Conducting Polymer Core

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Biological and Medical Applications of Materials and Interfaces

Controllable Synthesis of Gold Nanorod/Conducting Polymer Core/Shell Hybrids Towards In Vitro and In Vivo Near-Infrared Photothermal Therapy Juan Wang, Chunhua Zhu, Jie Han, Na Han, Juqun Xi, Lei Fan, and Rong Guo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16784 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

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ACS Applied Materials & Interfaces

Controllable Synthesis of Gold Nanorod/Conducting Polymer Core/Shell Hybrids Towards In Vitro and In Vivo Near-Infrared Photothermal Therapy Juan Wang,† Chunhua Zhu,‡ Jie Han,*,† Na Han,† Juqun Xi,*,‡ Lei Fan,† Rong Guo*,† †

School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, Jiangsu,

225002, P. R. China. ‡

School of Medicine, Yangzhou University, Yangzhou, Jiangsu, 225002, P. R. China.

KEYWORDS: gold nanorod; conducting polymer; core/shell structure; photothermal therapy ABSTRACT: Photothermal therapy (PTT) is a minimally invasive tumor treatment technology, and is regarded as a potential anticancer strategy due to its targeted destruction and low toxicity. Specifically, near-infrared light-induced PTT has attracted intriguing interest due to the high transparency of tissue, blood and water. However, effective PTT generally requires the assistance of photothermal agents. Gold nanorods (GNRs) and conducting polymer are often used as photothermal materials because of their high absorption efficiency and photothermal conversion efficiency. Herein, we combined GNRs with poly(o-methoxyaniline) (POMA, a polyaniline derivative) to form GNRs/POMA core/shell hybrids through the surfactant-assisted chemical oxidative polymerization route and studied their photothermal conversion properties. The configuration of GNRs/POMA core/shell hybrids has been precisely controlled through adjusting the monomer concentration, and the relationship between morphology and absorption band of GNRs/POMA core/shell hybrids has been revealed. Finally, the in vitro and in vivo

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experiments were performed, and the results indicated that the GNRs/POMA core/shell hybrids with optimized absorbance at around 808 nm exhibited the best performance on photothermal therapy under 808 nm NIR laser irradiation.

1. INTRODUCTION Photothermal therapy (PTT) based on the conversion of light into local heat by photothermal nanomaterials can lead to the thermal ablation of cancer cells, which has aroused significant interest in the forefront of medical science and material science because of high specificity, minimal invasiveness and low systemic toxicity, as compared with traditional cancer therapies.1-9 Specifically, near-infrared (NIR) light-induced PTT is an intriguing choice due to the high transparency of tissue and blood with in the NIR light range of 650 to 950 nm.10-20 Until now, the various photothermal nanomaterials for PTT ablation of cancer have been widely carried out. Among the various photothermal nanomaterials, gold nanomaterials, including nanoparticles21-25 and nanorods26-31 are the most widely explored class of PTT agents. Gold nanorods (GNRs) have been frequently used in PTT and relevant medical science owing to their high absorption efficiency and photothermal conversion efficiency in the NIR wavelength range. However, GNRs exhibit low photostability due to the "melting effect".28 Besides, the clustering and aggregating of GNRs have also restricted their application in PTT.28 In order to take advantage of the excellent optical performance and overcome shortcomings of GNRs, GNRs are often hybridized with other materials. For instance, GNRs coated with silica as core/shell hybrids enables GNRs with excellent stability.32-35 However, the silica shells may hamper the penetration of the electric field and heat transfer.36 Conducting polymers, such as polyaniline (PANI) and polypyrrole (PPY), have been intensively studied for decades because of


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their novel properties and potential applications in fields of sensor, catalysis, environmental remediation, etc.37-41 More interesting, it has been shown that PANI and PPY can generate a large amount of heat energy through the photothermal conversion upon NIR irradiation and therefore showing potential as photothermal agents.26,

42-44

In addition, as polymeric

photothermal agents, PANI and PPY have also been established to show good biocompatibility and biodegradability.28, 31, 43-45 As a result, the incorporation of conducting polymers into GNRs will not only enables high stability of GNRs, but also potentially contributes to improve photothermal conversion efficiency of GNRs. Although PPY stabilized urchin-like gold nanoparticles and GNRs have been reported for photothermal therapy, the rational design of GNRs/conducting polymer hybrids with optimized photothermal effect and the in vitro and in particular in vivo NIR photothermal therapy of cancer have not been revealed, which are crucial from the points of both fundamental understanding and practical applications. Herein, we reported the controllable synthesis of GNRs/poly(o-methoxyaniline) (POMA) core/shell hybrids through surfactant-assisted chemical oxidative polymerization of monomers to ensure the even coating of polymer shells on surfaces of GNRs. 46-47 By adjusting the amount of monomers, the coating thickness of POMA on surfaces of GNRs could be precisely controlled. The relationship between the core/shell structure and absorption property of GNRs/POMA core/shell hybrids were revealed. The in vitro and in vivo NIR photothermal therapy was also investigated. The results revealed that GNRs/POMA core/shell hybrids showed enhanced photothermal property and photostability, as compared to bare GNRs upon NIR laser exposure, and the GNRs/POMA core/shell with optimized photothermal effect had been exploited. In addition, the in vivo photothermal ablation of tumors showed excellent treatment efficacy.


