Biomaterials xxx (2014) 1e11

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Engineered, self-assembled near-infrared photothermal agents for combined tumor immunotherapy and chemo-photothermal therapy Yu Tao, Enguo Ju, Zhen Liu, Kai Dong, Jinsong Ren*, Xiaogang Qu* State Key Laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Graduate School of The Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin 130022, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 March 2014 Accepted 17 April 2014 Available online xxx

New combination therapy strategy, which takes the advantages of co-delivery two or more therapeutic agents in one nanocarrier platform, has been widely used in the clinic and achieved immense popularity in cancer treatment. Here, we have rationally developed a multifunctional platform using a self-assembly strategy to incorporate materials with specific functions of chemotherapeutics, hyperthermia, and especially immunotherapy, which can collectively contribute to the effective cancer treatment. We design the immunomodulatory CpG ODNs based platform that is conjugated with NIR-responsive gold nanorods and doxorubicin for cancer therapy. The gold nanorods can be applied as the nanocarrier to simultaneously address the three kinds of treatments, which lead to a significant benefit relative to the use of each method alone. Both in vitro and in vivo assays reveal that this engineered vehicle exhibits significant antitumor efficacy. Our studies provide strong evidence that the AuNRs-CpG-Dox conjugates can be utilized as efficient antitumor agents. Ó 2014 Published by Elsevier Ltd.

Keywords: Immunotherapy Hyperthermia Chemotherapy CpG ODNs Combination therapy

1. Introduction New combination therapy strategy, which takes the advantages of co-delivery two or more therapeutic agents in one nanocarrier platform, has been widely used in the clinic and achieved immense popularity in cancer treatment [1e3]. Such strategies may not only achieve the synergistic effects of different treatment mechanisms to dramatically improve overall therapeutic outcomes, but also can overcome the drawbacks of using a single therapeutic modality [4e 7]. As one of the most widely studied therapeutic strategies in the treatment of cancer, the combination of photothermal therapy and chemotherapy has attracted immense research attention in the recent past [8e23]. Heat treatment, called hyperthermia, can be effective for local cancer treatment due to the sensitivity of tumor cells to temperature elevation [24]. However, complete tumor eradication by photothermal therapy alone is difficult because heterogeneous heat distribution can lead to sublethal thermal dose in some areas of the tumor [11]. Therefore, successful cancer therapy requires the ability to combine photothermal therapy with other therapy modalities, such as chemotherapy. The combinational photothermal therapy/chemotherapy strategies, which can

* Corresponding authors. Tel./fax: þ86 431 85262625. E-mail addresses: [email protected] (J. Ren), [email protected] (X. Qu).

induce synergistic efficiencies that are greater than the two treatments alone [25], have been reported to be able to significantly enhance the efficacy against resistant tumors. Despite their potential advantages, weak immunogenicity of tumors still seriously affects the cancer therapy effects [26]. As one of the most effective strategies for preventing diseases [27], increasing immunogenicity of tumors can significantly enhance therapeutic effects of any treatment modality. Immunotherapy is a relatively new and promising concept for the treatment of cancer, by which the host immune system is activated to destroy cancer cells. Dendritic cells (DCs) represent important targets for immunotherapeutics in cancer because they can efficiently activate T cells that contribute to tumor rejection [28e32]. However, because cancer cells can prevent the maturation and function of DCs by a variety of mechanisms, DCs that infiltrate into tumor microenvironments usually exhibit an immature phenotype [33,34]. Whereas mature DCs can induce potent antigen-specific antitumor immunity, immature DCs do not promote T cell responsiveness and instead induce infiltration of regulatory T cells [35]. Therefore, it is necessary that DCs in tumor microenvironments are matured with immunostimulatory factors such as inflammatory cytokines, Toll-like receptor (TLR) ligands [36,37]. As an immunostimulatory TLR agonist, TLR9-specific unmethylated cytosineeguanosine (CpG) oligodeoxynucleotides (ODNs) are already in clinical trials for melanoma [38]. Unmethylated CpG

http://dx.doi.org/10.1016/j.biomaterials.2014.04.073 0142-9612/Ó 2014 Published by Elsevier Ltd.

