Improved Anticancer Photothermal Therapy ... - Wiley Online Library

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Improved Anticancer Photothermal Therapy Using the Bystander Effect Enhanced by Antiarrhythmic Peptide Conjugated Dopamine-Modified Reduced Graphene Oxide Nanocomposite Jiantao Yu, Yu-Hsin Lin, Lingyan Yang, Chih-Ching Huang, Liliang Chen, Wen-Cheng Wang, Guan-Wen Chen, Junyan Yan, Saranta Sawettanun, and Chia-Hua Lin*

Despite tremendous efforts toward developing novel near-infrared (NIR)absorbing nanomaterials, improvement in therapeutic efficiency remains a formidable challenge in photothermal cancer therapy. This study aims to synthesize a specific peptide conjugated polydopamine-modified reduced graphene oxide (pDA/rGO) nanocomposite that promotes the bystander effect to facilitate cancer treatment using NIR-activated photothermal therapy. To prepare a nanoplatform capable of promoting the bystander effect in cancer cells, we immobilized antiarrhythmic peptide 10 (AAP10) on the surface of dopaminemodified rGO (AAP10-pDA/rGO). Our AAP10-pDA/rGO could promote the bystander effect by increasing the expression of connexin 43 protein in MCF-7 breast-cancer cells. Because of its tremendous ability to absorb NIR absorption, AAP10-pDA/rGO offers a high photothermal effect under NIR irradiation. This leads to a massive death of MCF-7 cells via the bystander effect. Using tumorbearing mice as the model, it is found that NIR radiation effectively ablates breast tumor in the presence of AAP10-pDA/rGO and inhibits tumor growth by ≈100%. Therefore, this research integrates the bystander and photothermal effects into a single nanoplatform in order to facilitate an efficient photothermal therapy. Furthermore, our AAP10-pDA/rGO, which exhibits both hyperthermia and the bystander effect, can prevent breast-cancer recurrence and, therefore, has great potential for future clinical and research applications.

J. Yu, L. Yang, J. Yan Key Laboratory of Nano-Bio Interface Division of Nanobiomedicine Suzhou Institute of Nano-Tech and Nano-Bionics Chinese Academy of Sciences Suzhou 215123, China Dr. Y.-H. Lin Department of Food and Beverage Management Taipei College of Maritime Technology Taipei 11174, Taiwan Dr. C.-C. Huang Institute of Bioscience and Biotechnology National Taiwan Ocean University Keelung 20224, Taiwan Dr. L. Chen The National Key Laboratory of Shock Wave and Detonation Physics Institute of Fluid Physics CAEP, Mianyang 621900, China

1. Introduction The use of nanotechnology in cancer treatment has received extensive attention in recent years. Nanotechnologybased cancer photothermal therapy (PTT) involves using nanomaterials that can generate heat by absorbing near-infrared (NIR) radiation and, consequently, improve the therapeutic efficiency of the conventional thermal therapy.[1–4] NIR radiation has already been used in PTT for cancer treatment because biological tissues are transparent in the NIR region (780–2500 nm).[5] Therefore, PTT has great potential as a noninvasive cancer therapy in which the absorbed radiation is converted to thermal energy; this leads to irreversible tumor destruction at temperatures of 42–46 °C.[2,6] Engineered nano materials that exhibit NIR absorption, such as graphene oxide (GO), gold nanomaterials, and iron-oxide nanoparticles, have already been developed for use in anticancer PTT.[2,7–10]

Dr. W.-C. Wang Research Center for Environmental Changes Academia Sinica Taipei 11529, Taiwan Dr. G.-W. Chen Department of Food Science National Taiwan Ocean University Keelung 20224, Taiwan S. Sawettanun, Dr. C.-H. Lin Department of Biotechnology National Formosa University Yunlin 63208, Taiwan E-mail: vicchlin@nfu.edu.tw

