Overcoming the Heat Endurance of Tumor Cells by ... - ACS Publications

Overcoming the Heat Endurance of Tumor Cells by Interfering with the Anaerobic Glycolysis Metabolism for Improved Photothermal Therapy Wei-Hai Chen,†,§ Guo-Feng Luo,†,§ Qi Lei,† Sheng Hong,† Wen-Xiu Qiu,† Li-Han Liu,† Si-Xue Cheng,† and Xian-Zheng Zhang*,†,‡ †

Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry and ‡The Institute for Advanced Studies, Wuhan University, Wuhan 430072, People’s Republic of China S Supporting Information *

ABSTRACT: In this study, we developed a general method to decorate plasmonic gold nanorods (GNRs) with a CD44-targeting functional polymer, containing a hyaluronic acid (HA)-targeting moiety and a small molecule Glut1 inhibitor of diclofenac (DC), to obtain GNR/HA-DC. This nanosystem exhibited the superiority of selectively sensitizing tumor cells for photothermal therapy (PTT) by inhibiting anaerobic glycolysis. Upon specifically targeting CD44, sequentially time-dependent DC release could be achieved by the trigger of hyaluronidase (HAase), which abundantly existed in tumor tissues. The released DC depleted the Glut1 level in tumor cells and induced a cascade effect on cellular metabolism by inhibiting glucose uptake, blocking glycolysis, decreasing ATP levels, hampering heat shock protein (HSP) expression, and ultimately leaving malignant cells out from the protection of HSPs to stress (e.g., heat), and then tumor cells were more easy to kill. Owing to the sensitization effect of GNR/HA-DC, CD44 overexpressed tumor cells could be significantly damaged by PTT with an enhanced therapeutic efficiency in vitro and in vivo. KEYWORDS: tumor targeting, anaerobic glycolysis, photothermal therapy, gold nanorod

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tumor cells’ susceptibility to heat, specific HSP inhibitors and small-interfering RNA (siRNA) have been employed in hyperthermia treatment.13−18 However, the use of small molecular inhibitors is a hysteretic process because they only act on the already existing HSPs after heat stimuli rather than before treatment. Additionally, inhibitors lacked universality as one kind of inhibitor targets one specific HSP. Moreover, for siRNA delivery to target cells, there are various barriers including serum stability, cellular uptake, and endosomal escape that must be overcome.19,20 All of these issues severely hinder the inhibition efficiency of HSPs. Hence, there in an urgent need to develop a strategy to increase the effective therapeutic window of hyperthermia by depleting HSPs. As described by Warburg, a hallmark of human cancers is the anaerobic glycolysis that produces adequate adenosine 5′-triphosphate (ATP) and biological components to meet the rapid metabolic requirements.21,22 Such a metabolic

hotothermal therapy (PTT), as a noninvasive therapeutic model, has attracted considerable research attention in tumor therapy. PTT is a hyperthermia (over 41 °C) treatment of tumorigenic cells to eliminate them with spatiotemporal selectivity.1−6 Unfortunately, hyperthermia-treated cells have been confirmed to readily acquire tolerance to heat stress, which markedly enhances their survival ability, termed as thermoresistance,7−9 resulting in ineffective therapy in PTT. As a result, high-power laser or repeated light irradiation has been suggested to produce a useful level of therapeutic efficiency. However, this may cause adverse risks including inflammatory disease and tumor metastasis. Therefore, imperative development of a general strategy to reverse thermoresistance in PTT for improved therapeutic efficacy has been challenging. Heat shock proteins (HSPs) have been recognized as the key factor in thermoresistance to initiate the defense mechanism of tumors. When it comes to treating tumorigenic cells with heat, HSPs (e.g., HSP70, HSP90, and HSP110) are produced rapidly at an enhanced level that chaperone denatured proteins, which exert central antiapoptotic and cytoprotective effects in preventing cell death.10−12 With the expectation of elevating © 2017 American Chemical Society

Received: October 4, 2016 Accepted: January 20, 2017 Published: January 20, 2017 1419

DOI: 10.1021/acsnano.6b06658 ACS Nano 2017, 11, 1419−1431

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Scheme 1. Schematic Illustration of GNR/HA-DC for Selectively Sensitizing Tumor Cells to Photothermal Therapy by Interfering with the Anaerobic Glycolysis Metabolism

via esterification (Scheme S1 and Figure S1) in advance. Thereafter, the tumor-targeting polymer of HA-LA or HA-LA/ DC was conveniently decorated on GNRs by the formation of Au−S bonds to obtain GNR/HA or GNR/HA-DC to study the tumor-targeted metabolic therapy-promoted PTT. The transmission electronic microscopy (TEM) images in Figure 1A displayed good dispersion of nanoparticles with or without the polymer coating, guaranteeing the feasibility of GNR/HA-DC for biological applications. Moreover, only small changes in hydrodynamic diameter and polydispersity index (PDI) were detected during 4 days (Table S1 and Figure S2), indicating that both GNR/HA and GNR/HA-DC were stable at normal physiological conditions. Additionally, both GNR/HA and GNR/HA-DC were negatively charged, and the ζ-potential was −26.8 ± 1.2 mV for GNR/HA and −20.4 ± 1.4 mV for GNR/ HA-DC (Table S1). After being coated with HA-LA and HA-LA/DC, the longitudinal surface plasmon resonance (SPR) peak of GNRs (750 nm) showed a slight red shift to 765 nm for GNR/HA and 768 nm for GNR/HA-DC (Figure 1B). For GNR/HA-DC, an additional peak at 270 nm from DC absorbance appeared, confirming the existence of DC in the nanosystem. As further key evidence, X-ray photoelectron spectroscopy (XPS) was applied and the spectra (Figure 1C and Figure S3) revealed that signals of O 1s strongly increased after polymer coating with the appearance of an additional characteristic peak of S 2p at 168.8 eV in GNR/HA (vs GNRs) and Cl 2p at 198.6 eV in GNR/HA-DC (vs GNR/HA), validating the introduction of LA in GNR/HA and DC in GNR/ HA-DC, respectively. From thermogravimetric (TG) curves (Figure 1D), the amount of DC loaded in GNR/HA-DC was calculated to be 21.3 wt %, which was close to the result obtained from NMR data (19 wt %). An enzyme-triggered DC release was observed with more than 87% of DC liberating from GNR/HA-DC after post-treatment with HAase for 24 h (Figure 1E). Additionally, the release rate increased with HAase concentration. However, in the absence of HAase, no significant amount of DC was released. Similarly, significant DC release was detected in the extracts of tumor tissues (Figure S4), implying that DC could be liberated in the tumor. Since HAase is specifically overexpressed in the tumor microenvironment and effectually breaks down HA,41,42 it provides a promising