Please cite this article in press as: Tao Y, et al., Engineered, self-assembled near-infrared photothermal agents for combined tumor immunotherapy and chemo-photothermal therapy, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.073

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ODNs binding to TLR9 are efficiently internalized by various antigen-presenting cells and induce release of various cytokines, which can initiate a cascade of innate and adaptive immune responses [39,40]. These responses have been shown to be effective in inhibiting tumor growth and metastasis in tumor-bearing animal models [41,42], which shed light on potential applications of the CpG ODNs in adoptive cancer immunotherapy. Inspired by these advances, we sought to develop a drug carrier vehicle that has the ability to increase immunogenicity, remotely control the drug release, and thermally ablate cancer cells by utilizing the CpG ODNs and doxorubicin conjugated gold nanorods. 2. Materials and methods 2.1. Materials and measurements Sodium borohydride (NaBH4), ascorbic acid, silver nitrate (AgNO3), tetrachloroauric acid (HAuCl4), thiazolyl blue tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS) and Hoechst 33258 were purchased from SigmaeAldrich. Ncetyltrimethylammonium bromide (CTAB) was obtained from Alfa Aesar. Purified anti-mouse TNF-a, biotin conjugated anti-mouse TNF-a cocktail, anti-mouse IL-6, biotin anti-mouse IL-6 were purchased from eBioscience. OPD (o-phenylenediamine) substrate was obtained from DingGuo. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen. The mouse leukemic monocyte macrophage cell line (RAW264.7 cell line) was purchased from Cell Bank of Chinese Academy of Sciences (Shanghai). All the chemicals were used as received without further purification. Nanopure water (18.2 MU; Millpore Co., USA) was used in all experiments and to prepare all buffers. DNA oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Technology & Services (Shanghai, China). The sequences are as follows: CpG ODN-1: 50 -TCG ACG TTT CCG CAT GAC ATT CGC CGA ACG-30 Cy3-CpG ODN-1: 50 -TCG ACG TTT CCG CAT GAC ATT CGC CGA ACG-30 -Cy3 CpG ODN-2: 50 -TCG ACG TTT GAT TCG GTT CAT GCG GAA ACG-30 SH-CpG ODN-3: 50 -TCG ACG TTC GGC GAA TGA CCG AAT CAA ACG AAA AAeSHe30 Fluorescence measurements were carried out by using a JASCO FP-6500 spectrofluorometer (Jasco International Co., Japan). UV-vis spectroscopy was carried out with a JASCO V-550 UV/vis spectrometer. TEM images were recorded using a FEI TECNAI G2 20 high-resolution transmission electron microscope operating at 200 kV. Fluorescence images were captured using an Olympus BX-51 optical equiped with a CCD camera. 2.2. Preparation of gold nanorods CTAB solution (1.0 mL, 0.20 M) was mixed with 1.0 mL of 0.5 mM HAuCl4. To the stirred solution, 0.12 mL of ice-cold 0.01 M NaBH4 was added, which resulted in the formation of a brownish-yellow solution. Vigorous stirring of the seed solution was continued for 2 min. After the solution was stirred, it was kept at 25  C. The growth solution was prepared by mixing together in 250 mL flask 100 mL of 0.2 M CTAB, 5.6 mL of 4 mM AgNO3, 6.5 mL of 23 mM HAuCl4 and 95 mL of Milli-Q water. Ascorbic acid (0.08 M) approximately 2.5 mL was slowly added to the mixture. The addition of ascorbic acid was conducted dropwise, until the mixture became colorless after which one quarter more of the total number of droplets to that point was added. The final step was the addition of 1.8 mL of the seed solution to the growth solution at 27e30  C. The color of the solution gradually changed within 10e20 min. The temperature of the growth medium was kept constant at 27e30  C during the full procedure.