DOI: 10.1002/adhm.201600804

Adv. Healthcare Mater. 2016, DOI: 10.1002/adhm.201600804

© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Recently, GO has attracted significant interest in the field of biomedicine and bioengineering.[2,11,12] In addition to its superior biocompatibility, thermal conductivity, and photothermalconversion efficiency, GO offers an excellent drug-loading capacity and facile surface modification.[13,14] Because of these properties, chemically modified GO can be used as a multifunctional theranostic agent in photothermal cancer treatment.[15–17] However, the NIR absorption of GO is significantly less than that of other photothermal-conversion nanomaterials. Thus, in order to use GO-based nanomaterials in PTT, reduced GO (rGO) was prepared using the reducing reagents obtained from GO.[18–21] Although most of the reagents used for the reduction of GO are toxic or hazardous substances, dopamine is an exception.[22–24] Polydopamine (pDA) was prepared via the oxidative polymerization of dopamine in the GOreduction process;[22] pDA coated the GO surface. Simultaneously, GO was reduced by dopamine to obtain pDA/rGO composites. Surface modification in pDA can improve the stability and dispersity of rGOs.[25] Furthermore, both pDA and rGOs exhibit stronger NIR absorption than GO.[26–29] We, therefore, expect that pDA/rGO composites are potential nanomaterials for PTT. The bystander effect, in which individual damaged cells transfer toxic signals to adjacent cells, has been demonstrated in a previous study.[30] The bystander effect is mainly mediated by gap-junction intercellular communication (GJIC).[31] Unfortunately, a deficiency in GJIC, which can limit the effectiveness of the bystander effect, appears in most cancer cells.[32] Nevertheless, the bystander effect may enhance the diffusion of cytotoxic signals and increase the sensitivity of the cells to chemotherapy.[33–35] In addition to a reduced bystander effect, another shortcoming of PTT is the fact that actual tumors are three-dimensional structures; this allows tumors to shade some tumor cells from NIR irradiation, thereby preventing PTT from completely exterminating all tumor cells. Such a scenario is highly undesirable because residual tumor cells significantly increase the post-therapy cancer-recurrence rate. To overcome this challenge and improve the efficiency of PTT, such nonirradiated cells must be destroyed indirectly. A promising way to achieve this is to enhance the bystander effect within human tumor cells during PTT to increase the rate at which cancer cells are destroyed. Connexin-mediated GJIC has been demonstrated previously as a major mechanism in the transfer of toxic signal or toxicant to adjacent cells.[36] Many different types of connexins (Cxs) exist, such as Cx43, Cx32, and Cx26 (they are named on the basis of their molecular weight). Cx43 is the most widely used and well-studied member of this family. In cancer, loss of GJIC and Cx has been shown to facilitate tumorigenesis, and the upregulation of Cx43 by antitumor agents is related to their ability to suppress carcinogen-induced neoplastic transformation.[37] Collectively, these results imply that inducing Cx43 expression may be a promising technique to increase the efficiency of anticancer PTT. Antiarrhythmic peptide 10 (AAP10) peptide (H2N-Gly-AlaGly-4Hyp-Pro-Tyr-CONH2) was used in a previous study to elevate the protein activity of Cx43 in cancer cells.[38] In this study, we attempt to develop AAP10-functionalized pDA/rGO (AAP10pDA/rGO) to enhance the bystander effect, which in turn allows 2


more efficient PTT with less NIR irradiation. Attaching AAP10 to pDA/rGO results in greater Cx43 expression and GJIC than those obtained with AAP10 alone. The modification of APP10 not only enhances the cancer-treatment efficiency of pDA/rGO but also reduces the risk of cancer recurrence. On the basis of these results, we conclude that APP10-pDA/rGO displays excellent anticancer activity by combining photothermal and Cx43mediated bystander effects, thereby displaying a significant potential for application as a photothermal agent for combatting cancer.