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Results confirmed that the GNRs/POMA core/shell hybrids showed potential as advanced photothermal agents.

2. EXPERIMENTAL METHODS 2.1. Materials: Amphiphilic poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) triblock copolymer (molecular weight of 12,600) was purchased from Sigma-Aldrich. MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Thermo Fisher Scientific (USA). Other reagents were obtained from Sinopharm Chemical Reagent Co. Ltd. o-methoxyaniline monomer were distilled under reduced pressure before use. Water used in this study was purified with Milli-Q Plus system (Millipore, France). 2.2. Synthesis of GNRs GNRs with an aspect ratio of ∼3.0 were synthesized as reported.48 The seed solution for GNRs was prepared by mixing 5.0 mL of 0.2 mol L-1 hexadecyltrimethylammonium bromide (CTAB) aqueous solution with 5.0 mL of 0.5 mmol L-1 HAuCl4•3H2O, after which 0.6 mL of ice-cold 0.01 mol L-1 NaBH4 aqueous solution was quickly added into the mixture solution under vigorous stirring for 2 min. For the GNR growth solution, 4.5 g CTAB, 0.772 g sodium oleate (NaOL), 6.0 ml of 4 mmol L-1 AgNO3 aqueous solution and 125.0 mL of 1.0 mmol L-1 HAuCl4•3H2O aqueous solution were first mixed, 1.05 mL concentrated HCl was then employed to adjust the pH. Second, 0.625 mL of 0.064 mol L-1 ascorbic acid (AA) and 0.2 mL seed solution was added. Finally, the resultant mixture was aged at 30 °C for 12 h without stirring. The as-synthesized GNRs were centrifuged with 40 mL aliquots at a time for 5 min, and were dispersed in 2.0 mL water for further usage.


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2.3. Synthesis of GNRs/POMA Core/Shell Hybrids The even coating of POMA on GNRs was realized using a similar method for the synthesis of POMA coating on Au nanoparticles through the surfactant-assisted chemical oxidative polymerization route as we previously reported.46-47 The thickness of the POMA shell was adjustable by altering the amounts of o-methoxyaniline monomer. The typical synthesis of GNRs/POMA core/shell hybrids were as follows: after addition of 0.03 g F127 into 8.0 mL water with vigorous stirring to form a transparent solution, 2.0 mL of GNRs colloidal solution was subsequently added under stirring for 1 h. Then a certain volume (1, 2, 5, or 10 µL) of omethoxyaniline monomer was injected into above mixture with stirring for 1 h. After that, 1.0 mL of APS (the molar ratio of monomer to APS was set at 1:1) aqueous solution was added. The chemical oxidative polymerization reaction was proceeded with agitation for 2 h. Finally, the assynthesized GNRs/POMA core/shell hybrids were centrifuged and dispersed in 2.0 mL water. For the purpose of distinction, the as-synthesized GNRs/POMA core/shell hybrids at the monomer amount of 1, 2, 5, and 10 µL were denoted as GNRs/POMA(1), GNRs/POMA(2), GNRs/POMA(5), and GNRs/POMA(10), respectively. 2.4. In Vitro Photothermal Evaluation The photothermal effect of GNRs and GNRs/POMA core/shell hybrids (50 µg mL-1) were investigated by monitoring the temperature of colloidal solution containing photothermal nanomaterials irradiated by the 808 nm-laser (3.0 W cm-2, MW-GX-808, Changchun Laser Technology Co., Ltd.). The temperature of the colloidal solution was measured by a thermocouple thermometer. The colloidal solution containing GNRs or GNRs/POMA core/shell hybrids were exposed to the 808 nm-laser for 10 min (laser on), then the laser was turned off till


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the solution was cooled to room temperature (laser off). Six cycles of irradiation on/off with the laser were performed. 2.5. In Vitro PTT The CT26 mouse colon cancer cell line was provided by the Cell Resource Center, Shanghai Institutes for Biological Sciences. The cell line was cultured in DMEM medium at 37 °C in 5% CO2-humidified environment with 10% fetal bovine serum and 1% penicillin/streptomycin. For testing the cytotoxicity, the CT26 cells were seeded in 96-well plates at a density of 1×104 cells per well at 37 °C with 5% CO2 overnight. The medium was replaced by a fresh medium containing the photothermal agents of GNRs or GNRs/POMA core/shell hybrids at different concentrations. Then the standard MTT assays were measured to determine the relative cell viability. For in vitro PTT, CT26 cells were seeded in 96-well plates at a density of 1×104 cells per well and cultured for 24 h followed by treatment with the same concentration of photothermal agents. After incubation for 4 h, the cells were exposed to the 808 nm NIR laser (3.0 W cm-2) for 10 min, followed by measuring the relative cell viabilities using the similar method. For the observation of laser scanning confocal microscopy, the CT26 cells were seeded in 24well plate at a density of 1×105 per well and incubated for 12 h. Then the culture medium in each well was replaced with 1.0 mL of GNRs or GNRs/POMA(5) (20 µg mL-1) diluted with culture medium. After the incubation for 2 h, the cells were irradiated under 808 nm-laser (3 W cm-2) for 10 min, and then incubated at 37 °C for 24 h. The cells were then rinsed twice with 0.01% phosphate buffer saline (PBS), fixed with 4% paraformaldehyde for 15 min, 0.4% Triton for 5 min and stained with Calcein-AM and PI for 30 min. After rinsing with PBS three times, cell climbing slices were placed on the glass slides and imaged by laser scanning confocal