quartz cell and exposed to the 808 nm laser source at a distance of 2 cm. The original temperature was maintained at w25  C. To determine the impact of material concentrations on the photothermal heating effect, a series of solutions of the AuNRse CpG conjugates with different AuNRs concentrations from 100 pM to 1 nM were irradiated by NIR laser (wavelength, 808 nm; power density, 1.5 W/cm2) for different time periods. To determine the impact of NIR power density, the AuNRseCpG conjugates with AuNRs concentration fixed at 250 pM were irradiated under different power densities. As a control, the temperatures of distilled water (1.0 mL) irradiated by the same laser as described above were measured. The solution temperatures were determined using a digital thermometer. 2.5. Cell culture The murine macrophage-like RAW264.7 cells were grown at 37  C in an atmosphere of 5% (v/v) CO2 in air, in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS, 1.5 g/L NaHCO3, 100 units/mL penicillin, 100 mg/ml streptomycin, 4.5 g/L glucose and 4 mM glutamine. The media was changed every three days, and the cells were digested by trypsin and resuspended in fresh complete medium before plating. The mouse hepatocellular carcinoma cells (H22) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Gibco). The cells were kept at 37  C in a humidified atmosphere containing 5% CO2. 2.6. Cytotoxicity assays MTT assays were used to probe cellular viability. H22 cells were seeded at a density of 5000 cells/well (100 mL total volume/well) in 96-well assay plates. After 24 h incubation, the as-prepared AuNRseCpG conjugates at the indicated concentrations, were added for further incubation of 24 h. To determine toxicity, 10 mL of MTT solution (BBI) was added to each well of the microtiter plate and the plate was incubated in the CO2 incubator for an additional 4 h. Then the cells were lysed by the addition of 100 mL of DMSO. Absorbance values of formazan were determined with Bio-Rad model-680 microplate reader at 490 nm (corrected for background absorbance at 630 nm). Three replicates were done for each treatment group. 2.7. In vitro photothermal heat triggered drug delivery H22 cells were plated in 96-well culture plates (5000 cells/well) separately. After 24 h, drugs (suspended in 2% DMEM) at the indicated concentrations were added and control treatments received only 2% DMEM. At 4 h after incubation, excess unbound AuNRs were removed by rinsing three times with phosphate buffered saline (PBS). Fresh culture medium was then added to the wells. The cells were exposed to NIR light (1.5 W/cm2) for 10 min for photothermal and chemophothermal treatments, and then incubated again at 37  C with 5% CO2 for 24 h. After treatment, MTT solution (5 mg/mL) was added to each well of the microtiter plate and the plate was incubated in the CO2 incubator for an additional 4 h. The cells then were lysed by the addition of 100 mL of DMSO. Absorbance values of formazan were determined with Bio-Rad model-680 microplate reader at 490 nm (corrected for background absorbance at 630 nm). Three replicates were done for each treatment group. 2.8. Proliferation of tumor cells co-cultured with RAW264.7 cells RAW264.7 cells (1  105 cells/ml) and H22 cells (5  104 cells/ml) were placed in the upper and lower chambers of Transwell plates, respectively. AuNRs, CpG ODNs, AuNRseCpG, AuNRs-CpG-Dox and Dox were added to the upper side, respectively, and cells were incubated at 37  C for 48 h. Then, MTT solution was added to the H22 cells in the lower chambers and the plate was incubated in the CO2 incubator for an additional 4 h. The cells then were lysed by the addition of DMSO. Absorbance values of formazan were determined with Bio-Rad model-680 microplate reader at 490 nm (corrected for background absorbance at 630 nm). Three replicates were done for each treatment group.

2.3. Functionalization of gold nanorods Before DNA loading, the thiol functionality on the oligonucleotides was deprotected. The 30 -thiol DNA (0.1 mM) was deprotected by 5.0 mM TCEP in 50 mM Trise HCl (pH 7.5) buffer for 1 h at room temperature. The as-prepared AuNRs solutions (100.0 mL) were subjected to two wash-centrifugation cycles to remove excess CTAB before DNA conjugation. Centrifugation was conducted at 12 000 rpm for 10 min, and deionized water was used for washing in each cycle. To stabilize and functionalize the AuNRs, fresh mPEG-SH stock solution (2.0 mM) was added to the AuNRs suspension (1 nM, 1.0 mL). The mixture sat for 1 h at room temperature, followed by addition of alkanethiol capture DNA (100 nM). Mixtures were incubated for 12 h, after which the solution was buffered at pH 7 (10 mM phosphate), and NaCl solution was added (final concentration of 0.1 M); the entire mixture was allowed to “age” under these conditions for additional 12 h, and excess reagents were finally removed by centrifugation at 12000 rpm for 5 min. 2.4. Photothermal conversion experiments To measure the photothermal conversion performance of the AuNRseCpG conjugates, the solution of the AuNRseCpG conjugates (1.0 mL) was placed in a

2.9. Fluorescence microscopy To examine the characteristics of the AuNRseCpG and AuNRs-CpG-Dox conjugates as the effective photothermal therapy probes, the selective killing of cancer cells with the AuNRseCpG and AuNRs-CpG-Dox were investigated. H22 cells were incubated with the samples in solution for 4 h, washed with PBS solution, and then irradiated by a continuous-wave NIR laser (l ¼ 808 nm, power ¼ 1.5 W/cm2) for 10 min. After irradiation, cells were restored in an incubator for another 2 h, and then stained with both calcein AM and propidium iodide to verify the photothermal effect on cancer cells (where green fluorescence from calcein and red fluorescence from propidium iodide indicate live and dead cells, respectively). Then, the images were captured using an Olympus BX-51 optical equipped with a CCD camera. 2.10. Cytokine assays RAW264.7 cells were seeded on 24-well culture plates at a density of 5  105 cells/well. After 24 h incubation, cells were washed with 0.5 ml PBS before treatment with indicated conditions for 8 h (TNF-a) or 24 h (IL-6). The supernatants were collected and stored at 80  C until use. The levels of TNF-a and IL-6 in the

Please cite this article in press as: Tao Y, et al., Engineered, self-assembled near-infrared photothermal agents for combined tumor immunotherapy and chemo-photothermal therapy, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.073