2. Results and Discussion 2.1. Preparation of pDA/rGO and AAP10-pDA/rGO The synthesis routes for PDA/rGO and AAP10-PDA/rGO are shown in Scheme 1. GO was first synthesized according to the method employed in Yang et al.[2] The atomic force micro scopy (AFM) image in Figure 1a shows that the monolayered GO is ≈300 nm long and 1.1 nm thick. Additionally, pDA/ rGO was synthesized according to the method of Xu,[22] and mussel-inspired pDA was coated onto the GO surface to form a uniform 3.5 nm thick film (Figure 1a). Initiated by the reduction property of dopamine,[39] electronic conjugation of GO was restored, as indicated by the redshift of the 228 nm absorbance peak in the UV spectra (π–π* transition in CC bond) to 278 nm and the disappearance of the 300 nm absorbance peak (n→π* electronic transition of peroxide and/or epoxide functional groups) (Figure 1b). The absorbance also increased over the entire spectrum, especially in the NIR region (Figure 1b). In addition, the photothermal effect of pDA/rGO significantly increased. To confirm the amount by which dopamine reduces GO, we applied Raman and Fourier-transform IR (FTIR) spectroscopy. In the Raman spectra of GO, the in-phase vibration of the graphene lattice (G band, sp2) appeared at 1585 cm−1 whereas the disorder band associated with graphene edges (D band, sp3) appeared at ≈1343 cm−1 (Figure 1c).[40] The D-to-G intensity ratio, ID/IG, was calculated to be 0.99. After dopamine reduction, the D-to-G ratio decreased to 0.88 (Figure 1c). The significant decrease in the relative D-band intensity means that the disorder on the GO surface essentially disappears. Furthermore, the G band of the in-phase vibration of the graphene lattice redshifted to 1599 cm−1, which was possibly due to the pDA attached to the rGO surface (Figure 1c). In the FTIR spectra of GO, multiple peaks appeared in the range of 900–1500 cm−1; these peaks could be assigned to CO, COC, COH, and CO in carboxylic acid and carbonyl moieties (Figure 1d).[41] After dopamine reduction, the peak intensity between 900 and 1500 cm−1 in pDA/rGO and AAP10-pDA/rGO decreased significantly (Figure 1d). These results confirm that most oxygen functionality was removed from the GO surface in pDA/rGO and AAP10-pDA/rGO (Figure 1c,d). When the peptide AAP10 was functionalized onto the pDA surface using the Michael addition reaction/Schiff base reaction,[42] the D-to-G intensity ratio increased to 0.96 and the wavenumber of the characteristic absorbance peak of pDA-rGO remained constant (Figure 1c). According to the AFM result,



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FULL PAPER Scheme 1. Schematic of the route for preparing AAP10-pDA/rGO (top row). Destruction of cancer cells with the aid of the bystander effect upon exposure to NIR irradiation during PTT (bottom row).

when peptide AAP10 was attached to the pDA/rGO surface, the thickness of the AAP10-pDA/rGO layer is not significantly larger than pDA/rGO (Figure 1a). We changed the synthesis procedure to adjust the AAP10-to-pDA/rGO mass ratio to 416 × 10−9 m AAP10 per milligram pDA/rGO (50 × 10−9 m AAP10/120 µg pDA/rGO) (see Supporting Information S3).

2.2. Photothermal Properties and Biocompatibility of AAP10-pDA/rGO To examine the photothermal capabilities of AAP10-pDA/ rGO nanocomposites, we used a thermocouple and a thermalimaging camera to measure the temperature variation in the

Figure 1. Physicochemical properties. a) Tapping-mode AFM image of GO, pDA/rGO, and AAP10-pDA/rGO. b) UV–vis absorption spectra of GO and pDA/rGO. Absorbance (Abs) is plotted in arbitrary units (a.u.). c) Raman spectra of GO, pDA/rGO, and AAP10-pDA/rGO. d) FTIR spectra of GO, pDA/rGO, and AAP10-pDA/rGO.




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Figure 2. Photothermal effects and biocompatibility of AAP10-pDA/rGO. a) Photothermal heating of GO (120 µg mL−1), pDA/rGO (120 µg mL−1), and AAP10-pDA/rGO (50 × 10−9 m AAP10/120 µg pDA/rGO/mL) under NIR illumination (880 nm, 1.5 W cm−2). b) Photothermal conversion efficiency of AAP10-pDA/rGO under NIR illumination. Cytotoxicity of AAP10-pDA/rGO (40–160 µg mL−1) in c) MCF-10A cells and d) MCF-7 cells (*p < 0.05, **p < 0.01).