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microscopy (LSCM, TLS SP8 STED). 2.6. In Vivo PTT Female Balb/c mice were purchased from Yangzhou University Comparative Medicine Centre. The procedures were approved by Yangzhou University Laboratory Animal Center and the Medical Ethics Committee of Yangzhou University Medical Academy. To create CT26 tumor model, 2×106 cells in 200 µL of serum-free DMEM medium were subcutaneously injected into the back of each female Balb/c mouse. The mice were used for in vivo experiments when the tumor volumes reached ~60 mm3. The tumor-bearing mice were intravenously administrated with 50 µL of GNRs or GNRs/POMA core/shell hybrids with the concentration of 2 mg mL-1 at the agent dose of 100 µg per mouse, and mice treated with the same volume of saline were used as the control. On day 1, 4, 6, 9, 12, and 15, each group was intravenously injected with the photothermal solutions. For the in vivo PTT, an 808 nm-laser (3 W cm-2) was employed to irradiate the tumor areas after 3 h of intravenous injection for 10 min. The temperature of the tumor sites was measured by an IR 7320 thermal camera (Fluke, Ti-959 Hz). The tumor volume was measured by a caliper and calculated based on the following equation: tumor volume (mm3) = length × width2 × 0.5. The relative tumor volumes were calculated as V/V0 (V0 was the initial volume). When the tumor disappeared, the tumor volume was recorded as ‘zero’. In order to study the survival rate of the mice after various treatment, five groups of the tumor-bearing mice (n=8) were used for in vivo experiments again. 2.7. Characterization The morphologies were revealed by a transmission electron microscopy (TEM, Tecnai-12 Philip Apparatus Co.). The Ultraviolet-visible (UV-vis) spectra of the colloidal solutions were acquired in the range between 400 and 850 nm using UV-2501 (Shimadzu Corp., Japan). Fourier


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transform infrared (FTIR) spectra were recorded in the range 400-4000 cm-1 using FTIR spectroscopy (Bruker Tensor 27). The samples were prepared in pellet form with spectroscopicgrade KBr.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of GNRs/POMA Core/Shell Hybrids Figure 1 illustrates the synthesis of GNRs/POMA core/shell hybrids. Firstly, the CTABcapped GNRs nanoparticles were synthesized through a seed-mediated approach,48 followed by the addition of F127 to change the surface properties for the preparation of subsequent reaction. Next, GNRs were mixed with o-methoxyaniline monomers, followed by the addition of APS oxidant to initiate the chemical oxidative polymerization. Finally, the polymerized POMA polymers located on surfaces of GNRs to form GNRs/POMA core/shell hybrids. At the low amount of monomer, the POMA polymer is preferred to deposit on surfaces instead of the tips of GNRs to low down the surface energy of the GNRs. With increasing monomer concentration, the polymer coating extends to the tips of GNRs with the ellipsoid configuration. Further increase in monomer concentration leads to the spherical configuration of GNRs/POMA core/shell hybrids. Direct visualization of the polymer coating on GNRs can be achieved by TEM observations. Figure 2a and Figure 2b-e show the TEM images of GNRs and GNRs/POMA core/shell hybrids, respectively. The diameter of the GNRs is about 25 nm and the length of the GNRs is about 75 nm (Figure 2a). As for GNRs/POMA(1) core/shell hybrids, POMA polymer was coated on side surfaces of GNRs, rather than the tip surfaces (Figure 2b). The morphological transformation from rod to ellipsoid should be ascribed to the surface energy demand. The polymer coating covered the side surfaces in the case of GNRs/POMA(2) core/shell hybrids (Figure 2c). The GNRs were totally embedded in polymer with spherical configuration in GNRs/POMA(5)


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core/shell hybrids (Figure 2d), and the diameter increases further in spherical GNRs/POMA(10) core/shell hybrids (Figure 2e). The coating thickness of POMA polymer were summarized in Figure 2f. FTIR spectrum was applied to establish the existence of POMA in GNRs/POMA core/shell hybrids. As shown in Figure S1, the absorption peak at 3400 cm-1 was ascribed to the N–H stretching vibration, the characteristic bands at 1560 and 1460 cm-1 were attributed to the C=C stretching vibration of the quinonoid and benzenoid rings, respectively, the band at 1275 cm-1 was assigned to the C–N stretching vibration, the band at 1100 cm-1 was due to the C–O stretching vibration. Results confirmed the successful formation of POMA polymer.28 The UV-vis absorption spectra of the GNRs and GNRs/POMA core/shell hybrids were shown in Figure 3a. The spectrum of GNRs showed the typical longitudinal plasmon resonance at 715 nm.48 However, the absorption spectra showed obvious red shift after POMA coating on surfaces of GNRs. For instance, the absorption peaks for GNRs/POMA(1), GNRs/POMA(2), GNRs/POMA(5), and GNRs/POMA(10) core/shell hybrids were at 740 nm, 786 nm, 809 nm, and 788 nm, respectively. Based on the Mie theory, it is reasonable that the extent of electronic vibration is shortened when the electronic vibration in the GNRs is confined to a smaller space after polymer coating.49 Besides, additional electronic oscillation can be provided when POMA polymer is exposed to an external irradiation owing to the conductive nature of POMA polymer. Both the shortened extent of electronic vibration and the enhanced restore force promote the red shift of absorption bands for GNRs/POMA core/shell hybrids. However, thicker polymer coating may be unfavorable for shortening the extent of electronic vibration, and thus leading to adverse effect.28, 49-50 As 808 nm NIR light is popular by the current biomedical diagnosis and therapy due to its