Y. Tao et al. / Biomaterials xxx (2014) 1e11 supernatants were determined by enzyme-linked immunosorbent assay (ELISA) using antibody pairs specific to these cytokines following protocols recommended by the manufacturer. 2.11. In vivo anti-tumor efficacy studies Subcutaneous tumors were initiated in the flank of the BALB/c mice by injecting one million H22 cells. When the tumor volume reached w100 mm3, mice were divided into six groups consisting of 5 mice in each group, with weight and tumor size differences minimized among the groups. Prior to drug injection, mice were anesthetized with isofluorane. After that, mice in groups 1 to 3 were injected intratumorally with Dox (1.75 mM, 50 mL), Y-shaped CpG ODNs (116 nM, 50 mL) and the AuNRs-CpG-Dox conjugates (ACD, 5  1010 NR particles, 1.65 nM and 50 mL), respectively. Mice in group 4e6 received the injections of PBS (50 mL), AuNRs (5  1010 NR particles, 1.65 nM and 50 mL) as well as the AuNRs-CpG-Dox conjugates (ACD, 5  1010 NR particles, 1.65 nM and 50 mL), and were followed by NIR laser treatment (1.5 W/cm2, for 10 min) 2 h after each drug injection. Then the tumor development was monitored by measuring the tumor size at regular intervals for 12 days. The tumor dimensions were measured with a caliper, and the tumor volume was calculated according to the equation: Volume ¼ (Tumor Length)  (Tumor Width)2/2. In addition, the body weights of the mice in these groups were also recorded at regular intervals for 12 days. All animal procedures were in accord with the guidelines of the Institutional Animal Care and Use Committee. 2.12. Statistical analysis All data were expressed in this article as mean result  standard deviation (SD). All figures shown in this article were obtained from three independent experiments with similar results. The statistical analysis was performed by using Origin 8.0 software.

3. Results and discussion 3.1. Synthesis of the AuNRs-CpG-Dox conjugates Herein, we design the immunomodulatory CpG ODNs based platform that is conjugated with NIR-responsive gold nanorods (AuNRs) and doxorubicin (Dox) for cancer therapy (Scheme 1). Due to tunable localized surface plasmon resonance (LSPR), AuNRs can become highly localized heat sources when irradiated with a laser through the photothermal effect, which can be utilized to provide hyperthermal cancer therapy [43e46]. In addition, the immobilization of the Y-shaped CpG motifs on the AuNRs can largely improve the immune response [47], which offers great potential for effective cancer immunotherapy. Moreover, Dox molecules, which

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are able to intercalate into the CpG ODNs [48], can be controllably released from the CpG ODNs/Dox complex upon NIR irradiation. Therefore, with AuNRs serving as the CpG ODNs and anticancerdrug carrier, NIR laseremodulated photothermal effects can not only provide hyperthermal cancer therapy, but also trigger the immunostimulatory signals and anticancer agents release for chemo/immunotherapy. Development of such multimodal theranostic systems with individual functions acting in a coordinated way is critical to optimize therapeutic efficacy and safety of therapeutic regimes, and would provide more opportunities for ondemand therapy and pave the road toward personalized medicine. To synthesize AuNRs, a seed-mediated surfactant-directed approach was used [49]. The synthesized AuNRs were characterized by the TEM image and UVvis absorption spectrum analysis. As shown in Fig. 1a, the as-prepared AuNRs were well dispersed and showed good uniformity. The characteristic transverse and longitudinal bands were observed at 520 and 800 nm, respectively (Fig. 1b). Next, to prevent aggregation of AuNRs during the conjugation process and reduce the toxicity of AuNRs, thiol-terminated poly-(ethylene glycol) (PEG-SH, molecular weight (MW) 5000)) was conjugated on the AuNR surfaces via thiol chemistry [50]. After that, the surface modifications of the AuNRs with the thiol-labeled CpG ODNs were performed by the same procedures as previously reported [51,52]. Then, the other two complementary strands were mixed with the thiol-labeled CpG ODNs on AuNRs, and DNA hybridization led to the assembly of the Y-shaped CpG ODNs (Fig. S1), which were beneficial for Dox loading. Zeta potential measurements showed that the CTAB bilayer formed around AuNRs made them highly positively charged (42.3 mV) (Fig. S2). After DNA attachment, the zeta potential of AuNRs was changed to negatively charged (18.6 mV) [12,53]. Dynamic light scattering revealed that the AuNRseCpG conjugates had a hydrodynamic size of 91.5 nm (Fig. S3), compared to AuNRs which had a hydrodynamic size of 25.0 nm. We took these results as the evidence for the covalent DNA binding to the AuNRs surface. In addition, the CpG ODNs-modified AuNRs showed a 6 nm blue (in the web version) shift of the longitudinal peak as compared to CTAB-stabilized AuNRs with no peak broadening (Fig. 1b). The observed spectral shifts could be attributed to changes in the refractive index of the local environment

Scheme 1. Schematic diagram of a co-delivery system based on gold nanorods to deliver doxorubicin and CpG ODNs simultaneously to tumor tissue for enhanced therapy efficacy.