DMEM cell medium solution (100 µL) containing GO, pDA/ rGO, or AAP10-pDA/rGO (120 µg mL−1) after exposure to irradiation by the NIR 808 nm laser at 1.5 W cm−2. NIR irradiation caused a significant temperature increase in the GO-, pDA/ rGO-, and AAP10-pDA/rGO-containing solutions, which all contained the same amount of GO (Figure 2a and Figure S1, Supporting Information). The temperature increase in these nanocomposite-containing solutions was the result of NIR absorption (Figure 1b). As shown in Figure 2a, the temperature of the pDA/rGO- and AAP10-pDA/rGO-containing solutions increased by ≈4 °C min−1, which was greater than the temperature increase for GO (≈1 °C min−1) and the control solution (≈0.1 °C min−1). Reduction of GO using dopamine thus increases NIR absorption of the GO-derived nanocomposites; this, in turn, increases their potential for anticancer PTT (Figures 1b and 2a).[42] The photothermal conversion efficiency (η) of AAP10-pDA/ rGO was calculated using the equation given in Supporting Information S4. The aqueous dispersion of AAP10-pDA/rGO exhibited the highest temperature (Tmax) of 55 °C when the surrounding temperature (Tsur) is at 25 °C (Figure 2b). The temperature difference, Tmax − Tsur, was calculated to be 30 °C. The value of hS, the absorbance of AAP10-pDA/rGO at 808 nm, and Qdis were obtained from Supporting Information S4. The laser intensity, I, was 1.5 W cm−2. Therefore, the photothermal conversion efficiency of AAP10-pDA/rGO was estimated to be 49.1% (Supporting Information S4), which is much higher than that previously reported for GO and rGO (25.4% and 41%, respectively).[2,43] The biocompatibility of AAP10-pDA/rGO is an important factor when it is used in biomedical applications. Therefore, 4


we also evaluated the cytotoxicity of AAP10-pDA/rGO (40−160 µg mL−1) in both human normal MCF-10A cells and breast-cancer MCF-7 cells. After incubation with GO for 24 h, the cell viability decreased upon increasing the GO concentration (Figure 2c,d). It has been demonstrated that high concentration of GO may induce adverse effects in biological systems.[44–46] However, when GO is modified with dopamine and AAP10, the viability of MCF-10A and MCF-7 cells remained above ≈95% after incubation with AAP10-pDA/rGO for 24 h (Figure 2c,d). The inherent toxicity of AAP10-pDA/ rGO is significantly low. Collectively, these results imply that pDA surface modification can improve the biocompatibility of GO (Figure 2c,d). The changes in the physicochemical properties, such as solubility and dispersibility of AAP10-pDA/rGO, may be used to describe the difference in cytotoxicity between GO and AAP10-pDA/rGO.[47] Overall, our results suggest that AAP10-pDA/rGO can serve as a biocompatible material for anticancer PTT because it exhibits an excellent NIR photothermal heating efficiency.

2.3. Bystander Effect Induced by AAP10-pDA/rGO Demonstrated previous research showed that overexpression of Cx43 results in increased GJIC, thereby leading to an increased bystander effect.[37] AAP10 has been shown to elevate the protein activity of Cx43.[38] Therefore, we expect that using AAP10-functionalized pDA/rGO might be a useful approach for enhancing the bystander effect. To determine whether this is true for MCF-7 cells, we conducted an immunohistochemistry (IHC) assay and the scrape load/dye transfer analysis of



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FULL PAPER Figure 3. Bystander effect induced by AAP10-pDA/rGO. a) IHC analysis of the Cx43 expression. b) Scrape load-dye transfer analysis of activity of gap-junction channels in MCF-7 cells after treatment with AAP10 (50 × 10−9 m), pDA/rGO (120 µg mL−1), and AAP10-pDA/rGO (50 × 10−9 m AAP10/120 µg pDA/rGO/mL).

the Cx43 expression and of the activity of gap-junction channels. As shown in Figure 3a, a significant increase in the level of Cx43 protein occurs upon treating MCF-7 cells with AAP10 and AAP10-pDA/rGO. Furthermore, AAP10-pDA/rGO induces higher levels of Cx43 compared with AAP10. Similar results also observed for the activity of gap-junction channels (Figure 3b). AAP10-pDA/rGO induced a greater increase in dye-transfer distance compared with AAP10 (Figure 3b). Conversely, no significant increase was detected in Cx43 protein expression and in the activity of gap-junction channels in the pDA/rGO-treated group (Figure 3). These results suggest that surface modification of AAP10 on pDA/rGO enhances the bystander effect of AAP10.[38] Thus, because the bystander effect is widely considered to improve radiotherapeutic efficacy in cancer treatment,[48] these results suggest that nanotechnology may boost the functional role and application potential of the bystander effect in cancer therapy by increasing Cx expression and GJIC in cancer cells.