pathway needs high rates of glucose uptake, which greatly relies on the up-regulated glucose transporters (Gluts). One of the typical representatives is Glut1, which is ubiquitously overexpressed in tumor cells.23−25 Thus, targeting Glut1-mediated glucose transport has been suggested as an effective strategy to depress tumor cell progression.26−28 More importantly, the deprivation of intracellular glucose by specific inhibition of Glut1 leads to the suppression of glucose metabolism in cells, thereby decreasing the production of metabolites (e.g., lactate and ATP).29,30 As the most important and indispensable energy sources of living entities, enough intracellular ATP production is crucially important for cell growth and protein synthesis, with no exception for HSPs. Thus, the intracellular ATP depletion is proposed to block the HSP generation in cells. This means tumor-specific Glut1 inhibitors with the capability to decrease ATP induces HSP depletion in tumor cells, which is expected to overcome the heat endurance of tumor cells. In this study, we introduced a Glut1 inhibitor delivery nanosystem to elevate PTT efficacy for high-performance tumor therapy. Gold nanorods (GNRs) were chosen as ideal PTT agents because of their strong surface plasmon resonance and outstanding photothermal conversion efficiency.31−34 Diclofenac (DC), a small molecule inhibitor with high selectivity toward Glut1,35,36 was conjugated to a targeting polymer of hyaluronic acid (HA),37−40 which was further decorated on GNRs (GNR/HA-DC). The tumor overexpressing hyaluronidase (HAase) could trigger the release of DC. Subsequently, the liberated DC would induce down-regulation of Glut1 and thereby inhibit the glucose metabolism and ATP-dependent HSP synthesis. Both in vitro and in vivo studies demonstrated that the promoted photothermal eradication of tumor cells by such a cascade effect could be achieved (Scheme 1).

RESULTS Preparation and Characterization of GNR/HA-DC. Rod shaped GNRs (length = 39.6 ± 4.1 nm and width = 10.5 ± 2.3 nm) with an aspect ratio of about 3.8 were prepared as the heat generator for PTT (Figure 1A). To facilitate the coating of a subsequent layer, lipoic acid (LA)-conjugated HA (HA-LA) and LA/DC-conjugated HA (HA-LA/DC) were synthesized 1420


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Figure 1. (A) TEM images of GNRs, GNR/HA, and GNR/HA-DC. (B) UV−vis absorption spectra of GNRs, GNR/HA, GNR/HA-DC, and free DC. (C) XPS spectrum of GNR/HA-DC. Inset: Magnified XPS signal of the Au 4f region. (D) TG weight loss profiles of GNR/HA and GNR/HA-DC. (E) DC release profile of GNR/HA-DC under different conditions. (F) Thermal images of different samples recorded by an IR camera during laser irradiation. (G) Heating curves of GNR/HA-DC at different concentrations irradiated with fixed laser power (808 nm, 1.0 W cm−2). (H) Heating curves of GNR/HA-DC at the fixed concentration of 20 μg mL−1 with different 808 nm laser power.

opportunity for GNR/HA-DC to realize tumor-targeted drug release. Moreover, as the most extensively studied photothermal conversion agent, heat generation from GNR/HA-DC under NIR laser irradiation was monitored by using an IR camera. As displayed in Figure 1F−H, with the increasing nanoparticle concentration and laser exposure power, the ultimate temperature and temperature increase rate of the laser-irradiated nanoparticle suspension increased accordingly, indicating that heat generation could be finely turned with an exact control. Specific Cell Uptake and GNR/HA-DC-Mediated Metabolism Inhibition. One desired property of GNR/ HA-DC is that it can inhibit Glut1, decrease glucose uptake, and subsequently block glycolysis in specific tumor cells. To evaluate the tumor-targeting efficacy of GNR/HA-DC, CD44 positive cell lines (HeLa (human cervical carcinoma cell) and MCF-7 (breast cancer cell)) and CD44 negative normal cell lines (COS7 (kidney fibroblast cell) and 293T (human embryonic kidney transformed cells)) were used in this study. Due to the specific role of HA in targeting its receptors in tumor cells, intracellular gold content was detected by inductively coupled plasma mass spectrometry (ICP-MS) to evaluate the tumor-targeting capacity of GNR/HA-DC. As displayed in Figure 2A, for cells co-incubated with GNR/HA-DC nanoparticles, the amount of gold in CD44 overexpressed tumor cells (HeLa and MCF-7 cells) was much greater than that in normal cells (COS7 and 293T cells), but only a small

amount was detected in CD44 negative COS7 and 293T cells. For example, gold content in HeLa cells was 13.2-fold higher than that in COS7 cells. By blocking the CD44 receptor with free HA, the cellular uptake of GNR/HA-DC in HeLa and MCF-7 cells was inhibited. Similar cellular uptake was observed for GNR/HA nanoparticles (Figure S5). However, it was difficult for the nontargeted nanoparticles (GNRs) to be internalized by all cells lines (Figure S6). These results strongly demonstrated that the remarkably enhanced internalization of GNR/HA-DC by CD44 overexpressed tumor cells was attributed to the CD44 receptor-mediated endocytosis. After specific internalization by tumor cells, the intracellular fate of GNR/HA-DC in affecting cellular metabolism was investigated. According to the “Warburg effect”, glucose transporters, typically Glut1, are widely overexpressed in tumors to adapt to the increased glucose transport for cell survival and proliferation. As confirmed by Western blotting analysis, up-regulated level of Glut1 was detected in tumorigenic cells (HeLa and MCF-7) but not in normal cells (COS7 and 293T) (Figure 2B,C and Figure S7). Note that the great significance of introducing DC in our designed GNR/HA-DC nanosystem is to down-regulate the level of Glut1 expression in tumor cells and consequently to move the targets downstream to further promote the therapy. To evaluate the influence of GNR/HA-DC on Glut1’s expression, HeLa and MCF-7 cells were incubated with GNR/HA-DC for 12, 24, or 48 h. As expected, the level of Glut1 in HeLa and MCF-7 cells was significantly decreased 1421


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Figure 2. (A) ICP-MS analysis of the Au amount in GNR/HA-DC-treated HeLa, MCF-7, COS7, and 293T cells with or without the pretreatment of free HA. (B) Western blotting analysis of Glut1 expression in HeLa and COS7 cells and (C) corresponding gray values. (D) Evaluation of Glut1 expression in HeLa cells after being treated with GNR/HA-DC by Western blotting analysis and (E) corresponding gray values. (F) Confocal laser scanning microscopy images of HeLa cells and COS7 cells treated with GNR/HA-DC for 12, 24, and 48 h. Scale bar: 20 μm. Flow cytometry measurement of intracellular glucose levels in (G) HeLa cells and (H) COS7 cells treated with GNR/HA-DC. Inset: Corresponding mean fluorescence intensity.

over time (Figure 2D,E and Figure S8), which was proposed to hamper the cellular functions including glucose uptake and anaerobic glycolysis. Thereafter, we analyzed the amount of glucose in cells under the treatment of GNR/HA-DC. For confocal laser scanning microscopy (CLSM) observation, 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-2-deoxyglucose, a widely used green fluorescent probe, was utilized to directly image the intracellular glucose uptake. As displayed in Figure 2F, HeLa cells without the treatment of GNR/HA-DC displayed strong green fluorescence, indicating sufficient glucose uptake by HeLa cells due to their high expression of Glut1. By treating cells with GNR/HA-DC, a remarkable inhibition of glucose uptake with gradually decayed fluorescence intensity over time was observed. After 48 h incubation, very weak fluorescence was visible inside HeLa cells. In contrast, the change of fluorescent signal in COS7 cells with the same treatment was negligible, due to limited cellular internalization of GNR/HA-DC by