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Fig. 1. (a) TEM image of the gold nanorods. Scale bars, 20 nm. (b) Absorption spectrum of original AuNRs stabilized with CTAB, PEG-modified AuNRs, and AuNRseCpG conjugates. NIR-induced heat generation of (c) AuNRseCpG conjugates (250 pM) at different power densities, (d) AuNRseCpG conjugates at different concentrations at the same power density of 1.5 W/cm2.

surrounding the nanorods. This effect had been well documented in the literature [54,55]. TEM result (Fig. S4) indicated that after DNA attachment, the morphology (aspect ratio) of AuNRs was almost not changed (3.79  0.36 vs 3.81  0.32) and the aggregation of AuNRs did not occur. Quantification of the immobilized oligonucleotides was performed by a fluorescence based method using Cy3-labeled complementary oligonucleotides CpG ODN-1 (Cy3CpG ODN-1). The self-complementary Y-shaped CpG ODNs (Cy3CpG ODN-1, CpG ODN-2 and SH-CpG ODN-3) were anchored on the AuNRs surface. The amount of immobilization was measured from the decrease in fluorescence intensity of Cy3-CpG ODN-1 left in supernatants and calculated based on a calibration curve according to Cy3-CpG ODN-1 concentration (Fig. S5) [20,50,52,56]. It was estimated that about 70 Y-shaped CpG ODNs molecules were bound on each AuNRs. After that, Dox could be efficiently intercalated into the Y-shaped CpG ODNs due to its flat aromatic rings and positive charge [57]. Previous studies showed that the fluorescence of Dox could be quenched after intercalation into the CG base pairs [58,59]. Here, we used this finding to identify the intercalation efficiency of Dox loaded onto the designed AuNRseCpG conjugates platform (Figs. S6, S7) [12,60]. Fig. S6a showed a sequential decrease in the Dox fluorescence intensity, when a fixed concentration of Dox was incubated with an increasing molar ratio of the Y-shaped CpG ODNs. The maximum Dox value was confirmed using a Hill plot as described in Fig. S6b [12]. Theoretical evaluation

indicated that the loading capacity of the designed DNA sequence was about 15 Dox per Y-shaped CpG ODNs. Based on the loading of 15 Dox molecules per Y-shaped CpG ODNs, and 70 copies of CpG ODNs per AuNRs, we inferred that each AuNRs could load about 1050 Dox molecules. 3.2. Photothermal effect of the AuNRseCpG conjugates and NIR light-triggered drug release Prior to construct the multifunctional platform for cancer therapy, it was necessary to evaluate the efficiency of photothermal conversion. Temperature changes were shown in Fig. 1ced, demonstrating that photothermal heating effect increased monotonically with AuNRs concentration and radiant energy, whereas, the temperature of nanoparticle-free distilled water increased only slightly. These results revealed that the temperature delivered by our material could be effectively regulated, indicating that it had great potential for thermal ablation of malignant tissues. Next, to demonstrate the photothermal remote controlled actuation of the AuNRs-CpG-Dox platform (ACD), the NIR laser-controlled release of Dox and CpG ODNs was tested. As can be seen in Fig. S8, a very clear and highly effective photothermal-operable gating effect was demonstrated by monitoring the fluorescence intensity (555 nm) of Dox as a function of time. The results showed that, without NIR irradiation, only negligible release occurs (less than 5% of the entire

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Fig. 2. (a) Cytotoxicity assays of H22 cells upon 808 nm laser irradiation and those incubated with Dox, AuNRseCpG and AuNRs-CpG-Dox conjugates upon 808 nm laser irradiation with a power density of 1.5 W/cm2 for 10 min. Error bars represent standard deviation of three independent measurements. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.005, ***P < 0.001). (bee) Fluorescence microscopy images of H22 cells incubated with (b) AuNRseCpG, (c) control þ 808 nm NIR light, (d) AuNRseCpG þ 808 nm NIR light and (e) AuNRs-CpG-Dox þ 808 nm NIR light. Viable cells were stained green with calcein AM, dead cells were stained red with propidium iodide (PI). Scale bar ¼ 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

payload) at the beginning 10 min, indicating good capping efficiency. However, when the suspension of ACD was exposed to 808 nm laser (1.5 W/cm2), a burst release of Dox molecules could be observed within 10 min. As irradiation was prolonged, the amount of Dox released into the solution kept increasing, and eventually leveled off. The burst release of Dox from the ACD was ascribed to the remote heating generating by AuNRs through photothermal conversion. In addition, the CpG ODNs could also be efficiently released from the gold surfaces with NIR irradiation (Fig. S9). Thus, we inferred that the ACD with the photothermal effects could also be used as an efficient remote controlled delivery system for effective release of Dox and the CpG ODNs [61]. Furthermore, it was possible to fine-tune the delivery rate by simply controlling the NIR

irradiation power density and the irradiation time. Therefore, with the NIR laseremodulated photothermal effects, the AuNRs could not only be applied for photothermal therapy, but also be used as a promising system for delivery of immunostimulatory signals and anticancer agents due to its transport function, a combination that would be highly effective for cancer therapy. 3.3. In vitro combined chemo-photothermal therapy Before the AuNRseCpG conjugates (AC) to be applied in biological systems, the potential cytotoxicity of AC was evaluated by a conventional methyl thiazolyl tetrazolium (MTT) assay (Fig. S10). The viability of H22 cells was measured in the presence of AC at