2.4. Enhancing the Anticancer Efficiency of Photothermal Therapy Using AAP10-pDA/rGO along with an In Vitro Bystander Effect After demonstrating excellent NIR photothermal properties and an enhanced bystander effect of AAP10-pDA/rGO, we performed in vitro studies to determine whether AAP10-pDA/ rGO can enhance the bystander effect and thereby improve


the efficiency of PTT to destroy MCF-7 breast-cancer cells. To examine the therapeutic response to PTT, MCF-7 cells were treated with AAP10-pDA/rGO, pDA/rGO, or AAP10 (120 µg mL−1) and were exposed for 5 min to NIR irradiation (880 nm, 0.32–2.2 W cm−2). The viability of MCF-7 cells was then determined via a thiazolyl blue tetrazolium bromide (MTT) assay. The group treated with AAP10-pDA/rGO had 76% cell viability after 5 min of NIR 808 nm irradiation at 0.32 W cm−2 (Figure 4a). At the same concentration of AAP10pDA/rGO, the cell viability decreased to 49%, 21%, 14%, and 1% after 5 min exposure to NIR 808 nm irradiation at intensities of 1.0, 1.5, 2.0, and 2.2 W cm−2, respectively (Figure 4a). These results demonstrate that the AAP10-pDA/rGO has a strong potential for cancer treatment. Under the same conditions, the percent viabilities of pDA/rGO-treated cells were 81%, 58%, 43%, 24%, and 11% for NIR intensities of 0.32, 1.0, 1.5, 2.0, and 2.2 W cm−2, respectively (Figure 4a). Overall, AAP10pDA/rGO induced greater therapeutic effect than pDA/rGO in MCF-7 cells (Figure 4a). The increase in temperature under NIR irradiation was almost the same for AAP10-pDA/rGOand pDA/rGO-containing media (Figure 2a). Thus, any differences in therapeutic efficiency between AAP10-pDA/rGO and pDA/rGO were possibly due to the AAP10-pDA/rGO-induced bystander effects. A key characteristic of the bystander effect was observed in a previous study and is shown in Figure S2[33] (Supporting Information)—the bystander response in AAP10pDA/rGO-treated cells saturates under an 808 nm irradiation at 1.5 W cm−2 (Figure S2, Supporting Information). This indicates



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Figure 4. Effect of PTT. a) Cell viability of MCF-7 cells after incubation with AAP10 (50 × 10−9 m), pDA/rGO (120 µg mL−1), AAP10-pDA/rGO (50 × 10−9 m AAP10/120 µg pDA/rGO/mL), and the DMEM medium as a control under NIR illumination (808 nm, 1.5 W cm−2, 5 min). (b) Bystander effect in MCF-7 cells after treatment with pDA/rGO (120 µg/mL) or AAP10-pDA/rGO (50 × 10−9 m AAP10/120 µg pDA/rGO/mL) coupled with NIR 808-nm irradiation at 1.5 W cm−2 for 5 min. (*p < 0.05, **p < 0.01).

that above a certain dose, no additional effect occurs. In practice, this means that for any given endpoint, not every cell experiences the bystander effect.[33] Another method to verify the bystander effect induced by AAP10-pDA/rGO during PTT is to use fluorescent dye for identifying dead cells. The results of this approach (shown in Figure 4b) indicate that cell death expands beyond the NIRirradiated zone in the pDA/rGO-treated group because the irradiated-pDA/rGO nanoparticles reach sufficiently high temperatures to transfer significant thermal energy to the adjacent cells outside the irradiated zone. However, the expansion of cell death is much higher in the AAP10-pDA/rGO-treated group than in the pDA/rGO-treated group (Figure 4b). We tentatively attribute this phenomenon to the combination of the diffusion of thermal energy and the bystander effect induced by AAP10pDA/rGO. AAP10-pDA/rGO may effectively destroy cancer cells via not only photothermal effects but also the bystander effect (Figure 4). The use of AAP10-pDA/rGO, therefore, has a strong potential to improve the efficacy of anticancer PTT.