CD44 negative COS7 cells to inhibit their cellular glucose uptake. Moreover, flow cytometry analysis also revealed a remarkably inhibited glucose uptake in HeLa cells by the impact of GNR/HA-DC (Figure 2G,H), which was in line with the CLSM results. Furthermore, a quantitative glucose uptake assay was performed to estimate the exact inhibition efficacy of GNR/ HA-DC. As indicated in Figure 3A, compared with the control cells without any treatment, there was a 27.1, 41.5, and 52.7% reduction of cellular glucose in HeLa cells after GNR/HA-DC treatment for 12, 24, and 48 h, respectively. However, for COS7 normal cells under the same treatment, the intracellular glucose level did not change. Such a serious reduction of intracellular glucose uptake in tumor cells would hinder the glucose metabolism, leading to the decrease of ATP (one of the main metabolites of glycolysis) generation. As shown in Figure 3B, after GNR/HA-DC treatment, significant decrease of the cellular ATP level was detected in HeLa cells. On the contrary, 1422


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Figure 3. (A) Quantitative glucose uptake and (B) ATP level in HeLa cells and COS7 cells after being treated with GNR/HA-DC. The cells without any treatment were used as the control. (C) Western blotting analysis of HSP70 and HSP90 expression in HeLa cells with the treatment of (1) blank control, (2) incubating at 40 °C, (3) free DC, (4) GNR/HA-DC, (5) GNR/HA-DC with NIR irradiation, and (6) GNR/HA-DC under incubation at 40 °C. (D) Quantitative determination of the relative protein expression from Western blotting results.

almost no influence on the ATP production was found in GNR/HA-DC-treated COS7 cells. Thus, these results strongly demonstrated that GNR/HA-DC could specifically inhibit the tumor cell uptake of glucose and glycolysis by reducing the transporters in cells. To further analyze whether inhibition of ATP generation could interfere with cellular HSP level, we detected two main HSPs production (i.e., HSP70 and HSP90) in HeLa cells under different conditions by Western blotting analysis (Figure 3C,D). In comparison to cells without treatment, a strong expression of HSP70 and HSP90 were clearly observed for HeLa cells incubated at defined mild temperature (∼40 °C), validating that a heat shock response could be triggered in cells to elevate the HSP generation. For cells treated with free DC or GNR/HA-DC without NIR irradiation, the HSP expression was significantly prevented, which confirmed our prognostication that interference of glucose metabolism by the small molecule Glut1 inhibitor would eventually lead to HSP depletion in cells. Similarly, when exposed to NIR laser or incubated at 40 °C, GNR/HA-DC-treated HeLa cells showed obvious inhibited expression of HSP70 and HSP90, which was expected to make malignant cells more vulnerable to heat and therefore hold great potential in improving therapeutic efficiency of hyperthermia treatment. In Vitro Cell Killing Evaluation. Next, the synergistic effect of Glut1 inhibitor and PTT for killing tumor cells by GNR/HA-DC was investigated, and the results are summarized in Figure 4. With the methylthiazolyl tetrazolium assay (Figure 4A,B), the viabilities of both HeLa cells and COS7 cells treated with GNR/HA were greater than 85% even at a relatively high nanoparticle concentration, indicating good biocompatibility of our designed nanoplatform. For HeLa cells, the cell viability remarkably changed with the treatment pattern. Compared with GNR/HA-treated HeLa cells, there was a moderate decrease in cell viability for cells treated with the

DC-loaded nanosystem (GNR/HA-DC) because the DC released from GNR/HA-DC displayed a certain extent of antitumor effect similar to that with free DC (Figure S9 and Figure S10).35 Upon NIR laser exposure, more cells were killed by the treatment of GNR/HA, with 31.9% of cells remaining alive. Strikingly, a much more enhanced cell killing was observed for HeLa cells treated with GNR/HA-DC under NIR irradiation. The cell survival ratio was only 8% at a GNR concentration up to 200 μg mL−1. With the above metabolism analysis results considered (Figure 3), such an enhancement of therapeutic effect under mild PTT temperature (Figure S11) could be attributed to the collaboration of DC to significantly increase tumor cell sensitivity to hyperthermia. Conversely, the variation of cell lethality in CD44 negative COS7 cells with the different treatments was negligible because the restricted CD44-mediated cellular uptake of HA-coated nanoparticles strictly limited their damage to COS7 cells. Thus, GNR/HA-DC showed favorable capacity to recognize tumor cells and subsequently kill them without damaging normal healthy cells. Fluorescence live/dead cell assay was utilized to directly visualize the cell death induced by GNR/HA-DC in HeLa cells. As displayed in Figure 4C, the amount of dead HeLa cells was dependent on the laser irradiation time. More tumor cells were killed by GNR/HA-DC with a longer laser irradiation time. Compared with the cells that were not treated with GNR/HA-DC or not exposed to NIR, significant red fluorescence (dead cells) was observed at 1.5 min post-irradiation. More importantly, the percentage of apoptosis/necrotic cells was determined to be remarkably increased from 3.9% (blank control) to 83.1% (cells treated with GNR/HA-DC for 1.5 min irradiation) (Figure 4D), further confirming the powerful apoptosis-inducing capacity of GNR/HA-DC in tumor cells. However, less cells were killed or in apoptosis/necrotic status by the treatment of GNR/HA under laser irradiation (Figure S12), which may be ascribed to the lack of DC for thermal therapy enhancement. 1423


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Figure 4. Cytotoxicity of GNR/HA and GNR/HA-DC with or without NIR irradiation against (A) HeLa cells (**p < 0.01 and ***p < 0.001) and (B) COS7 cells. (C) Fluorescence live/dead cell images of HeLa cells with blank control treatment, GNR/HA-DC preirradiation, and GNR/HA-DC with NIR light irradiation for 0.5, 1.0, and 1.5 min. Scale bar: 200 μm. (D) Flow cytometry analysis of apoptosis and necrosis of HeLa cells with blank control treatment, GNR/HA-DC, and GNR/HA-DC with NIR light irradiation for 0.5, 1.0, and 1.5 min.

In Vivo Photothermal Conversion, Biodistribution, and Blood Circulation Time. To explore the potential photothermal conversion of GNR/HA-DC in vivo, NIR-lasertriggered tumor temperature change was evaluated on HeLa-tumor-bearing mice and recorded in real time by an IR thermal imaging camera. As shown in thermal images and the time−temperature curves (Figure 5A,B), upon 808 nm laser irradiation, the local temperature of the tumors treated with GNR/HA-DC increased rapidly over the course of photoirradiation. The ultimate tumor temperature reached ∼50 °C at 6 min post-irradiation, which was high enough to ablate malignant cells in hyperthermia therapy. For the mice treated with GNRs, a slower temperature increase rate and a lower tumor temperature (up to ∼46 °C) were detected. More importantly, the tumor temperature curves of GNR/HA and GNR/HA-DC groups behaved similar to those in the comparable photothermal effect. In marked contrast, the tumor temperature on PBS-treated mice under the same irradiation conditions showed no significant temperature change with ΔT less than 4 °C. Furthermore, to confirm the in vivo tumor-targeted efficiency of GNR/HA-DC, the biodistribution and blood circulation time of GNR/HA-DC were evaluated by using ICP-MS analysis

