potential for future clinical translation. KEYWORDS: graphitic carbon nanocages, enhancing initial body temperature, improved photothermal effect, improved in vivo cancer phototherapy, biosafety

1. INTRODUCTION Cancer therapy remains one of the greatest challenges in medicine. To improve therapeutic efficiency and overcome clinical relapse, combination therapy is often used to efficiently treat numerous types of tumors.1 Nanocarriers are ideal platforms for incorporating different drug molecules into a single system for combination therapy. Nanocarriers with therapeutic functions have attracted significant attention for drug delivery, as such drug-loaded nanosystems exhibit dualor multi-modal therapeutic properties. These nanocarriers include gold nanocages, carbon nanotubes (CNTs), graphene oxide (GO), and reduced graphene oxide (rGO), graphene quantum dots (GQDs), activated carbon (AC), black mesoporous silicon nanoparticles, black polymer nanospheres, and hollow magnetic Fe3O4 nanoparticles, among others. These nanoparticles not only have hollow and/or porous structures or large surface areas with aromatic rings on the particle surface for efficient drug loading; but can also inhibit tumor growth through photothermal therapy (PTT) (e.g., 2

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all nanoparticles as described above) and/or photodynamic effects (e.g., GQDs). Among these numerous nanoparticles, graphene-based nanoparticles, such as CNTs,2-7 GO,8-12 rGO,13-16 and GQDs,17-19 have attracted significant interest for drug loading and cancer combination therapy. Graphitic carbon nanocages (GCNCs) are an important member of the graphene-based nanomaterial family as they have a large internal space and graphitic shell. These promising cage-like nanomaterials have many interesting physical and chemical properties such as excellent electrical conductivity and high chemical and thermal stability.20-24 During the past decades, the applications of GCNCs mainly focus on hydrogen production and storage,25,26 and as electrode materials,27,28 catalyst supports29,30 and super capacitors.31,32 GCNCs absorb red and near-infrared light owing to their graphitic walls, which could make them a potential photothermal agent for cancer PTT. In addition, both the graphitic walls and internal cage of GCNCs are suitable for entrapping drugs, which could make them a promising drug delivery platform for cancer therapy. However, no reports on using GCNCs for enhanced in vivo photothermal laser ablation through increasing the initial body temperature, or in vivo biosafety have been published. In this work, we found that GCNCs are excellent photothermal agents in the NIR region for local ablation of solid mouse tumors, and the tumor inhibition efficiency was significantly improved by increasing the initial body temperature of the mice. We selected nasopharyngeal carcinoma (NPC) as a cancer model, as it is a common type of head and neck cancer which results in a superficial tumor located in nasal cavity, which provides easy access for local therapy such as laser-induced photodynamic therapy.33-36 Local PTT may therefore also be suitable for this type of cancer. To enhance the total amount of drug loaded in the carrier and improve the sustained drug release effect, chitosan (CS), a polymer with good biocompatibility and biodegradability, was used as a GCNC coating. 5-fluorouracil (5FU) was then incorporated into the GCNCs/CS nanospheres. 5FU was selected as a chemotherapeutic drug model, as it is commonly used for clinical NPC therapy. These 3

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nanomaterials, particularly the 5FU-loaded GCNCs/CS, exhibited high phototoxicity under 808-nm laser irradiation. Mouse NPC tumors were no longer detected after being exposed to the phototoxicity, and 5FU-loaded GCNCs/CS showed a strong synergistic effect between PTT and chemotherapy in inhibiting tumor growth (Figure 1). When the initial body temperature was enhanced, the temperature reached by GCNCs upon NIR laser irradiation was also higher compared with the unenhanced case, and the mouse tumors treated with the photothermal effect of GCNCs disappeared more rapidly. Owing to the biocompatibility and high efficiency of the cancer therapy, the 5FU-loaded GCNCs/CS nanosystem developed in this work may have significant potential for clinical cancer treatment.