2.5. In Vivo Enhancement in the Anticancer PhotothermalTherapy Efficiency Using AAP10-pDA/rGO We used a mouse 4T1 breast-tumor model to determine the efficiency of cancer PTT using AAP10-pDA/rGO. When the volume of the 4T1 tumor at the right back of the Balb/c female mice grew to 80 mm3, they were intratumorally injected with phosphate buffered saline (PBS), pDA/rGO, or AAP10-pDA/ rGO (50 µL, 6 mg mL−1). After injection, the tumors were irradiated for 5 min using the NIR 808 nm laser at 1.5 W cm−2. The surface temperature of pDA/rGO- and AAP10-pDA/rGOinjected tumors in the mice approximately reached 52 °C after 6


NIR irradiation (Figure 5a). Conversely, the surface temperature of PBS-injected tumors in the mice was approximately reached 37 °C (Figure 5a). The tumor size was measured using a caliper, and the tumor volume was calculated as (length × width × width)/2 on a daily basis, starting from day 1 till day 17. The irradiated tumors injected with PBS and the nonirradiated tumors in the mice rapidly grew (Figure 5b). However, the irradiated tumors in the mice injected with pDA/rGO almost disappeared 15 d after treatment (Figure 5b). However, the tumor reappeared after day 27 (Figure 5c). Under the same irradiation conditions, tumor growth was significantly inhibited for the AAP10-pDA/rGOinjected group compared with the pDA/rGO-injected group. After NIR irradiation, the tumors in the AAP10-pDA/rGO-injected group extremely slowly grew after 7 d with a tumor-inhibition rate of 99.3% and disappeared by day 9 (Figure 5b). Twenty-seven days after the treatment, the AAP10-pDA/rGO-injected group displayed only residual scar tissue with no evidence of tumor recurrence (Figure 5c and Figure S3a, Supporting Information). To further clarify the effects of in vivo PTT at the cellular level, tumor sections were stained with hematoxylin-eosin and were examined using an optical microscopic. The AAP10pDA/rGO-injected group exhibited severe organ damage in tumor tissue compared with the pDA/rGO-injected and control groups, thereby providing evidence of the effective therapeutic effect of AAP10-pDA/rGO on tumors (Figure 6). These results imply that the bystander effect induced by AAP10-pDA/rGO not only enhances the efficiency of the cancer treatment but also reduces the risk of cancer recurrence. Note that despite the excellent antitumor effect of AAP10-pDA/rGO, the total body mass of AAP10-pDA/rGO-treated mice was comparable to that of the other mice (Figure S3b, Supporting Information). These results indicate that AAP10-pDA/rGO-mediated PTT cancer therapy caused no harmful side effects.



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FULL PAPER Figure 5. Efficiency of anticancer PTT. a) Thermal images of tumor-bearing mice under NIR 808 nm irradiation at 1.5 W cm−2 for 5 min after intratumor injections of 50 µL PBS; 50 µL, 6 mg mL−1 pDA/rGO; and 50 µL, 6 mg mL−1 AAP10-pDA/rGO. b) Normalized tumor size in tumor-bearing mice as a function of days after intratumor injection of 50 µL PBS; 50 µL, 6 mg mL−1 pDA/rGO; and 50 µL, 6 mg mL−1 AAP10-pDA/rGO with and without NIR 808 nm irradiation at 1.5 W cm−2 for 5 min. c) Photograph showing the tumors from mice 28 d after intratumor injection of 50 µL, 6 mg mL−1 pDA/ rGO and 50 µL, 6 mg mL−1 AAP10-pDA/rGO with and without NIR 808 nm irradiation at 1.5 W cm−2 for 5 min.

3. Conclusion Cancer PTT relies on irradiating cancer cells with NIR radiation, which heats and destroys the cancer cells. However, because tumors are 3D structures, some tumor cells are shaded

and prevent PTT from destroying all the cancer cells. For this reason, the recurrence rate of cancer after PTT is significantly high. Therefore, this study uses AAP10-pDA/rGO to increase the bystander effect in cancer cells and to consequently improve the therapeutic efficiency of cancer PTT. We use in vitro and

Figure 6. Hematoxylin and eosin staining of the tumor-tissue section after intratumor injection of pDA/rGO (50 µL, 6 mg mL−1) and AAP10-pDA/rGO (50 µL, 6 mg mL−1) with and without NIR 808 nm irradiation at 1.5 W cm−2 for 5 min). Red arrows show undamaged tumor tissue.