of Au contents in the tumor tissues, major organs (liver, spleen, lung, kidney, heart, and brain), and blood. As shown in Figure 5C, GNR/HA-DC was prominently enriched in tumor, liver, and spleen tissues, and the lung, kidney, heart, and brain have lower Au contents. The accumulated gold contents of GNR/HA-DC in tumor tissues was much greater than that of GNR during the entire period. Twenty-four hours after injection, the accumulation of GNR/HA-DC in tumor tissues was 23.2% ID/g, which was 2.3-fold higher than that of GNR (10.1% ID/g). Due to the advantage of passive and active targeting effects, GNR/HA-DC could effectively accumulate in tumor sites to achieve tumor-targeted therapy. In particular, the blood circulation time of GNR/HA-DC was obviously longer than that of GNR (Figure 5D). Since GNR/HA-DC was negatively charged (Table S1), which benefitted from the resistance of protein adsorption in blood and retarded metabolic clearance in the circulation time. Moreover, GNR/ HA showed blood retention and biodistribution similar to that of GNR/HA-DC due to their similar chemical properties. In Vivo Tumor Therapy. Encouraged by the favorable therapeutic effect in vitro and the satisfactory NIR-induced photothermal conversion result in vivo, the in vivo antitumor effect of GNR/HA-DC was further investigated. After the 1424


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Figure 5. (A) In vivo photothermal images of mice after intravenous injection of PBS, GNR, GNR/HA, and GNR/HA-DC. (B) Temperature increase curve of the NIR-laser-irradiated tumor tissues as a function of irradiation time; **p < 0.05 and ***p < 0.01. (C) Biodistribution of GNR, GNR/HA, or GNR/HA-DC at 1, 12, and 24 h after intravenous injection. The data were based on ICP-MS analysis and expressed as a percentage of the injected dose per gram of tissue (% ID/g). (D) Pharmacokinetic profiles of GNR, GNR/HA, or GNR/HA-DC after intravenous injection. Au contents in blood samples were analyzed by ICP-MS at predestined time intervals after injection.

Figure 6. In vivo antitumor study. (A) Relative tumor volume after different treatments. (B) Average tumor weight obtained on the 12th day. (C) Representative photographs of tumor tissues. Immunofluorescent staining of (D) HSP70 and (F) HSP90 of tumors after different treatments. Quantitative analysis of (E) HSP70 and (G) HSP90 expression. (H) Apoptotic cell detection by TUNEL immunofluorescence staining and (I) quantitative evaluation of the percentage of TUNEL positive apoptotic cells in tumors from different groups. The blue signal of DAPI and green signal represent the nuclei and apoptotic cells, respectively. (J) H&E staining of tumors (**p < 0.01 and ***p < 0.05).

tumor volumes reached ∼150 mm3, the nude mice were divided into six experimental groups and treated with (1) PBS, (2) PBS+NIR, (3) GNR/HA, (4) GNR/HA+NIR, (5) GNR/

HA-DC, and (6) GNR/HA-DC+NIR. As evaluated by the relative tumor volume (Figure 6A), tumors in the PBS+NIR group increased rapidly over the course of therapy, similar to 1425


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the biocompatibility of GNR/HA-DC with no acute toxicity in vivo and supported its potential application in tumor treatment.

that in the PBS-treated group, indicating that NIR irradiation nearly had no effect on the tumor suppression. In comparison with the insignificant suppression effect in group 3, the tumor growth in the GNR/HA-treated mice with laser irradiation (group 4) was inhibited to some extent, owing to the tumor cell killing effect of PTT. For the mice in the GNR/HA-DC-treated group, the growth of the tumor was only inhibited slightly. Compared to GNR/HA (group 3), the introduction of DC did not promote tumor suppression. In striking contrast, by the integration of the DC inhibitor and PTT, the GNR/HA-DC+NIR group exhibited a therapeutic efficacy significantly higher than that in other groups. Extraordinary tumor regression was observed with the relative tumor volume value of 0.17 on the 12th day. This satisfactory therapeutic effect may be ascribed to the tumor-triggered release of DC from GNR/HA-DC to inhibit Glut1, resulting in inhibited HSP synthesis and improved sensitivity of hyperthermia, which led to elevated efficacy of PTT. All mice were euthanized on the 12th day because extensive tumor growth in the PBS group became too burdensome. Tumors were excised and weighed. The average tumor weight (Figure 6B) and the corresponding tumor graphs (Figure 6C) also proved the optimal antitumor performance of GNR/HA-DC under NIR irradiation. These results suggested that combining a Glut1 inhibitor with PTT was much more effective than PTT alone. Immunofluorescence staining results showed that HSP70 (red fluorescence) (Figure 6D,E) and HSP90 (green fluorescence) (Figure 6F,G), two of the typical thermoresistant-related HSPs that protect tumor cells from destruction, were markedly up-regulated under hyperthermia treatment (GNR/HA+NIR group), which weakened the therapeutic effect of individual PTT. The expression of these HSPs in the GNR/HA-DC+NIR group dramatically decreased. These results suggested that GNR/HA-DC could block HSP (the cellular protection protein) expression in vivo, leading to the tumor cells being more easily killed under heat stimulation, thereby enhancing the PTT efficacy. As further demonstrated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Figure 6H,I), the maximum amount of apoptotic cells was detected in the GNR/HA-DC +NIR group, which was significantly more than that in other treatment groups. Hematoxylin and eosin (H&E) staining (Figure 6J) also revealed that GNR/HA-DC+NIR-treated tumor tissues showed obvious tumor cell damage, such as a severe degree of cell shrinkage and nuclear condensation, which were the typical characteristics of apoptosis. Collectively, these results confirmed that GNR/DC-HA integrated tumor metabolic therapy with photothermal therapy exhibited superior efficacy in suppressing tumors. In Vivo Biosafety Evaluation. Of particular note, the body weight of mice in all six experimental groups did not change obviously (Figure S13), indicating negligible systemic toxicity of GNR/HA-DC. Moreover, H&E images of major organs (lung, liver, spleen, kidney, heart) revealed no noticeable inflammation lesions or tissue damage in all of the organ structures (Figure S14A), demonstrating the favorable biocompatibility as well as significantly reduced side effects of our designed nanosystem. Furthermore, complete blood panel test and serum biochemistry assay showed that the content of specific indicators of liver and kidney functions, including total protein, aspartate aminotransferase, alanine aminotransferase, albumin, creatinine, and blood urea nitrogen, fell within normal ranges (Figure S14B−G). There were no significant differences between all the tested groups. These results demonstrated