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weak autofluorescence and strong penetration in live tissues,51-54 the absorbance of GNRs and GNRs/POMA core/shell hybrids at 808 nm was exhibited in Figure 3b, where GNRs/POMA(5) core/shell hybrids showed the highest value. 3.2. Photothermal Effect and Photostability An important feature of GNRs is their NIR light-induced thermal effect because of the tunable surface plasmon bands in the NIR region, which can be used for NIR absorption photothermal therapy.27, 30, 33 GNRs and GNRs/POMA core/shell hybrids were exposed to the 808 nm-laser (3 W cm-2) to evaluate the photothermal conversion effect. The temperature of the colloidal aqueous solution containing GNRs, GNRs/POMA(1), GNRs/POMA(2), GNRs/POMA(5), and GNRs/POMA(10) core/shell hybrids were increased by 13.5, 14.9, 17.7, 20.3 and 18.8 °C after 10 min irradiation, respectively (Figure 4a). However, there was no significant temperature increase observed for water. Results showed that GNRs/POMA(5) core/shell hybrids showed the highest photothermal performance. Apparently, the extent of temperature increment was in accordance with the absorbance at the wavelength of 808 nm (Figure 3b). We then examined the phototheraml conversion efficiency (η) of these materials based on the time-dependent temperature increment and energy exchange equilibrium.55 According to Roper’s report,56 the η and the absorption efficiency (ηAbs) at 808 nm of GNRs and GNRs/POMA were calculated, which were shown in Table S1. The GNRs (η = 22.3%) and GNRs/POMA(5) (η = 18.7%) showed higher η than some reported photothermal agents, such as gold nanoshells (η = 13%),21 gold vesicle (η = 18%),21 and Cu2-xS nanocrystals (η = 16.3%).57 As for the value of ηAbs, it was increased after the polymer coating, and reached as high as >99% for GNRs/POMA(2), GNRs/POMA(5) and GNRs/POMA(10). As the photothermal effect is dependent on both η and ηAbs, therefore, GNRs/POMA(5) core/shell hybrids showed the best photothermal effect.


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The photostability of GNRs and GNRs/POMA(5) core/shell hybrids were then compared. GNRs and GNRs/POMA(5) core/shell hybrids were exposed to the 808 nm-laser (3 W cm-2) for 10 min. Then the NIR laser was turned off till the solution was cooled to room temperature. The temperature elevation of GNRs changed from 13.5 to 9.4 oC after six cycles, whereas there was no significant decrease in temperature for GNRs/POMA(5) core/shell hybrids (Figure 4b). TEM images of the GNRs/POMA after irradiation were shown in Figure S2, from which we could find that there was no significant change in morphology of GNRs/POMA core/shell hybrids before and after irradiation. Results indicated that the POMA polymer coating on surfaces of GNRs not only increased the NIR absorbance (Figure 3), but also led to enhanced photostability. 3.3 Cytotoxicity Assay and In Vitro PTT MTT assay was employed to justify the viability of CT26 cells. CT26 cells were pre-incubated with GNRs and GNRs/POMA core/shell hybrids with concentration ranges from 0 to 25 µg mL-1 for 24 h. The results showed a negligible influence on cell viability when the concentration of GNRs and GNRs/POMA core/shell hybrids was below 20 µg mL-1 (Figure 5a). It was noted that GNRs/POMA(10) core/shell hybrids exhibited obvious toxicity to the cells as compared to other samples. As the concentration of photothermal agents used was based on the concentration of GNRs, the GNRs/POMA(10) core/shell hybrids had the highest POMA polymer concentration among the tested GNRs/POMA core/shell hybrids, which caused the cytotoxicity.58 The photothermal cytotoxicity of GNRs and GNRs/POMA core/shell hybrids was then detected. CT26 cells were pre-incubated with GNRs and GNRs/POMA core/shell hybrids at the concentration of 20 µg mL-1. As shown in Figure 5b, more cells were killed after 10 min irradiation and cancer cells were almost destroyed by incubation with GNRs/POMA(5) and GNRs/POMA(10) core/shell hybrids. However, due to the higher cytotoxicity to the cells under