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Fig. 3. Cytokine release from RAW264.7 cells stimulated by AuNRseCpG conjugates. Comparison of (a) TNF-a and (b) IL-6 release stimulated by single-stranded CpG ODNs (ssCpG), Y-shaped CpG ODNs (Y-CpG), AuNRs and AuNRseCpG conjugates. Error bars represent standard deviation of three independent measurements.

various concentrations (12.5e250 pM). No apparent cellular toxicity of AC could be observed, even at the highest concentration (250 pM). These data indicated that AC exhibited extremely low cytotoxicity toward H22 cells, revealing that AC had high biocompatibility. In the following, the synergistically enhanced anticancer effect of the combined photothermal-/chemo- therapy was quantified. To demonstrate the detailed effect of NIR irradiation, a power density of 1.5 W/cm2 was introduced to treat the H22 cells. As shown in Fig. 2a, no obvious cytotoxicity was observed when cells were treated with the NIR light only. However, when cells were incubated with Dox, the viability decreased from 88 to 52%, depending on the concentration of Dox. In addition, the viability of cells treated with AC also decreased to 58% upon NIR light irradiation, demonstrating the AuNRs could function as the photothermal agent to effectively kill the cancer cells under the irradiation of the NIR laser. As expected, when the two treatments were combined under a single irradiation, the cell viability was remarkably reduced to 12% (P < 0.005). The dramatically decreased cell viability suggested that ACD could not only be utilized for photothermal ablation of cancer cells but also act as the drug delivery vehicle for cancer chemotherapy, and importantly the combination of chemotherapy and photothermal therapy demonstrated better effects on cancer treatment, overmatching individual treatment. These results revealed that ACD was a powerful agent for combined thermo-chemotherapy of cancer cells. The fluorescent microscopic images further confirmed that the cell viability was distinguishing following different treatments and after being stained with the LIVE (green)/DEAD (red) stains. As shown in Fig. 2d, a clear demarcation line between dead (red) and live cell (green) regions could be observed in the presence of AC under laser irradiation. By contrast, neither the nanoparticles themselves (Fig. 2b) nor laser irradiation alone (Fig. 2c) led to cell death. These results indicated that the overheating of the cells resulted from the photothermal conversion of AuNRs in cells would contribute for the cell death (Fig. S11, S12), which implied that the AuNRs could act as a promising candidate for photothermal therapy. Furthermore, almost all the cells were destroyed after being incubated with ACD and exposed to the NIR laser at 1.5 W/cm2 for 10 min (Fig. 2e). All these results clearly demonstrated that, by integration of the advantages of chemotherapy and photothermal therapy, the ACD could improve the anticancer efficiency relative to the use of each approach independently.

3.4. In vitro combined chemo-immunotherapy On the other hand, to investigate the differences of the immune stimulation with treatments, we tested the immunostimulatory activities of AC by measuring secreted cytokine levels. We cultured the single-stranded CpG ODNs, Y-shaped CpG ODNs, AuNRs and AC with RAW264.7 cells and measured the release of cytokines by using ELISA assays. As can be seen in Fig. 3a, AC dramatically induced the production of tumor necrosis factor alpha (TNF-a) [39,62], excelling that of Y-shaped CpG ODNs by more than 5 times. In addition, AuNRs alone had nearly no effect on cytokine secretion, thus indicating that the observed immunostimulatory activities were indeed caused by the CpG sequence. Similarly, compared with the Y-shaped CpG control, the level of the other cytokine interleukin 6 (IL-6) [62] (Fig. 3b) induced by AC was also greatly increased. It was exciting to see that by simply conjugating the CpG ODNs on AuNRs, this proposed system was capable of producing

Fig. 4. Antiproliferative activity of AuNRs-CpG-Dox conjugates against H22 cells. RAW264.7 cells and H22 cells were placed in the upper and lower chambers of Transwell plates, respectively, and CpG ODNs, AuNRs, Dox or their mixture were added to the upper sides. After 48 h, the viability of the H22 cells was estimated by MTT assays. Error bars represent standard deviation of three independent measurements. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.005, ***P < 0.001).