DISCUSSION It is well-known that acquired thermotolerance in PTT comes mainly from the stress-induced generation of heat shock proteins, which protects tumor cells from heat, enhances cell survival ability, and suppresses cell apoptosis, ultimately leading to the failure of PTT.7−9 As a consequence, studies aimed at the thermotolerance are intensively pursued to increase therapeutic efficiency in hyperthermia treatment. Recently, several nanosystems were developed to selectively sensitize tumor cells to heat by using HSP inhibitors or siRNA for enhanced therapeutic efficacy both in vitro and in vivo.13−18 Liu et al. developed a thermoresponsive nanoplatform with the entrapment of the HSP70 inhibitor for PTT of deeper tumor cells. Assisted by the thermo-induced release of inhibitor, the function of HSP70 was inhibited and photothermal ablation of tumor cells in deeper tissues was achieved.13 In addition, they further attached HSP70−siRNA onto porous upconversion nanoparticles by loading cypate to achieve combinational effects of gene silencing and PTT.18 These reports highlighted the key role of HSPs for activating thermotolerance in hyperthermia therapy. However, as derived from the former efforts, it is challenging to sensitize tumor cells before heat treatment by using inhibitor/siRNA, and the delivery of siRNA crossing the extra-/cellular barriers remains difficult.20 Therefore, searching for alternative approaches that induce the downregulation of HSPs before heat treatment may provide an alternative solution to broaden the thermo-therapeutic window. Increasing evidence suggested that inhibition of anaerobic glycolysis, as the key glycolytic pathway in fast-growing cancers for fulfilling diverse cellular requirements, could cause metabolic dysfunction including blocking the synthesis of amino acids, fatty acids, proteins, membrane lipids, decreasing the production of ATP, and ultimately inhibiting the generation of downstream metabolites such as HSPs.29,47,48 Thus, a specific glycolysis inhibitor was proposed to deplete HSPs to cooperate with PTT. In this study, diclofenac, a classic nonsteroidal anti-inflammatory drug that inhibits glycolysis, was specifically delivered to CD44 overexpressed tumor cells by a well-designed nanosystem (GNR/HA-DC). Since glucose transporters (Gluts) are responsible for a high rate of glucose uptake in many malignant tumors to meet the glycolysis requirement, the effect of GNR/HA-DC on Glut1 (one of the key Gluts) was particularly evaluated. By targeting CD44 and subsequently releasing DC in specific HeLa cells, the level of Glut1 was demonstrated to be down-regulated (Figure 2), resulting in significantly decreased glucose uptake (Figure 3). Originating from such a metabolism inhibition effect, the generation of HSP70/90 in GNR/HA-DC-treated HeLa cells was significantly reduced (Figure 3). As a consequence, selective sensitization of HeLa cells to hyperthermia was achieved both in vitro (Figure 4) and in vivo (Figure 5). In contrast, the cascade metabolism inhibition and sensitization effect was not detected in normal cells due to their low-level expression of CD44 and Glut1. Compared with previously reported works, thermotolerance induced by HSPs could be surmounted ahead of heat treatment by this well-designed nanosystem, leading to greatly enhanced photothermal therapeutic efficacy. Due to the multiple functions of DC, including antiinflammation effects, antiangiogenic effect, and direct tumor 1426


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ACS Nano apoptosis,35,49−51 additional antitumor mechanisms of sensitizing tumor cells by DC to thermotherapy would also contribute to the enhanced antitumor effect. As assessed by real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) of pro-inflammatory cytokines (TNF-α, IL-6, IL-1) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells, the concentration of these cytokines was significantly reduced and the corresponding mRNA expression levels were remarkably down-regulated by GNR/HA-DC (Figure S15), suggesting their anti-inflammatory properties. Moreover, free DC or DC released from GNR/ HA-DC showed similar cytotoxicity to cells, demonstrating their cell killing ability in inducing apoptosis (Figure 4 and Figures S9 and S10). Besides the direct effect on tumor cell viability, antitumoral capacity of DC has been demonstrated to be additionally attributed to its antiangiogenic effect.51,52 However, these effects were obtained by using relatively high concentrations of DC. For in vivo evaluation in our study, the nanoparticles were administrated only twice rather than repeated dosing, so that the local tumor DC concentration was relatively low, which was not expected to achieve an effective inhibition of tumor growth, an anti-inflammation effect, or an antiangiogenic effect by DC. Only with the cooperation of DC and thermal therapy could achieve significant tumor inhibition. Furthermore, it has been suggested that glucose metabolism is critical for cancer stem cells (CSCs) that correspond to many failures of antitumor therapy.26 The superiority effect of GNR/HA-DC on CSCs was evaluated in MCF-7 cells, a cell line which has been well-shown to have a small population of CSCs with CD44high/CD24low surface markers.53,54 As obtained from flow cytometry analysis (Figure S16), the fraction of CD44high/CD24low cells (i.e., CSCs) was remarkably reduced by the treatment of GNR/HA-DC with laser irradiation. Thus, both CSCs and nonstem cancer cells could be effectively killed by the combination of DC and PTT, which would also contribute to the enhanced antitumor effect because CSCs have been proposed to be responsible for therapy resistance, tumor progression, and metastasis.

(CTAB) were purchased from Sigma-Aldrich. 1-(3-(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Chemical Co. (China) and used as received. Hyaluronic acid (HA, 35 kDa) was obtained from Freda Biochem Co. Ltd. (Shandong, China). Dulbecco’s modified Eagle’s medium (DMEM), trypsin, penicillin− streptomycin, fetal bovine serum (FBS), 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT), adenosine 5′-triphosphate (ATP) bioluminescent assay kit, and Dulbecco’s phosphate buffered saline (PBS) were provided by Invitrogen. 2-Deoxy-2-[(7-nitro-2, 1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), calcein-AM, and propidium iodide (PI) were obtained from Sigma-Aldrich. All other reagents and solvents were of analytical grade and used directly. Apparatus. Transmission electronic microscopy (TEM, JEOL2100) was used to study the morphology of GNRs, GNR/HA, and GNR/HA-DC. Absorbance spectra of different samples were collected using a UV−vis spectrophotometer (Lambda Bio40, PerkinElmer). XPS were obtained on a Thermo Fisher ESCALAB 250Xi multitechnique surface analysis instrument to analyze the chemical composition of different samples. Thermogravimetric analysis (TGA) was performed with a thermal analyzer (TGS-II, PerkinElmer) at 5 °C min−1 under nitrogen flow of 40 mL min−1. The ζ-potentials, hydrodynamic sizes, and colloidal stability of the nanoparticles were measured on a Malvern Zetasizer Nano-ZS ZEM3600 (UK). The 808 nm NIR laser (STL808T1-7.0W, Beijing STONE Laser) and the FLIR Ax5 camera (FLIR Systems AB, Sweden) were used to record the photothermal conversion effects. Synthesis of HA-LA. In brief, LA (0.26 g, 3 mmol), NHS (0.22 g, 1.88 mmol), and EDC (0.36 g, 1.88 mmol) were dissolved in 15 mL of anhydrous N,N-dimethylformamide (DMF) followed by addition of 1.0 g of HA. The reaction mixture was stirred at room temperature for 24 h. The resulting HA-LA was obtained by dialyzing (MWCO = 14 000 Da) the reaction solution against deionized water for 3 days and was then lyophilized. Synthesis of HA-LA/DC. In brief, DC (1.12 g, 3.75 mmol), NHS (0.66 g, 5.64 mmol), and EDC (1.08 g, 5.64 mmol) were dissolved in 25 mL of anhydrous DMF followed by addition of 1.0 g of HA-LA. The reaction mixture was stirred at room temperature for 24 h. The resulting HA-LA/DC was obtained by dialyzing (MWCO = 14 000 Da) the reaction solution against deionized water for 3 days and was then lyophilized. The substitution degree of DC in HA-DC was determined to be ∼22.8% according to the 1H NMR spectrum by comparing the integrals of the benzene proton (δ 6−8) relative to the methyl singlet of HA at δ 1.95. Synthesis of Gold Nanorods. GNRs were synthesized according to a modified seed-mediated method.34 Briefly, the seeds were prepared first. Specifically, CTAB (2.0 mL, 0.20 M) was mixed with 2.0 mL (0.5 mM) of HAuCl4. With continuous stirring, 0.24 mL (10 mM) of ice-cold NaBH4 was added to the above mixture, resulting in the formation of a brownish-yellow solution. The seeds formed immediately and were used within 2−3 h when kept at 25 °C. The growth solution of GNRs was obtained by mixing the solution of 100 mL (0.2 M) of CTAB, 5.6 mL (4 mM) of AgNO3, 6.5 mL (23 mM) of HAuCl4, and 95 mL of Milli-Q water. Thereafter, ascorbic acid (2.5 mL, 0.08 M) was added dropwise. The final step was the addition of 1.8 mL of the seed solution to the above growth mixture. The temperature of the growth medium was kept constant at 27−30 °C during the full process. The color of the solution gradually changed within 10−20 min. To reduce excess CTAB, the as-synthesized GNRs were centrifuged at 11 000 rpm for 25 min, resuspended in deionized water, and followed by dialyzing against distilled water (MWCO = 14 000 Da) for 6 h. Then the well-dispersed GNR solution was stored at 4 °C before use. Preparation of GNR/HA and GNR/HA-DC. GNR/HA and GNR/HA-DC were prepared by reacting HA-LA and HA-LA/DC with GNRs via the classic Au−thiol bond.43 The concentrations of HA-LA and HA-LA/DC were fixed at 2.0 mg mL−1 and reacted with GNRs (0.5 mg mL−1) at room temperature for 24 h. The excess HA-LA and HA-DC were removed by repeated centrifugation (11 000 rpm for 25 min) and washed with distilled water. Finally,