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the same conditions, the GNRs/POMA(5) core/shell hybrids were regarded as the best choice among the tested photothermal agents. Correspondingly, the PTT efficacy in vitro was also assessed by live/dead staining, where live cells were stained by calcein AM (green fluorescence) and dead cells were differentiated by co-staining with propidium iodide (PI) (red fluorescence). As shown in Figure S3, in PBS group and laser alone treated groups, the cells exhibited green fluorescence, suggesting negligible damage effect. Meanwhile, compared with the group of GNRs plus NIR, almost all of the cells incubated with GNRs/POMA(5) were killed and stained in red fluorescence after laser irradiation. Thus, the POMA polymer coating on surfaces of GNRs coating could improve the photothermal efficiency of the materials. 3.4 In Vivo PPT In vivo experiments were then conducted by assessing the tumor growth inhibition. In the treatment groups using GNRs, GNRs/POMA(5) and GNRs/POMA(10) core/shell hybrids with 808 nm-laser irradiation, each mouse was intravenously injected via tail vein followed by irradiation for 10 min. The temperature increment of the tumor site was recorded by an IR thermal camera every two minutes. The other four control groups included the injection with PBS, GNRs, GNRs/POMA(5) and GNRs/POMA(10) core/shell hybrids without laser irradiation. As shown in Figure 6, the tumor temperature of the treatment groups increased rapidly under irradiation, and the tumor temperature showed the highest within 10 min in the case of GNRs/POMA(5) core/shell hybrids. In contrast, the tumor temperature almost unchanged with PBS. The tumor volumes were monitored and recorded every three days during the subsequent 15 days. As shown in Figure S4, tumors intravenously injected with GNRs, GNRs/POMA(5) and GNRs/POMA(10) plus irradiation were greatly inhibited and only small black scars were left in the original tumor sites at 15 days post-irradiation. Figure 7a showed that all the tumors in the


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treatment groups were effectively decreased, while the control groups showed no tumor regression but continuous tumor growth. The tumor photographs (Figure 7b) and tumor weights (Figure 7c) further verified the above results. Furthermore, it was found that the tumor volume and weight in group of GNRs/POMA(5) with irradiation showed the lowest values, proving the best photothermal effect during the treatment. The body weights of the mice were also measured. As shown in Figure 7d, there were no obvious weight loss, illustrating that the toxic side effects caused by GNRs and GNRs/POMA core/shell hybrids were unnoticeable because high toxicity usually leads to a drop in body weight. These preliminary investigations verified the biosafety of the GNRs and GNRs/POMA in vivo. Furthermore, Figure 8 showed that the mice in the PTT treatment groups were cured and survived for more than 40 days, while the mice in control groups died in less than 20 days. Taken all the results together, GNRs/POMA(5) core/shell hybrids had efficient photothermal performance, demonstrating an excellent tumor elimination effect upon NIR irradiation.

4. CONCLUSION In summary, GNRs/POMA core/shell hybrids with controlled POMA polymer coating through surfactant-assisted chemical oxidative polymerization route have been developed. In comparison with GNRs and other GNRs/POMA core/shell hybrids, GNRs/POMA(5) core/shell hybrids exhibited the best photothermal performance due to their excellent NIR absorption and photostability. By intratumoral injection with NIR irradiation, GNRs/POMA(5) core/shell hybrids successfully inhibited the tumor growth, demonstrating their superior anticancer efficacy. We believe the results will be instructive for the synthesis of superior NIR photothermal agents.


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ASSOCIATED CONTENT Supporting Information. Additional figures about material. This information is available free of charge via the Internet at http://pubs.acs.org/. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]; [email protected] ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (21673202 and 21703198), Qing Lan Project and the Priority Academic Program Development of Jiangsu Higher Education Institutions. We would also like to acknowledge the technical support received at the Testing Center of Yangzhou University.

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Isoindigo Derivative and Bithiophene Units for Near-Infrared Modulated Cancer ThermoChemotherapy. ACS Appl. Mater. Interfaces 2016, 8, 19312-19320. (4) Liu, T.; Wang, C.; Gu, X.; Gong, H.; Cheng, L.; Shi, X.; Feng, L.; Sun, B.; Liu, Z. Drug Delivery with PEGylated MoS2 Nano-sheets for Combined Photothermal and Chemotherapy of Cancer. Adv. Mater. 2014, 26, 3433-3440. (5) Yang, K.; Zhang, S.; Zhang, G.; Sun, X.; Lee, S. T.; Liu, Z. Graphene in Mice: Ultrahigh In Vivo Tumor Uptake and Efficient Photothermal Therapy. Nano Lett. 2010, 10, 3318-3323. (6) MacNeill, C. M.; Graham, E. G.; Levi-Polyachenko, N. H. Soft Template Synthesis of Donor-Acceptor Conjugated Polymer Nanoparticles: Structural Effects, Stability, and Photothermal Studies. J. Polym. Sci. Part A: Polym. Chem. 2014, 52, 1622-1632. (7) Liu, Y. L.; Ai, K. L.; Liu, J. H.; Deng, M.; He, Y. Y.; Lu, L. H. Dopamine-Melanin Colloidal Nanospheres: An Efficient Near-Infrared Photothermal Therapeutic Agent for In Vivo Cancer Therapy. Adv. Mater. 2013, 25, 1353-1359. (8) Zheng, M.; Yue, C.; Ma, Y.; Gong, P.; Zhao, P.; Zheng, C.; Sheng, Z.; Zhang, P.; Wang, Z.; Cai, L. Single-Step Assembly of DOX/ICG Loaded Lipid--Polymer Nanoparticles for Highly Effective Chemo-Photothermal Combination Therapy. ACS Nano 2013, 7, 20562067. (9) Jaque, D.; Maestro, L. M.; del Rosal, B.; Haro-Gonzalez, P.; Benayas, A.; Plaza, J. L.; Rodriguez, E. M.; Sole, J. G. Nanoparticles for Photothermal Therapies. Nanoscale 2014, 6, 9494-9530. (10) Cui, W.; Li, J.; Decher, G. Self-Assembled Smart Nanocarriers for Targeted Drug Delivery. Adv. Mater. 2016, 28, 1302-1311.