Figure 1. Schematic diagram: developing 5FU-loaded GCNCs/CS nanospheres for cancer chemo-photothermal combination therapy and improving combination therapy through enhancing the initial mouse body temperature. 4

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2. EXPERIMENTAL SECTION 2.1. Chemicals. Ferrous oxalate was purchased from Xiya Chemical Industry Co., Ltd. (China). The RPMI-1640 culture medium and fetal calf serum was purchased from Hylcone (Logan, UT, USA). 5-fluorouracil and HEPES buffer were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents including absolute ethanol, CS, sodium tripolyphosphate (TPP), hydrochloric acid, n-hexanol, cyclohexane, Triton X-100, sodium hydroxide and distilled water were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All reagents were used as received without any further purification.

2.2. Cell lines and Animals. Human nasopharyngeal carcinoma cells (CNE line) were purchased from the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Nude mice (6–7 weeks old, BALB/c) were purchased from the Shanghai Sipper-BK Lab Animal Co., Ltd. (Shanghai, China), and were housed in a pathogen free environment and provided with food and water ad libitum. All animal experiments were performed in accordance with the University of Tongji Institutional Animal Care and Use Committee Guidelines.

2.3. Preparation of GCNCs, GCNCs/CS and 5FU-loaded GCNCs/CS nanospheres. The GCNCs were synthesized according to the method reported in published paper with a slight modification.37 Briefly, ferrous oxalate (1.5 g) was dissolved in ethanol (15 mL) in a stainless-steel autoclave with a capacity of 20 mL. After the autoclave was sealed, the sample was heated from room temperature to 550 °C over 55 min in an electronic furnace. Following 12 h of reaction at 550 °C, the autoclave was cooled to room temperature naturally. The precipitate was collected and washed three times with ethanol. Hydrochloric acid solution (14%, 20 mL) was added to the precipitate and the mixture was stirred at 90 °C for 6 h. The black precipitate was then washed with distilled water until the 5

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suspension reached neutral pH. Finally, the precipitate (GCNCs) was collected and freeze-dried. To prepare GCNCs/CS nanospheres, GCNCs dry powder (1 mg) was added to an aqueous solution of CS (1 mg/mL, 3 mL) containing 50 µL of acetic acid, followed by ultrasonication for 10 min. The pH was adjusted to 7.0 using sodium hydroxide. This solution was then added to a mixture of n-hexanol (8.8 mL), cyclohexane (31.2 mL) and Triton X-100 (10 mL) with stirring at room temperature to produce a microemulsion system. TPP solution (1 mg/mL, 1 mL) was then added and the mixture was stirred at room temperature for 2 h. The mixture was then centrifuged, and the precipitate obtained was washed with ethanol followed by distilled water. As a control, CS nanospheres without GCNCs were prepared using the same methods. For preparation of 5FU-GCNCs/CS, GCNCs/CS nanospheres were mixed with 5FU in a 2 mL of aqueous solution. The concentrations of GCNCs and 5FU were 0.5 and 1 mg/mL, respectively. Forty eight hours later, the mixture was centrifuged and the precipitates were collected. This drug-loading experiment was repeated in triplicates. The loading efficiency (the mass ratio of 5FU in GCNCs/CS to GCNCs/CS) was calculated to be (22.4 ± 2.0) %.

2.4. Measurement of the photothermal conversion of nanoparticles at different initial temperatures. CNE cells (~1 × 106 for each mouse) were injected subcutaneously into eight mice on their right sides. When the tumor diameter reached ~4 mm, three tumors were intratumorally injected with 50 µL of HEPES buffer-dispersed GCNCs, GCNCs/CS and 5FU-GCNCs/CS, the remaining mouse was intratumorally treated with 50 µL of HEPES buffer. The initial temperature of the mouse tumor was maintained at the normal level (~36.9 ± 0.2 °C). The tumors were then irradiated with an 808-nm laser (Shanghai Inter-Diff Optoelectronics Tech. Co., Ltd., Shanghai, China) with a low power density of 0.25 W/cm2 for 0–20 min. The concentrations of GCNCs and 5FU were 0.2 and 0.17 mg/mL, respectively. The temperatures of the tumor-bearing mice were 6