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Fig. 5. In vivo photothermal tumor heating. (a) IR thermal images of tumor-bearing mice exposed (or not exposed) to the NIR laser (808 nm, 1.5 W/cm2, 10 min) after injection with PBS (50 mL), AuNRs (5  1010 NR particles, 1.65 nM and 50 mL) or AuNRs-CpG-Dox (5  1010 NR particles, 1.65 nM and 50 mL). Before IR thermal imaging, the mice were anesthetized and abdominal hair was removed with depilatory cream. (b) Temperature changes of mice tumors monitored by the IR thermal camera during laser irradiation as indicated in (a).

cytokines with productivity well above that of the CpG ODNs alone. We could attribute the enhanced cytokine expressions to two important parameters that were unique to the AuNRseCpG. One of the obvious factors was the greatly enhanced cellular uptake efficiency of AC as compared to CpG ODNs (Fig. S13) [63]. In addition, increased stability and high resistance to nuclease degradation was another key factor for the enhanced immunostimulatory activity [64].

It was well known that tumor tissues were enriched with immune cells, such as dendritic cells and macrophages [65]. CpG ODNs could be recognized by these cells and induced the release of anticancer cytokines from these cells. In contrast, Dox had been reported to be released from these cells in tumor tissues and reach adjacent cancer cells [66,67], which resulted in no significant cytotoxicity to immune cells. We therefore estimated the antiproliferative activity of ACD against H22 cells co-cultured with the

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Fig. 6. In vivo imaging and biodistribution of BALB/c mice bearing H22 tumors after intratumoral injection of free Dox (1 mg/kg) or AuNRs-CpG-Dox conjugates (containing 1 mg/kg Dox) with NIR irradiation. (a) Time-lapse NIR fluorescence images of BALB/c mice. Before the imaging, the mice were anesthetized and abdominal hair was removed with depilatory cream. The fluorescence of heads and feet was attributed to the strong tissue autofluorescence from the animal fur as the excitation/emission of Dox lies in the visible spectrum window. (b) NIR fluorescence images of major organs and tumors after injection of Dox or AuNRs-CpG-Dox conjugates with NIR irradiation at 24 h.

RAW264.7 cells (Fig. 4). AuNRseCpG-free ODNs had no significant effect on the viability of tumor cells. The higher cytotoxic activity of the AuNRseCpG platform (P < 0.001) compared with AuNRse CpG-free ODNs demonstrated that CpG motif-mediated production of soluble factors, probably antitumor cytokines, were effective in inhibiting the proliferation of H22 cells. In addition, the cytotoxicity of the AuNRs-based nanocarriers complexed with CpG ODNs and Dox demonstrated a higher cytotoxicity than their corresponding counterparts (ACD versus AC (P < 0.05) and Dox (P < 0.005)). It was probably because of the synergistic effect between the chemotherapy and immunotherapy [68], which significantly enhanced the selective tumor cell destruction and antitumor immune response. All these results indicated that ACD could be an effective platform that inhibited tumor cell growth by delivering both a proinflammatory signal and anticancer agent to tumor cells. 3.5. In vivo effects of the AuNRs-CpG-Dox conjugates Finally, to assess the in vivo antitumor efficacy of ACD, the tumor model (mouse hepatocellular carcinoma cell line H22) was employed. Subcutaneous tumors were initiated in the flank of the

BALB/c mice by injecting one million H22 cells. After the tumors had developed to about 100 mm3, the drug efficacy was studied in six groups of mice (n ¼ 5 per group), with weight and tumor size differences minimized among the groups. Therefore, the results of 5 mice in each group could accurately and comprehensively assess the in vivo antitumor efficacy of ACD. Five regimens (PBS, Dox, CpG, AuNRs and ACD) were administered by a single intratumoral injection, with a dose of 5  1010 NR particles per mouse in the H22 tumor model. The tumors were then irradiated by the 808 nm laser at a moderate power density of 1.5 W/cm2 for 10 min. An IR thermal camera was used to monitor the temperature changes on mice during laser irradiation (Fig. 5). Upon 808 nm laser irradiation, mice treated with AuNRs or ACD showed localized heating in the tumor regions, where the temperatures were greatly increased after 10 min. In marked contrast, the tumor temperatures of mice injected with PBS exhibited no significant increase during 10 min of laser irradiation. One of the important concerns after intratumoral injection was whether the Dox would accumulate in xenograft tumor or organs or clear out of the body. Fig. 6a showed the fluorescence signal of Dox around the tumor after intratumoral injections of free Dox and ACD. For free Dox, the fluorescence was greatly reduced at 24 h postinjection. In contrast,

Please cite this article in press as: Tao Y, et al., Engineered, self-assembled near-infrared photothermal agents for combined tumor immunotherapy and chemo-photothermal therapy, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.073