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(11) Qin, C.; Fei, J.; Wang, A.; Yang, Y.; Li, J. Rational Assembly of a Biointerfaced Core@Shell

Nanocomplex

Towards

Selective

and

Highly Efficient

Synergistic

Photothermal/Photodynamic Therapy. Nanoscale 2015, 7, 20197-20210. (12) Qin, C.; Fei, J.; Cai, P.; Zhao, J.; Li, J. Biomimetic Membrane-Conjugated Graphene Nanoarchitecture for Light-Manipulating Combined Cancer Treatment In Vitro. J. Colloid Interface Sci. 2016, 482, 121-130. (13) Cui, W.; Wang, A.; Zhao, J.; Li, J. Biomacromolecules Based Core/Shell Architecture Toward Biomedical Applications. Adv. Colloid Interface Sci. 2016, 237, 43-51. (14) Song, G.; Wang, Q.; Wang, Y.; Lv, G.; Li, C.; Zou, R.; Chen, Z.; Qin, Z.; Huo, K.; Hu, R.; Hu, J. A Low-Toxic Multifunctional Nanoplatform Based on Cu9S5@mSiO2 Core-Shell Nanocomposites: Combining Photothermal- and Chemotherapies with Infrared Thermal Imaging for Cancer Treatment. Adv. Funct. Mater. 2013, 23, 4281-4292. (15) Yang, Y.; Liu, J. J.; Liang, C.; Feng, L. Z.; Fu, T. T.; Dong, Z. L.; Chao, Y.; Li, Y. G.; Lu, G.; Chen, M. W.; Liu, Z. Nanoscale Metal-Organic Particles with Rapid Clearance for Magnetic Resonance Imaging-Guided Photothermal Therapy. ACS Nano 2016, 10, 27742781. (16) Guo, M.; Huang, J.; Deng, Y.; Shen, H.; Ma, Y.; Zhang, M.; Zhu, A.; Li, Y.; Hui, H.; Wang, Y.; Yang, X.; Zhang, Z.; Chen, H. pH-Responsive Cyanine-Grafted Graphene Oxide for Fluorescence Resonance Energy Transfer-Enhanced Photothermal Therapy. Adv. Funct. Mater. 2015, 25, 59-67. (17) Chen, L.; Zhong, X.; Yi, X.; Huang, M.; Ning, P.; Liu, T.; Ge, C.; Chai, Z.; Liu, Z.; Yang, K. Radionuclide

131

I Labeled Reduced Graphene Oxide for Nuclear Imaging Guided

Combined Radio- and Photothermal Therapy of Cancer. Biomaterials 2015, 66, 21-28.


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(18) Liang, C.; Diao, S.; Wang, C.; Gong, H.; Liu, T.; Hong, G.; Shi, X.; Dai, H.; Liu, Z. Tumor Metastasis Inhibition by Imaging-Guided Photothermal Therapy with Single-Walled Carbon Nanotubes. Adv. Mater. 2014, 26, 5646-5652. (19) Shi, J.; Wang, L.; Zhang, J.; Ma, R.; Gao, J.; Liu, Y.; Zhang, C.; Zhang, Z. A TumorTargeting Near-Infrared Laser-Triggered Drug Delivery System Based on GO@Ag Nanoparticles for Chemo-Photothermal Therapy and X-ray Imaging. Biomaterials 2014, 35, 5847-5861. (20) Cheng, L.; Yang, K.; Chen, Q.; Liu, Z. Organic Stealth Nanoparticles for Highly Effective In Vivo Near-Infrared Photothermal Therapy of Cancer. ACS Nano 2012, 6, 5605-5613. (21) Huang, P.; Lin, J.; Li, W.; Rong, P.; Wang, Z.; Wang, S.; Wang, X.; Sun, X.; Aronova, M.; Niu, G.; Leapman, R. D.; Nie, Z.; Chen, X. Biodegradable Gold Nanovesicles with an Ultrastrong Plasmonic Coupling Effect for Photoacoustic Imaging and Photothermal Therapy. Angew. Chem. Int. Ed. 2013, 52, 13958-13964. (22) Chen, M.; Tang, S.; Guo, Z.; Wang, X.; Mo, S.; Huang, X.; Liu, G.; Zheng, N. Core-Shell Pd@Au Nanoplates as Theranostic Agents for In-Vivo Photoacoustic Imaging, CT Imaging, and Photothermal Therapy. Adv. Mater. 2014, 26, 8210-8216. (23) Zhao, X. Q.; Wang, T. X.; Liu, W.; Wang, C. D.; Wang, D.; Shang, T.; Shen, L. H.; Ren, L. Multifunctional Au@IPN-pNIPAAm Nanogels for Cancer Cell Imaging and Combined Chemo-Photothermal Treatment. J. Mater. Chem. 2011, 21, 7240-7247. (24) Hu, Y.; Wang, R.; Wang, S.; Ding, L.; Li, J.; Luo, Y.; Wang, X.; Shen, M.; Shi, X. Multifunctional Fe3O4@Au Core/Shell Nanostars: A Unique Platform for Multimode Imaging and Photothermal Therapy of Tumors. Sci. Rep. 2016, 6, 28325-28336.