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measured using a Ti29 thermal imager (Fluke Corporation, Everett, WA, USA). To enhance the initial body temperature, the remaining four tumor-bearing mice (tumor diameter: ~4 mm) were intratumorally injected with HEPES buffer-dispersed GCNCs/CS (GCNCs: 0.2 mg/mL, 50 µL) or HEPES buffer alone (n=2 for each group), followed by heating with an infrared lamp (Philips, 60W) for ~15 min. The distance between the lamp and mouse was maintained at ~3.5 m. When the tumor temperature was increased to 38.1 ± 0.1 °C in ~15 min and did not obviously change throughout the subsequent ~20 min of continuous lamp irradiation, the initial tumor temperature or body temperature was considered to have stabilized. The two tumors injected with GCNCs/CS or HEPES buffer with initial temperature of 38.1 ± 0.1 °C were then immediately irradiated with an 808-nm laser for 20 min. During the laser irradiation, the mice were continuously exposed to the lamp. The remaining two tumors with initial temperature of 38.1 ± 0.1 °C were continuously irradiated with the lamp for 20 min. Each tumor was measured in triplicate. In addition, distilled water-dispersed GCNCs/CS (200 µL, 0.01 mg/mL) placed in small transparent glass tubes were irradiated with an 808-nm laser for 0–10 min. The initial temperature was maintained at 25.2 ± 0.3 °C, 27.5 ± 0.2 °C, or 29.7 ± 0.5 °C. Each experiment was repeated in triplicate.

2.5. In vivo anticancer activity of the nanoparticles under NIR laser irradiation and normal initial body temperature. Thirty five mice with similar tumor size (length≈width≈4 mm, grown from CNE cells) were selected and randomly divided into seven groups (n=5, for each group), as detailed

below:

the

mice

were

intratumorally

injected

with

HEPES

buffer-dispersed

5FU-GCNCs/CS, GCNCs/CS or GCNCs and then irradiated with the 808-nm laser (20 min/mouse/day);

the

mice

were

intratumorally

injected

with

HEPES

buffer-dispersed

5FU-GCNCs/CS, GCNCs/CS or 5FU, without laser irradiation; and the final group was untreated 7

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mice. The sample volume for injection was 50 µL, and the concentrations of GCNCs and 5FU were 0.2 and 0.17 mg/mL, respectively. After 3 days of irradiation, one mouse was selected at random from each group, and the tumors were resected after anaesthesia. After the sizes of the resected tumors were measured, the tumors were embedded in paraffin wax and cut into ultrathin sections (5 µm) using an ultramicrotome (Leica RM2126RT, Germany). These sections were then stained with H&E reagents, and examined using the upright microscope (Eclipse TE2000-S, Nikon, Japan). The remaining tumor-bearing mice were continuously irradiated with the laser or housed to record the tumor growth rate. When the tumors in the irradiation groups could no longer be detected, the irradiation was stopped. During the 18 days of treatment, the tumors were measured using a caliper every 3 days, and the tumor morphologies were taken every 3 days using an Apple iPad mini (Cupertino, CA, USA). Tumor volume was calculated using the formula: tumor volume = (length×width×width)/2. At 19 days post-injection, the mice were anaesthetized and tumors were resected, cut into ultrathin sections for histological analysis using the same method as described above. The mice were then subjected to blood and major organ analysis (see Sections 2.7 and 2.8)

2.6. In vivo anticancer activity of the nanoparticles under NIR laser irradiation and enhanced initial body temperature. Twelve mice with similar tumor size (length≈width≈4 mm, grown from CNE cells) were selected and randomly divided into three groups (n=4 for each group), as detailed below: the mice were intratumorally injected with HEPES buffer-dispersed 5FU-GCNCs/CS or GCNCs/CS and then exposed to a lamp. When the tumor temperature reached ~38 °C and maintained this temperature for ~20 min (total time of lamp exposure: ~35 min), the tumors were immediately irradiated with an 808-nm laser for 20 min. During the laser irradiation, the mice were continuously exposed to the lamp. Each mouse was treated with the lamp and laser 8