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in vivo models to explore cytotoxicity and the bystander effect in human cancer cells exposed to NIR-irradiated AAP10-pDA/ rGO. The results show that AAP10-pDA/rGO strongly enhances the NIR thermal energy-conversion efficiency and photothermal stability. Additionally, photothermal treatment with AAP10-pDA/rGO inhibits the growth of breast cancer cells both in vitro and in vivo. Thus, this newly designed nanocomposite strongly enhances the therapeutic efficiency against cancer by enhancing the bystander effect. This results in an elevated PTT efficiency with less NIR irradiation. The results also indicate that by enhancing the in vivo bystander effect, AAP10-pDA/rGO strongly reduces the recurrence rate of breast cancer. The AAP10-pDA/rGO-induced bystander effect occurs because cells that are not directly exposed to NIR radiation are destroyed by the toxic signals received (via the bystander effect) from nearby irradiated cells. These results could lead to more applications of the bystander effect in medical purposes. Moreover, the findings of this study offer a better understanding of the design of graphene-based nanocomposites for cancer PTT.

4. Experimental Section Chemicals: Graphite (7–11 nm), dopamine hydrochloride, potassium permanganate, hydrogen peroxide, Bisbenzimide Hoechst 33342, propidium iodide (PI), rhodamine B, and MTT were purchased from Sigma-Aldrich (Milwaukee, USA). Immune dye fixative was bought from the Beyotime Institute of Biotechnology (Shanghai, China). Peptide H2N-Gly-4Hyp-Pro-Tyr-CONH2 (AAP10) was bought from KareBayTM Biochem, Inc. (Shanghai, China). All other regents were purchased from Sigma-Aldrich or the China National Medicine Corporation (Beijing, China). All chemicals were used without further purification. Preparation of AAP10-pDA/rGO: First, pDA/rGO (20 mg) was dissolved in 10 mL deionized (DI) water and was sonicated for 10 min. Then, a 1 mL peptide aqueous solution (20 × 10−6 m) and 1.5 mL of Tris-HCl (80 × 10−3 m, pH 8.5) were added to this solution, and the mixture was stirred for 12 h at room temperature. The product was centrifuged and washed using DI water. All supernatants from each centrifugation were collected, and their APP10 peptide concentrations were measured using a NanoDrop 100 instrument. The final product was dissolved in 10 mL DI water and was stored at 4 °C for further use. Characterization of GO, pDA/rGO, and AAP10-pDA/rGO: The morphology of the synthesized materials was characterized via atomic force microscopy (AFM, USA). Ultraviolet–visible–NIR spectra were acquired using a PerkinElmer Lambda 750 spectrophotometer (PerkinElmer, USA). Raman spectra were acquired using a LabRam-HR spectrometer (Jobin Yvon, France). The peptide was quantified using the NanoDrop 100 instrument (AOSHENG, China). In vivo IR thermal images of the mice therapy were taken by using an IR thermal camera (FLIR SC325, USA). Cell Cultures: MCF-10A human breast cells, MCF-7 human breastcancer cells, and 4T1 mice breast-cancer cells were maintained in the DMEM-F12, DMEM, and RPMI 1640 cultures media containing 10% fetal bovine serum, 100 units mL−1 penicillin, and 100 µg mL−1 streptomycin, respectively, at 37 °C in an incubator with a humidified mixture of 5% CO2 and 95% ambient air. These media were changed twice a week, and the cells were subjected to a passage with trypsin every week. Measurement of the Cytotoxicity of GO, pDA/rGO, and AAP10-pDA/ rGO: MCF-10A and MCF-7 cells were treated with a gradient concentration of GO, AAP10, pDA/rGO, and AAP10-pDA/rGO diluted with the prepared culture media for 24 h. An MTT assay was conducted to measure cell viability.