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(32) Lee, J.; Jeong, C.; Kim, W. J. Facile Fabrication and Application of Near-IR LightResponsive Drug Release System Based on Gold Nanorods and Phase Change Material. J. Mater. Chem. B 2014, 2, 8338-8345. (33) Pérez-Juste, J.; Pastoriza-Santos, I.; Liz-Marzán, L. M.; Mulvaney, P. Gold Nanorods: Synthesis, Characterization and Applications. Coordin. Chem. Rev. 2005, 249, 1870-1901. (34) Wang, T. T.; Chai, F.; Wang, C. G.; Li, L.; Liu, H. Y.; Zhang, L. Y.; Su, Z. M.; Liao, Y. Fluorescent Hollow/Rattle-Type Mesoporous Au@SiO2 Nanocapsules for Drug Delivery and Fluorescence Imaging of Cancer Cells. J. Colloid Interface Sci. 2011, 358, 109-115. (35) Liu, Y.; Yang, M.; Zhang, J.; Zhi, X.; Li, C.; Zhang, C.; Pan, F.; Wang, K.; Yang, Y.; Martinez de la Fuentea, J.; Cui, D. Human Induced Pluripotent Stem Cells for Tumor Targeted Delivery of Gold Nanorods and Enhanced Photothermal Therapy. ACS Nano 2016, 10 , 2375-2385. (36) Li, J.; Han, J.; Xu, T.; Guo, C.; Bu, X.; Zhang, H.; Wang, L.; Sun, H.; Yang, B. Coating Urchinlike Gold Nanoparticles with Polypyrrole Thin Shells to Produce Photothermal Agents with High Stability and Photothermal Transduction Efficiency. Langmuir 2013, 29, 7102-7110. (37) Zhang, J.; Han, J.; Wang, M. G.; Guo, R. Fe3O4/PANI/MnO2 Core-Shell Hybrids as Advanced Adsorbents for Heavy Metal Ions. J. Mater. Chem. A 2017, 5, 4058-4066. (38) Jin, C. J.; Han, J.; Chu, F. Y.; Wang, X. X.; Guo, R. Fe3O4@PANI Hybrid Shell as a Multifunctional Support for Au Nanocatalysts with a Remarkably Improved Catalytic Performance. Langmuir 2017, 33, 4520-4527.


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(39) Cao, S. Y.; Han, N.; Han, J.; Hu, Y. M.; Fan, L.; Zhou, C. Q.; Guo, R. Mesoporous Hybrid Shells of Carbonized Polyaniline/Mn2O3 as Non-Precious Efficient Oxygen Reduction Reaction Catalyst. ACS Appl. Mater. Interfaces 2016, 8, 6040-6050. (40) Xu, Q.; Zhu, J. J.; Hu, X. Y. Ordered Mesoporous Polyaniline Film as a New Matrix for Enzyme Immobilization and Biosensor Construction. Anal. Chim. Acta. 2007, 597, 151156. (41) Liu, X. T.; Na, W. D.; Liu, H.; Sue, X. G. Fluorescence Turn-off-on Probe Based on Polypyrrole/Graphene Quantum Composites for Selective and Sensitive Detection of Paracetamol and Ascorbic Acid. Biosens. Bioelectron. 2017, 98, 222-226. (42) Lee, T.; Bang, D.; Park, Y.; Kim, S. H.; Choi, J.; Park, J.; Kim, D.; Kim, E.; Suh, J. S.; Huh, Y. M.; Haam, S. Gadolinium-Enriched Polyaniline Particles (GPAPs) for Simultaneous Diagnostic Imaging and Localized Photothermal Therapy of Epithelial Cancer. Adv. Healthcare Mater. 2014, 3, 1408-1414. (43) Bae, S. R.; Choi, J.; Kim, H.-O.; Kang, B.; Kim, M.-H.; Han, S.; Noh, I.; Lim, J.-W.; Suh, J.-S.; Huh, Y.-M.; Haam, S. Pseudo Metal Generation via Catalytic Oxidative Polymerization on the Surface of Reactive Template for Redox Switched off–on Photothermal Therapy. J. Mater. Chem. B 2015, 3, 505-513. (44) Jiang, B.-P.; Zhang, L.; Zhu, Y.; Shen, X.-C.; Ji, S.-C.; Tan, X.-Y.; Cheng, L.; Liang, H. Water-Soluble Hyaluronic Acid–Hybridized Polyaniline Nanoparticles for Effectively Targeted Photothermal Therapy. J. Mater. Chem. B 2015, 3, 3767-3776. (45) Yang, K.; Xu, H.; Cheng, L.; Sun, C. Y.; Wang, J.; Liu, Z. In Vitro and In Vivo NearInfrared Photothermal Therapy of Cancer Using Polypyrrole Organic Nanoparticles. Adv. Mater. 2012, 24, 5586-5592.