CONCLUSIONS In summary, we have developed a nanosystem of GNR/ HA-DC for improved tumor-targeting photothermal therapy, which can selectively sensitize malignancy to PTT by interfering with cellular glucose metabolism for complete destruction of tumors. The tumor-triggered release of DC from GNR/HA-DC played a critically important role in depleting Glut1 in tumor cells and hence inhibiting the glucose uptake to subsequently block glycolysis in generating ATP. Expression of thermoresistance HSPs, such as HSP70 and HSP90, was severely hampered as confirmed by Western blotting analysis. As a result, tumor cells became more sensitive to the damage induced by PTT, and a superior antitumor efficacy with reduced side effects was achieved both in vitro and in vivo. En route to elevate the oncology by traditional therapeutic modalities (hyperthermia, radiation, and chemotherapy), our nanosystem of combining a Glut1 inhibitor showed advanced development of an effective platform to ablate tumors. MATERIALS AND METHODS Chemicals. Lipoic acid (LA), diclofenac (DC), sodium borohydride (NaBH4), ascorbic acid, tetrachloroauric acid (HAuCl4·3H2O), silver nitrate (AgNO3), and N-cetyltrimethylammonium bromide 1427


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ICP-MS. The cells without any treatment were used as the blank control. Moreover, cellular uptake of nontargeted GNR and GNR/HA was also evaluated in these four kinds of cells. Determination the Intracellular Glucose Level by CLSM and Flow Cytometry. In brief, cells were seeded in a glass-bottom dish at a density of 1 × 105 cells per well for 24 h. Then HeLa cells and COS7 cells were incubated with GNR/HA-DC (containing 0.4 mM DC) in the cell culture media for 12, 24, and 48 h. The cells without any treatment were used as the control. After that, the cell culture medium was removed, and all cells were washed with PBS three times. For the glucose uptake assay, all cells were incubated with 2-NBDG at 37 °C for 30 min. Then all cells were observed by CLSM (Nikon C1-si). Excitation of 2-NBDG was performed with the laser at 488 nm, and the corresponding emission spectra were collected at 510−540 nm for green fluorescence. Moreover, flow cytometry analysis was also carried out to evaluate the intracellular glucose. After cells were treated with the same conditions as used with the CLSM observation, the intracellular glucose level was determined by flow cytometry (BD FACSAria TM III). The cells without any treatment were detected as the control. Quantitative Evaluation of the Inhibition of Glucose Uptake by GNR/HA-DC. In brief, HeLa cells and COS7 cells were seeded in 6-well plates at a density of 5 × 105 cells per well for 24 h. The inhibitory effect of GNR/HA-DC on glucose transport was evaluated by measuring the cell uptake of 2-deoxy-D-[3H] glucose as in previous reports.45,46 After being treated with GNR/HA-DC (containing 100 μg mL−1 GNR and 0.4 mM DC) for 12, 24, or 48 h, all cells were solubilized in 0.1% SDS before radioactivity measurement. The cells without any treatment were used as the control. Evaluation of Intracellular ATP Level. To examine whether the inhibition of glucose uptake can interfere with ATP production, the intracellular ATP levels were measured. In brief, HeLa cells and COS7 cells were seeded in 24-well plates at a density of 5 × 104 cells per well for 24 h. Then the cells were treated with GNR/HA-DC (containing 100 μg mL−1 GNR and 0.4 mM DC) for different incubation times (12, 24, or 48 h) and then washed with PBS three times. Subsequently, the cells were trypsinized and counted, centrifuged, and collected in bacteria-free centrifuge tube. The cell pallets were lysed, and the cellular ATP level was evaluated according to the protocol of the ATP bioluminescent assay kit. Western Blotting Analysis of HSP70 and HSP90. In brief, HeLa cells were seeded in 6-well plates at a density of 5 × 105 cells per well for 24 h. Then the cells were treated with different conditions: (1) without any treatment (blank control), (2) incubation at 40 °C for 30 min, (3) free DC (0.4 mM DC), (4) GNR/HA-DC (containing 0.4 mM DC), (5) GNR/HA-DC (containing 0.4 mM DC) with NIR irradiation, and (6) GNR/HA-DC under incubation at 40 °C. Afterward, the cells were lysed and treated as in our previous report.44 After the membranes were incubated with the primary antibody rabbit anti-human HSP70 or HSP90 overnight at 4 °C, the membranes were washed with TBST and incubated with the secondary antibody for 30 min. Specific proteins were detected using enhanced chemiluminescence. GAPDH was employed as a protein loading control. In Vitro Cytotoxicity Assay. The cytotoxicities of free DC, GNR/ HA, and GNR/HA-DC were estimated in HeLa cells and COS7 cells by MTT assay. In brief, HeLa cells and COS7 cells were seeded in a 96-well plate at a density of 6000 cells per well and incubated in 100 μL of DMEM containing 10% FBS and 1% antibiotics for 24 h. After that, free DC, GNR/HA, and GNR/HA-DC at different concentrations were added to each well. To evaluate the NIR-laserinduced cytotoxicity, the medium of some wells containing GNR/HA or GNR/HA-DC was replaced with fresh DMEM after 12 h incubation and then irradiated with NIR laser light (STL808T17.0W, Beijing STONE Laser). The 808 nm NIR laser with 1.3 mm diameter spot size and power density of 1 W cm−2 was exposed to each well for 1 min. After co-incubation for 48 h (additional 12 h incubation for laser-irradiated wells), 20 μL of MTT (5 mg mL−1 in PBS buffer solution) was added to each well and further incubated for another 4 h. Afterward, the medium was removed and replaced with