Y. Tao et al. / Biomaterials xxx (2014) 1e11

Dox fluorescence was significantly higher in the ACD-treated group compared to the free-Dox-treated group, especially at the time point of 24 h. The comparative major organs and tumors distribution of Dox in the ACD-treated group and the free-Doxtreated group were shown in Fig. 6b. The results demonstrated that most of Dox accumulated in the liver and kidneys 24 h after intratumoral injection of free Dox. By contrast, the AuNRs-CpGDox conjugates dramatically increased the accumulation of Dox in the tumor. These results indicated that ACD could efficiently enhance the Dox accumulation in tumor. Then the tumor development was monitored by measuring the tumor size at regular intervals for 12 days. In the H22 tumor model, the mean tumor volumes at different days were calibrated by normalizing the initial volume (at day 0) to 1 (Fig. 7a). Consistent with previous observations [69,70], the treatment with AuNRs plus NIR irradiation delayed tumor growth compared to treatments with PBS, due to the photothermal ablation of cancer cells. Moreover, the CpG group also showed higher efficacy in tumor reduction compared to PBS control group, presumably because CpG motif-mediated activation of the innate immune system, resulting in the production of proinflammatory factors, probably antitumor cytokines, was effective in inhibiting tumor growth. Remarkably, the ACD with NIR irradiation showed the highest efficacy in tumor reduction compared to other groups (P < 0.001), which indicated that the fusion of three kinds of treatments led to a significant benefit relative to the use of each method alone. Notably, the ACD group without NIR irradiation also demonstrated modest tumor reduction compared to the PBS group, possibly because of the gradual release of Dox and CpG ODNs from the intratumorally injected ACD overtime [15]. The advantage of ACD lied in the synergistic interaction among the photothermal reaction, chemotherapy and immunological stimulation at the same time during the tumor treatment. The AuNRs could effectively enhance the thermal destruction of cancer cells during NIR laser irradiation. In addition, the CpG motif-mediated production of soluble factors, such as antitumor cytokines, was effective in inhibiting the proliferation of H22 cells. Furthermore, Dox molecules that were released from ACD subsequent to NIR irradiation contributed to the additional chemotherapeutic efficacy. All these effects not only resulted in significant antitumor efficacy but also led to a long-term tumor-specific immunity. This triple combination of chemotherapy, thermotherapy, and immunotherapy, would achieve optimal therapeutic efficacy in cancer treatment, giving synergistic antitumor effects. Besides, no significant weight loss was observed in the mice, suggesting that the hyperthermia treatment with ACD was nearly nontoxic despite the pronounced tumoricidal effects (Fig. 7b). All these results implied that the integration of the three therapeutic methods in one vehicle has improved tumoricidal efficacy relative to the use of each approach independently. 4. Conclusion In summary, we have rationally developed a multifunctional platform using a self-assembly strategy to incorporate materials with specific functions of chemotherapeutics, hyperthermia, and especially immunotherapy, which collectively contributed to the effective cancer treatment. The AuNRs can be applied as the nanocarrier to simultaneously address the three kinds of treatments, which lead to a significant benefit relative to the use of each method alone. The induction of AuNRs here provides several critical advantages such as high biocompatibility, the high carrying capacity for immunostimulatory signals and anticancer agents, as well as the hyperthermal cancer therapy. In addition, the Y-shaped CpG ODNs have been shown to be effective in inducing the

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Fig. 7. Anti-tumor efficacy of the NIR-responsive NP platform. PBS, Dox, Y-shaped CpG ODNs, AuNRs, AuNRs-CpG-Dox were injected intratumorally in a single dose (5  1010 particles), followed by 10 min NIR irradiation or without NIR irradiation. (a) The volumetric changes in tumor size relative to that at day 0 are plotted over time. Asterisk on day 12 represents significant differences between tumor volumes of AuNRs-CpG-Dox- with NIR irradiation and other platforms-treated mice (*P < 0.05, **P < 0.005, ***P < 0.001). (b) Changes with time in body weight achieved from mice injected with PBS, Dox, Y-shaped CpG ODNs, AuNRs, AuNRs-CpG-Dox. Data are presented as mean  SE of five mice per group.

cytokines, such as TNF-a and IL-6, in macrophage-like TLR9-positive cells, which can greatly enhance the immunostimulatory activity. Furthermore, we have demonstrated that the anticancer agent Dox can efficiently intercalate into the Y-shaped CpG ODNs, which provide additional advantages for chemotherapy. Both in vitro and in vivo assays reveal that this engineered vehicle exhibits significant antitumor efficacy. Our studies provide strong evidence that the AuNRs-CpG-Dox conjugates can be utilized as efficient antitumor agents. Acknowledgments The authors are grateful for the referee’s helpful comments on the manuscript. Financial support was provided by the National Basic Research Program of China (2011CB936004 and 2012CB720602) and the National Natural Science Foundation of China (Grants 91213302, 21210002).

Please cite this article in press as: Tao Y, et al., Engineered, self-assembled near-infrared photothermal agents for combined tumor immunotherapy and chemo-photothermal therapy, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.073

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Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.04.073.

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