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FIGURE CAPTIONS Figure 1 Schematic illustration to show the fabrication process of GNRs/POMA with different morphologies. Figure 2 TEM images of (a) GNRs, (b) GNRs/POMA(1), (c) GNRs/POMA(2), (d) GNRs/POMA(5), and (e) GNRs/POMA(10) core/shell hybrids. (f) The longitudinal and horizontal shell thickness of GNRs and GNRs/POMA core/shell hybrids. The transverse thickness means the highest thickness of polymer coating form the side surface; the longitudinal thickness is calculated by the highest thickness of polymer coating form the tip surface. Figure 3 (a) UV−vis absorption spectra of GNRs and GNRs/POMA. (b) Diagram of the absorbance at 808 nm of GNRs and GNRs/POMA. Figure 4 (a) Temperature change curves GNRs and GNRs/POMA upon exposure to the NIR laser (808 nm, 3 W/cm2, 10 min). (b) Temperature elevation of GNRs and GNRs/POMA (5) over six laser on/off cycles of NIR irradiation. Figure 5 In vitro experiments. (a) Relative viabilities of CT26 cells incubated with GNRs and GNRs/POMA at various concentrations for 24 h. (1-5 represent GNRs, GNRs/POMA (1), GNRs/POMA (2), GNRs/POMA (5) and GNRs/POMA (10)), (b) Relative viabilities of CT26 cells incubated with the same concentration (20 µg mL-1) of GNRs and GNRs/POMA with and without the 808 nm laser irradiation (3 W/cm2).


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Figure 6 In vivo photothermal therapy of the CT26 tumor bearing mice after the injection of GNRs and GNRs/POMA through the tail vein: IR thermal images of the tumors irradiated under the 808 nm laser at the intensity of 3 W/cm2 for 10 min. Figure 7 In vivo photothermal therapy of the CT26 tumor bearing mice after the injection of GNRs and GNRs/POMA through the tail vein: (a) Relative tumor-growth curves after various treatments indicated in 15 days. (b) The final tumor weight after sacrifice of mice. (c) Representative photographs of tumor tissue on 15 d after sacrifice of mice. (d) The body weight after various treatments indicated in 15 days. Figure 8 Survival curves of various groups of the CT26 tumor bearing mice after various treatments.


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Figure 1


25

(f)

40

20

20

0

0

-20

-20

-40

-40

Transverse thickness (nm)

40

0) Rs (5) (2) (1) GN MA OMA(1 MA MA PO PO PO s/P Rs/ Rs/ Rs/ R N N N G G G GN

Figure 2

5 GNRs GNRs/POMA(1) GNRs/POMA(2) GNRs/POMA(5) GNRs/POMA(10)

4

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 33

Longitudinal thickness (nm)


715 nm 740 nm 786 nm 809 nm 788 nm

(a)

3 2 1 0 500

600

700

800

900

Wavelength (nm)


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Page 27 of 33

3.0

(b)

Absorbance

2.5

2.5

2.3

2.4

2.0 1.5

1.0 1.0

0.6 0.5 0.0

s 0) 2) 5) 1) GNR OMA( OMA ( OMA ( A (1 M O Rs/P NRs/P Rs/P NRs/P GN G GN G

Figure 3

25

(a)

GNRs GNRs/POMA(1) GNRs/POMA(2) GNRs/POMA(5) GNRs/POMA(10)

20

∆ T ( oC )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


water

15 10 5 0 0

2

4

6

8

10

Irradiation time (min)


27


25

GNRs GNRs/POMA(5)

(b) 20

∆ T ( oC )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 33

15 10 5 0 0

30

60

90

120

150

180

Time (min) Figure 4


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Page 29 of 33

150

GNRs GNRs/POMA(1) GNRs/POMA (2) GNRs/POMA (5) GNRs/POMA (10)

(a)

Cell Viability (%)

120 90 60 30 0 0

5

10

Concentration

15 20 ( µg/mL)

25

120

(b)

No laser 2

100

Cell Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3 W/cm

80 60 40 20 0 s 0) (2) (5) (1) GNR A (1 MA MA MA M O O O P O P P / s s/P Rs/ Rs/ GNR GN GN GNR

Figure 5


29


Page 30 of 33

Figure 6


30

8

(a)

GNRs GNRs+Laser GNRs/POMA(5) GNRs/POMA(5)+Laser GNRs/POMA(10) GNRs/POMA(10)+Laser PBS

4

Tumor weight (g)

6

(b) 1.5

2

1.0

0.5

zero

0 0.0

0

4

8

12

PBS

16

Time (day)

Rs (5) ser A(10) Laser ser GN s+La OMA 5)+La 0)+ R OM ( P GN NRs/ OMA NRs/P MA(1 G P G s/PO Rs/ R N G GN

30

Body weight (g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Relative Tumor Volume (V/V0)

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(d)

GNRs GNRs+Laser GNRs/POMA(5) GNRs/POMA(5)+Laser GNRs/POMA(10) GNRs/POMA(10)+Laser PBS

27

24

21

18 0

3

6

9

12

15

Time (day)

Figure 7


31


100

Survival rate (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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PBS GNRs GNRs+Laser GNRs/POMA(5) GNRs/POMA(5)+Laser

80 60 40 20 0 0

10

20

30

40

Time (day) Figure 8


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TOC Graphical


33

Conducting Polymer Core

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