GNR/HA and GNR/HA-DC were dialyzed against distilled water (MWCO = 14 000 Da), and the resultant nanoparticle solutions were stored at 4 °C before use. A part of the nanoparticle solution was lyophilized for TGA, and 3 mL of the nanoparticle solution was lyophilized to determine the amount of nanoparticles for further estimation of the nanoparticle concentration used for the in vitro and in vivo study. For in vitro and in vivo applications, GNRs were PEG-modified (mPEG-SH, Mw = 5000 Da) as in the previously reported method.55 As obtained from the TG results and 1H NMR spectra, the content of HA-DC in GNR/HA-DC was about 80.2%, and the substitution degree of DC in HA-DC was determined to be ∼22.8%. Thus, the DC loading amount could be calculated as mxy/m (where m is the weight of GNR/HA-DC, x is the amount of HA-DC in GNR/HA-DC, and y is the substitution degree of DC in HA-DC), and the result was estimated to be 19%. Evaluation the HAase-Triggered DC Release. To study the HAase-triggered DC release, 10 mg of GNR/HA-DC nanoparticles was immersed in 3 mL of acetate buffer solution (pH 5.5, 10 mM) containing different concentrations of HAase (0, 0.15, 0.3, 0.6 mg mL−1). As the control, an equal amount of nanoparticles was dispersed in 3 mL of acetate buffer solution (pH 5.5, 10 mM) or PBS (pH 7.4, 10 mM) solution. Two parallel samples were tested in each group, and all the nanoparticle dispersions were incubated in a water bath (temperature = ∼37 °C) with gentle shaking. At predetermined time points, the nanoparticles were withdrawn from the solution periodically by centrifugation followed by redispersion in the same amount of fresh incubation solution correspondingly. The DC concentration in the supernatant was detected by UV−vis measurement. Furthermore, blood samples and extracts of tumor tissue were collected from HeLa-tumor-bearing nude mice to evaluate the drug release. Blood was obtained by heart puncture followed by centrifugation (6000 rpm, 20 min) to obtain supernatants for use in the drug release study. Tumor tissues were harvested, dissected, and frozen for homogenization by using a tissue homogenizer followed by suspension in cold buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, 1% TritonX-100, 0.2 M NaCl). Supernatants of tumor extracts were collected by centrifugation (15 000 rpm, 15 min) for use in the drug release. Cell Culture. Human cervix carcinoma cells (HeLa), African green monkey SV40-transformed kidney fibroblast (COS7) cells, and RAW 264.7 cells were cultured in DMEM medium with 10% FBS and 1% antibiotics (penicillin−streptomycin, 10 000 U mL−1) at 37 °C in a humidified atmosphere containing 5% CO2. Human breast cancer cells (MCF-7) and human embryonic kidney transformed cells (293T) were incubated in 1640 medium with 10% FBS and 1% antibiotics (penicillin−streptomycin, 10 000 U mL−1) at 37 °C in a humidified atmosphere containing 5% CO2. Western Blotting Analysis of Glut1. In brief, HeLa, MCF-7, COS7, and 293-T cells were seeded in a glass-bottom dish at a density of 1 × 105 cells per well for 24 h. Then the cells in each group were lysed and treated as in our previous report.44 After the membranes were incubated with the primary antibody rabbit anti-human Glut1 overnight at 4 °C, the membranes were washed with TBST and incubated with the secondary antibody for 30 min. Specific proteins were detected using enhanced chemiluminescence. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was employed as a protein loading control. Evaluation the Tumor-Targeted Uptake of GNR/HA-DC by ICP-MS Assay. In brief, cells were seeded in 6-well plates at a density of 5 × 105 cells per well for 24 h. Specifically, some wells of HeLa and MCF-7 cells were preincubated with excess free HA (2 mg mL−1) for 4 h. To detect the tumor-targeted uptake of GNR/HA-DC, HeLa, MCF-7, COS7, and 293T cells were incubated with GNR/HA-DC (containing 0.4 mM DC) for 24 h. After being washed with PBS three times, the cells were collected and counted for ICP-MS analysis (model Agilent 7500a, Hewlett-Packard, Japan) to evaluate the cellular gold content. Then the cell samples were digested by aqua regia (extreme caution is required! Aqua regia is highly corrosive and damaging to skin and eyes!), and each sample was treated with H2O2 (2 mL 30%, w/w) at 150 °C for 3 h. After the solution was diluted with distilled water to 10 mL, gold content was analyzed by using 1428


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ACS Nano 150 μL of DMSO. The absorbance of the DMSO solution in the wells at the wavelength of 570 nm was measured by a microplate reader (model 550, Bio-Rad) to determine cell viability using the MTT assay. The relative cell viability was calculated as (OD570sample/OD570control) × 100%, where OD570control was obtained in the absence of therapeutic agents and OD570sample was obtained for cells treated with free DC, GNR/HA, and GNR/HA-DC. To evaluate the cytotoxicity of glycosaminoglycan-conjugated DC, samples were prepared by pretreatment of GNR/HA-DC with HAase (1 mg mL−1, pH 5.5) for 24 h followed by collection of the supernatant via centrifugation and lyophilization. To evaluate the cytotoixcity against RAW 264.7 macrogphages cells, GNR (25 μg mL−1), free DC (0.1 mM), GNR (25 μg mL−1) + free DC (0.1 mM), and GNR/HA-DC (corresponding to 25 μg mL−1 of GNR and 0.1 mM of DC) were incubated with RAW 264.7 cells for 12 h. Cells without any treatment were used as the control. Then, a similar procedure was carried out for the MTT assay. Live/Dead Cell Staining Assay. HeLa cells were seeded in 6-well plates at a density of 5 × 105 cells per well. After 24 h plating, the cells were incubated with GNR/HA-DC (containing 0.4 mM DC) or GNR/HA (100 μg mL−1) for 24 h. Then the culture medium was refreshed, and the cells were incubated with fresh DMEM at 37 °C without NIR laser irradiation or with NIR light irradiation (1.0 W cm−2) for 0.5, 1.0, or 1.5 min. The cells without treatment were tested as the negative control. Finally, all cells were treated with calcein-AM (4 μM) and PI solutions (4 μM) in PBS buffer solution and incubated for 15 min at 37 °C with 5% CO2. Finally, the cells were washed with PBS three times and visualized by CLSM (Nikon C1-si, Japan). Excitation of the calcein-AM and PI was performed with lasers at 488 and 543 nm, respectively. The corresponding emission spectra were collected using two different ranges of wavelength at 510− 540 nm (green) and 570−620 nm (red). Evaluation of Apoptosis by Flow Cytometry. HeLa cells were seeded in 24-well plates at a density of 5 × 104 cells per well. After 24 h incubation, the cells were incubated with GNR/HA-DC (containing 0.4 mM DC) or GNR/HA (100 μg mL−1) for 24 h. Then the culture medium was refreshed, and the cells were incubated with fresh DMEM medium at 37 °C without NIR laser irradiation or with NIR light irradiation (1.0 W cm−2) for 0.5, 1.0, or 1.5 min. The cells without any treatment were tested as the negative control. Then all of the cells were washed with PBS three times, digested by trypsin (EDTA depleted), and collected by centrifugation. After being washed with PBS three times, the cells were resuspended in 0.5 mL of annexin binding buffer. After that, all cells were stained in PI and Annexin-V-FITC containing binding buffer for 15 min and finally detected by flow cytometry (BD FACSAria TM III). Cell Killing Effect on Cancer Stem Cells in MCF-7 Cells. MCF-7 cells were seeded in a 6-well plate and treated under different conditions: (1) control, (2) GNR/HA, (3) GNR/HA-DC, (4) GNR/ HA+NIR, and (5) GNR/HA-DC+NIR. After 24 h incubation, cells were stained with anti-CD44 antibody (PE-conjugated) (BioLegend, San Diego, CA) and anti-CD24 antibody (FITC-conjugated) (Abcam, UK) for 20 min in the dark for analysis of CD44high/CD24low cells using flow cytometry. Anti-inflammatory Evaluation by RT-PCR and ELISA. For RT-PCR evaluation, RAW 264.7 cells incubated in a 6-well plate were pretreated with LPS (100 ng mL−1) for 6 h. GNR (25 μg mL−1), free DC (0.1 mM), GNR (25 μg mL−1) + free DC (0.1 mM), and GNR/HA-DC (corresponding to 25 μg mL−1 of GNR and 0.1 mM of DC) were then added to each well for further incubation of 12 h. Then, RAW 264.7 cells were treated with TRIzol reagent (Life Technologies, USA) to isolate the total RNA. Reverse transcription was conducted with 1 μg of total RNA using a M-MLV reverse transcriptase kit (Invitrogen) according to the manufacturer’s protocol. The PCR primers were used as follows: TNF-α (forward: 5′-CTCTTCTCCTTCCTGATCGTGG-3′ and reverse: 5′-CTTGTCACTCGGGGTTCGAG-3′); IL-6 (forward: 5′-TCAGCCCTGAGAAAGGAGACAT-3′ and reverse: 5′-GCTCTGGCTTGTTCCTCACTACT-3′); and IL-1β (forward: 5′-ACGATGCACCTGTACGATCACT-3′ and reverse: 5′-GAGAACACCACTTGTTGCTCCA-3′). GAPDH (forward: 5′-GGTCGGAGTCAACGGATTTG-3′ and reverse: 5′-GGAAGATGGTGATGGGATTTC-3′)