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under the same conditions each day. As a control, tumor-bearing mice that received no nanoparticle dose were exposed to the lamp for ~55 min every day (the first 35 min was used to heat the mice and keep the tumor temperature at ~38 °C, and the subsequent 20 min of exposure to the same lamp was used as a control to compare with the 20 min of laser irradiation). For convenient expression, the above groups were denoted “5FU-GCNCs/CS + lamp + laser”, “GCNCs/CS + lamp + laser” and “lamp only”. When the tumors in the irradiation groups could no longer be detected, the irradiation was stopped. The tumor size and morphology were recorded using the methods described above. At 19 days post-dosing, the mice were euthanized for tumor histological analysis (see Section 2.5), as well as blood and major organ analysis (see Sections 2.7 and 2.8) In addition, two tumor-bearing mice with similar tumor size (length≈width≈4 mm, grown from CNE cells) were intratumorally injected with HEPES buffer-dispersed GCNCs/CS and then exposed to a lamp until the tumor temperature increased and was maintained at ~38 °C. One mouse was irradiated with an 808-nm laser for 20 min, and the other was not exposed to the laser (both were continuously exposed to the lamp throughout the 20 min interval). One control included one tumor-bearing mouse being intratumorally injected with HEPES buffer-dispersed GCNCs/CS, and directly subjected to 808-nm laser irradiation for 20 min (i.e., initial temperature of ~37 °C). For another control one tumor-bearing mouse did not undergo any treatment. One day after treatment, the tumors were resected for histological analysis using the method described above.

2.7. Blood analysis. At 19 days post-injection, the mice described above (see Sections 2.5 and 2.6) were anaesthetized and blood was collected from the retro-orbital sinus (~0.8 mL for each mouse). Approximately half of the blood collected was centrifuged and the resulting serum was used for kidney and liver function analysis. The parameters for these analyses included albumin (ALB), globulin (GLOB), the ratio of albumin and globulin (A/G), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), total bilirubin 9

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(TBIL), creatinine (CRE) and urea. The remaining blood was used for the analysis of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelets (PLT). All blood analyses were performed at KingMed Diagnostics (Shanghai, China). As a control, the blood of four normal nude mice was analyzed using the same methods.

2.8. Histological analysis of organs. Following blood collection, the mice were euthanized and the major organs; including spleen, liver, lung, and kidney, were resected and weighed. Histological analysis was then carried out using the method described above. No tumor tissue sections were prepared for the groups in which tumors were no longer detected after treatment. The organ coefficients (the ratio of organ weight to mouse body weight) were calculated.

2.9. Statistical analysis. Unless otherwise specified, results are expressed as mean ± SD. In vivo experiments were compared by one-way analysis of variance (ANOVA), using the protected LSD test. The Student t-test was used for the comparison of the means of two samples. P < 0.05 was considered to be statistically significant, P < 0.01 was considered to be highly significant, and P < 0.001 was considered to be very highly significant.

3. RESULTS AND DISSCUSSION 3.1. The temperature increase of GCNCs or GCNCs/CS upon 808-nm laser irradiation was improved significantly by enhancing the initial temperature. The GCNCs synthesized in this work were hollow and spherical in shape with average diameter of 77.6 ± 11.7 nm, which has been reported in our previous work.38 GCNCs/CS nanocomposites were also spherical in shape with average diameter of 583.5 ± 129.0 nm (Figure S1). Both GCNCs and GCNCs/CS in mouse tumors could rapidly convert laser light energy into heat (Figure 2a,2c). 10

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When a tumor-bearing mouse was intratumorally injected with 50 µL of HEPES buffer-dispersed GCNCs with a concentration of only 0.2 mg/mL; the tumor temperature rapidly increased from ~37.0 °C (initial temperature) to 46.8 °C after 5 min of 808-nm laser irradiation, and further increased to 47.1 °C after 20 min of irradiation. The laser-triggered GCNCs/CS group showed a similar response, that is: the tumor temperatures reached 47.0 °C after 5 min of irradiation, and remained at around 47.0 °C in the subsequent 15 min of irradiation. For the tumor injected with 50 µL of HEPES buffer alone, however, the tumor temperature increased to no more than 39.6 °C after 20 min of irradiation. These results indicate that both GCNCs and GCNCs/CS nanospheres are excellent photothermal agents for cancer therapy. In addition, CS coating does not significantly reduce the photothermal effect of GCNCs in tumor tissue. When the initial mouse tumor temperature was increased to ~ 38.0 °C and was maintained at this temperature by lamp irradiation, the tumor temperature increased more rapidly after treatment with the photothermal effect of GCNCs (Figure 2a, 2c). For example, after 20 min of 808-nm laser irradiation, the temperature of tumors injected with GCNCs/CS increased from a initial temperature of ~38.0 °C to 49.8 ± 0.1 °C, which is ~2.6 °C higher than the case when the initial temperature was ~37.0 °C as described above. That is: only one degree of difference between the initial temperature, resulted in ~2.6 °C of difference between the final temperature after 20 min of laser irradiation. As a control, the temperature of tumors containing the same GCNCs/CS without 808-nm laser irradiation changed from 38.1 ± 0.1 °C to 38.2 ± 0.2 °C after 20 min of lamp irradiation only, which indicates that it is difficult to further increase the mouse body temperature by lamp irradiation after the temperature has reached 38.1 ± 0.1 °C through ~35 min of lamp irradiation (see experimental section).