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Temperature Monitoring of Laser-Induced Heat Generation: GO, pDA/ rGO, and AAP10-pDA/rGO suspensions in the cell culture medium were irradiated using an 808 nm NIR laser (PSU-III-LRD, CNI Optoelectronics Technology Co. Ltd., Changchun, China) at 1.5 W cm−2 NIR laser for 5 min. The real-time temperature was measured using a thermocouple or an IR thermal camera. In Vitro Photothermal Cancer Treatment: MCF-7 cells were treated with GO, pDA/rGO, AAP10-pDA/rGO, or NIR illumination alone or in combination. The well was irradiated using the NIR 808 nm laser at 1.5 W cm−2 for 5 min. The cells were then incubated for another 12 h, and cell viability was measured using the MTT assay according to the manufacturer’s protocol. The samples were analyzed using a PerkinElmer Victor X4 light plate reader (Victor X4, 2030 Multilabel Reader), and the results were compared with those of the controls. Visible absorbance was recorded from a 96-well plate reader at wavelength of 490 nm. Moreover, cell viability was expressed as the percent absorbance relative to that of the control cells. Immunohistochemistry and Scrape Load-Dye Assay: MCF-7 cells were treated with the culture medium, GO, pDA/rGO, or APP10-pDA/ rGO for 12 h. Cx43 expression was checked via an IHC assay, following the protocol of the product manufacturer (Beyotime Institute of Biotechnology, China). Intercellular communication was monitored via a scrape load-dye assay. Twelve hours after the treatment of the materials, 2.5 µL 1% (w/v) rhodamine B in a PBS drop was laid in the center of the well across a scrape prepared in the culture medium. Three minutes later, the cells were washed twice with the PBS solution and were incubated in a culture medium for another 20 min. All cells were fixed using an immune dye fixative for 10 min before taking fluorescence pictures. Double Fluorescence Mark Assay for Detection of Cell Death: MCF-7 cells were treated with pDA/rGO or APP10-pDA/rGO for 12 h and were irradiated using the NIR 808 nm laser at an intensity of 1.5 W cm−2 for 5 min. These cells were incubated for another 12 h at 37 °C and were washed with the PBS solution twice before the fluorescence assay was conducted. Mixtures of Hoechst 33342 (10 µg mL−1) and PI (5 µg mL−1) in PBS were used to incubate the cells for 10 min. The cells were then washed twice with the PBS solution and were fixed using the immune dye fixative for 10 min before fluorescence images were taken. Animal Studies: Five-week-old Bal/bc-8 mice were purchased from the Shanghai Laboratory Animal Center (Shanghai, China) and were acclimated for two weeks. Animal handling was conducted in accordance with standard animal husbandry practices and regulations. The animals were treated humanely and with regard for the alleviation of suffering throughout the study. All mice were maintained under a 12 h light–dark cycle at 23 ± 1 °C and 39%–43% relative humidity, with water and food available ad libitum. We selected six mice at random per time point for experimentation. All animal experiments were conducted according to the agreement with the Institutional Animal Use and Care Committee, Jiangsu Province, China, and experiments were conducted with appropriate ethical and humane concern. A total of 5 × 106 4T1 cells were injected subcutaneously into the backs of the mice, above the right leg. When the tumors grew to ≈80 mm3, the mice were injected with 100 µL of 10% chloral hydrate, and were then intratumorally injected with PBS, pDA/rGO, or APP10-pDA/rGO. Twenty minutes later, the tumor site was irradiated using the NIR 808 nm laser at 1.5 W cm−2 for 5 min. An IR camera was used to detect and record the real-time temperature of the tumor site. Nonirradiated tumor-bearing mice served as the control. Tumors were carefully removed and were prepared for other studies in this investigation. Histopathological Assessment and Tumor-Growth Measurement: Two mice from each group of treated mice were scarified 6 h after treatment, and the tumors were stained with hematoxylin and eosin for optical observation. The tumor size was measured the day after the treatment and every two subsequent days. The tumor volume V was calculated using Equation (1) V=

Lw 2 2


(1)


www.advhealthmat.de


Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements J.Y. and Y.-H.L. contributed equally to this work. The authors are grateful to the National Formosa University and Ministry of Science and Technology (MOST, Taiwan) for providing financial support for this study under contracts 105-2221-E-150-002. All the authors declare no conflict of interest. Received: July 25, 2016 Revised: October 12, 2016 Published online:

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where L is the tumor length and w is the tumor width. All mice were scarified on the 18th day. The tumors were then removed and the body mass was measured. Statistical Analysis: The results obtained from the experiments on various mice groups with differing treatments and their corresponding controls were compared using a Student’s t-test. All comparisons were considered significantly different for p < 0.05 or p < 0.01.

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