was used as the internal control. The cDNA was amplified using a SYBR Premix Ex Taq kit (TaKaRa) according to the manufacturer’s protocol, and quantitative RT-PCR was performed on a StepOne RT-PCR system (Life Technologies, USA) (denaturing at 95 °C for 3 min, followed by 40 cycles at 95 °C for 15 s, 58 °C for 40 s, and 72 °C for 45 s). The changes of gene expression were calculated using the ΔΔCT method. For ELISA study, RAW 264.7 cells incubated in 96-well plates (10 000 cells mL−1) were pretreated with LPS (100 ng mL−1) for 6 h. GNR (25 μg mL−1), free DC (0.1 mM), GNR (25 μg mL−1) + free DC (0.1 mM), and GNR/HA-DC (corresponding to 25 μg mL−1 of GNR and 0.1 mM of DC) were then added to each well for further incubation for 12 h. The levels of TNF-α, IL-6, and IL-1 in the medium of macrophages were quantified according to the manufacturer’s protocol by using the relevant ELISA kit (Elabscience). The concentrations were determined from the calibration curve generated by the recombinant TNF-α, IL-6, and IL-1 standard. Animals and Tumor Model. BALB/c mice (4−5 weeks old) were bought from Wuhan University Animal Biosafty Level III Lab and used for animal experiments directly. All animal experiments were performed in agreement with institutional animal use and care regulations from Wuhan University. The tumors were obtained by injecting female mice with HeLa cells (1.5 × 107 cells) subcutaneously on the right armpit region. In Vivo Tumor-Targeted Photothermal Imaging. When tumors reached a size of approximately 150 mm3 in volume, 200 μL of GNR (containing 200 μg mL−1 GNR), GNR/HA (containing 200 μg mL−1 GNR), or GNR/HA-DC (containing 200 μg mL−1 GNR and 0.8 mM DC) was intravenously injected into the tumor-bearing mice. The mice injected with PBS buffer were evaluated as the control. Infrared thermal images were recorded by using a FLIR Ax5 camera under irradiation with an 808 nm laser at a power density of 1 W cm−2, and the temperature was quantified by BM_IR software. In Vivo Biodistribution and Blood Circulation Time of GNR/ HA-DC. When tumors reached a size of approximately 150 mm3 in volume, 200 μL of GNR (containing 200 μg mL−1 GNR), GNR/HA (containing 200 μg mL−1 GNR), or GNR/HA-DC (containing 200 μg mL−1 GNR and 0.8 mM DC) was injected into the mice via the tail vein. Then mice were sacrificed 1, 12, and 24 h after systemic injection of GNR, GNR/HA, or GNR/HA-DC. Tumors and other main organs (liver, spleen, lung, kidney, heart, and brain) were harvested, weighed, and stored at −80 °C before ICP-MS analysis of Au element to evaluate the biodistribution of nanoparticles. To study the pharmacokinetic behavior of GNR, GNR/HA, or GNR/HA-DC, blood samples were collected at predestined time intervals (0.5, 2, 4, 8, 12, and 24 h) using a heparinized tube after injection of GNR, GNR/ HA, or GNR/HA-DC. All the tissues and blood samples were lysed in aqua regia, and the Au contents were evaluated by ICP-MS analysis. Evaluation the Antitumor Effect in Vivo. When the tumor volume grew to about 150 mm3, tumor-bearing mice were randomly divided into six groups with five mice in each group. The mice were treated with (1) PBS, (2) PBS with NIR laser irradiation (808 nm; 3 min, 1 W cm−2), (3) GNR/HA, (4) GNR/HA with NIR laser irradiation (808 nm; 3 min, 1 W cm−2), (5) GNR/HA-DC, and (6) GNR/HA-DC with NIR laser irradiation (808 nm; 3 min, 1 W cm−2). All mice were only injected via the tail vein on the first and third days. The weight of the mice and tumor volume was measured every day. Tumor size was measured by a caliper, and tumor volume was calculated as (tumor width)2 × (tumor length)/2. Relative tumor volume was determined as V/V0 (where V0 is the tumor volume before therapy). When the treatment was fulfilled, the tumors were excised and weighed. Simultaneously, the main organs (heart, liver, spleen, lung, and kidney) of the mice were also harvested and used for histological analysis. Moreover, the tumor tissues were treated with H&E staining, immunofluorescence staining, and immunohistochemical staining to evaluate the antitumor mechanism. Evaluation the Systemic Toxicity in Vivo. After all of the treatments were finished, five mice in each group were used to detect total protein, aspartate aminotransferase, alanine aminotransferase, 1429


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ACS Nano albumin, creatinine, and blood urea nitrogen in the serum at Union Hospital (Tongji Medical College, Wuhan, China). Statistical Analysis. Statistical significance was analyzed by a three-sample Student’s test. Statistical significance was inferred at a value of p < 0.05.

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b06658. Details for material preparation and additional experimental results (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Xian-Zheng Zhang: 0000-0001-6242-6005 Author Contributions §

W.-H.C. and G.-F.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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