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Figure 2. Photothermal conversion of the samples in mouse CNE tumors or in aqueous solution in response to 808-nm laser irradiation at different initial temperatures: (a, c) Temperature increase of mouse tumors intratumorally injected with 50 µL of HEPES buffer-dispersed GCNCs or GCNCs/CS (GCNCs: 0.2 mg/mL) under 808-nm laser irradiation at a initial tumor temperature of 36.9 ± 0.2 °C (laser group) or 38.1 ± 0.1 °C (lamp + laser group). (b, d) Temperature increase of mouse tumors intratumorally injected with 50 µL of HEPES buffer-dispersed GCNCs or HEPES buffer alone under lamp irradiation at initial body temperature of 38.1 ± 0.1 °C. (a, b) Infrared thermal images. (c, d) Average temperature vs. irradiation time based on the thermal images shown in (a, b). (e, f) Temperature increase of distilled water-dispersed GCNCs/CS under 808-nm laser irradiation varying with the initial temperature. (e) Infrared thermal images. (f) Average temperature vs. irradiation time 12

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based on the thermal images shown in (e). (IT : initial temperature)

The results described above show that the temperature increase of tumors containing nanoparticles upon laser irradiation was associated with the initial temperature; the higher the initial temperature, the greater the temperature increase following laser irradiation. To confirm this observation, the photothermal conversion of aqueous suspensions of GCNCs/CS under 808-nm laser irradiation at various initial temperatures was also measured. As shown in Figure 2e and 2f, when the initial temperatures were 25, 27.5, and 30°C, the temperatures of GCNCs/CS aqueous suspension increased to 33.6 ± 0.3, 37.4 ± 0.5, and 42.6 ± 0.5°C, respectively, after 10 min of 808-nm laser irradiation. This further indicates that the higher the initial temperature, the higher the final temperature, and the larger the difference between the initial and final temperature after laser irradiation. This interesting result implies that slightly enhancing the initial body temperature could noticeably improve the photothermal effect of GCNCs in tumor tissue. Although enhancing the laser power density can also improve the photothermal effect of nanoparticles,39-41 lasers with low power density can avoid damaging normal tissue.39,42-46 In this work, an 808-nm laser with only 0.25 W/cm2 was used for cancer PTT. The combination of a low power laser and a lamp is safe, simple, and convenient for improving the in vivo PTT effect of GCNCs.

3.2. Tumor tissue was seriously damaged after only 3 days of treatment with GCNCs, GCNCs/CS or 5FU-GCNCs/CS and NIR laser irradiation. Mouse tumor tissue damaged by GCNCs, GCNCs/CS or 5FU-GCNCs/CS upon 808-nm laser irradiation could be clearly identified, as the tumors had the appearance of having been burned. As shown in Figures S2-S3, when the tumor-bearing mice were intratumorally injected with 50 µL of the HEPES buffer-dispersed GCNCs 13

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(0.2 mg/mL) or GCNCs/CS (GCNCs: 0.2 mg/mL) and then irradiated with the 808-nm laser for 20 min every 24 h, dark spots appeared on the tumors after only 3 days of irradiation. This may have been caused by the photothermal stimulation of the GCNCs. For the mice intratumorally injected with 50 µL of HEPES buffer-dispersed 5FU-GCNCs/CS (GCNCs: 0.2 mg/mL, 5FU: 0.17 mg/mL) and then irradiated with the 808-nm laser every 24 h, all tumors also showed signs of significant damage after only 3 days of laser irradiation (Figure S4). For other groups including the tumors injected with GCNCs/CS, 5FU-GCNCs/CS (GCNCs: 0.2 mg/mL, 5FU: 0.17 mg/mL) or free 5FU (0.17 mg/mL) without laser irradiation, it was not possible to observe whether or not the tumors were damaged compared with the tumors which had received no treatment (Figures S5-S8). One mouse in each group was therefore selected at random after 3 days of treatment, and their tumors were resected for analysis. As shown in Figure 3a, the tumor size for the 5FU group without laser irradiation was similar to those for the ‘GCNCs + laser’ and ‘GCNCs/CS + laser’ groups, and these sizes were significantly smaller than those in the no treatment group. These findings indicate that the toxicity of 5FU and the photothermal effects of GCNCs or GCNCs/CS can all efficiently inhibit tumor growth. Compared with the photothermal conversion of GCNCs/CS in tumor (Figure 2a), the temperature increase of 5FU-GCNCs/CS in tumor upon 808-nm laser irradiation was not obviously influenced by the incorporation of 5FU (Figure 3b). Therefore, the tumor size in the ‘5FU-GCNCs/CS + laser’ group was the smallest among all groups, as both the photothermal effect and drug toxicity simultaneously acted on the tumor tissue. H&E stained microscopy images showed that the cancer cells in the tumor tissue treated with the 5FU-GCNCs/CS and laser were the most seriously damaged among the groups, and the intercellular spaces were significantly larger than those in the untreated tumor tissue (Figure 3c). Upon careful observation, some cell nuclei in this group were lost. The cells in the ‘GCNCs + laser’, ‘GCNCs/CS + laser’ or 5FU and 5FU-GCNCs/CS without laser irradiation groups were also significantly 14

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damaged compared with those in the untreated group. GCNCs/CS without laser irradiation may have little influence on tumor growth, as the H&E stained images for these groups is similar to that in the untreated group. The results presented indicate that the combination of the photothermal effect of GCNCs and the toxicity of 5FU could significantly improve the efficiency of cancer therapy.

Figure 3. Mouse CNE tumors were damaged by 5FU-GCNCs/CS, GCNCs/CS, or GCNCs, combined with 808-nm laser irradiation for 3 days, and controls. (a) Resected mouse tumors from the following groups: (1) 5FU-GCNCs/CS + laser; (2) GCNCs/CS + laser; (3) GCNCs + laser; (4) 5FU-GCNCs/CS; (5) GCNCs/CS; (6) 5FU and (7) no treatment. (b) Photothermal conversion of 5FU-GCNCs/CS in tumors upon laser irradiation determined using infrared thermal imaging (the photothermal conversions of GCNCs and GCNCs/CS in tumors are shown in the previous Figure 2). (c) H&E staining images of tumor tissues. The doses of GCNCs and 5FU were maintained at 0.2 and 0.17 mg/mL (volume: 50 µL), respectively. Scale bar: 100 µm. 15

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3.3. Tumors no longer detected after treatment with GCNCs, GCNCs/CS or 5FU-GCNCs/CS and NIR laser. The remaining tumor-bearing mice in each group described above (section 3.2) were continuously irradiated using an 808-nm laser and/or the tumor growths were continuously monitored. As shown in Figure 4a,4b and Figures S2-S3, no tumors were detected in the mice treated with GCNCs or GCNCs/CS combined with 808-nm laser irradiation, after 9 days of treatment. In addition, the tumors did not recover in the following 9 days when no laser irradiation was applied. However, the tumors injected with GCNCs/CS without laser treatment, gradually grew, and the average tumor growth rate was not statistically different from that of tumors without any treatment (Figure 4a,4c and Figure S5). For example, at 18 days of treatment, the average tumor volume in the GCNCs/CS and no treatment groups were 366.9 ± 64.8 and 398.4 ± 68.0 mm3, respectively. These results indicate that the tumor tissue was very sensitive to the photothermal effect generated by the GCNCs, but may not be affected by the nanoparticle system in the absence of irradiation. As shown in Figure 4a,4c and Figure S6, for the 5FU group, although all tumors were still detected following 18 days of treatment, there was a clear reduction in the average tumor volume compared with tumors that received no treatment (P