Enhanced Efficient NIR Photothermal Therapy Using Pleurocidin NRC

Feb 14, 2019 - Department of Civil Engineering, National Chi Nan University, Nantou ... have been widely studied but are still in the early stages of ...
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Article Cite This: ACS Omega 2019, 4, 3298−3305

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Enhanced Efficient NIR Photothermal Therapy Using Pleurocidin NRC-03 Peptide-Conjugated Dopamine-Modified Reduced Graphene Oxide Nanocomposite Yi-Chun Chen,†,§ Saranta Sawettanun,† Ku-Fan Chen,§ Chao-Yu Lee,‡ Junyan Yan,∥ Hung-Hsiang Chen,† Guan-Wen Chen,⊥ and Chia-Hua Lin*,†

ACS Omega 2019.4:3298-3305. Downloaded from pubs.acs.org by 5.62.154.125 on 02/19/19. For personal use only.

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Department of Biotechnology and ‡Department of Materials Science and Engineering, National Formosa University, Yunlin 63208, Taiwan § Department of Civil Engineering, National Chi Nan University, Nantou 54561, Taiwan ∥ School of Life Science, Shaoxing University, Zhejiang 312000, China ⊥ Department of Food Science, National Taiwan Ocean University, Keelung 20224, Taiwan S Supporting Information *

ABSTRACT: Breast cancer remains the leading cause of morbidity and mortality among women. Therefore, there is an urgent need to develop effective treatments for breast cancer. Peptide-based therapies have been applied to treat various diseases, particularly cancer. Peptides that exhibit antimicrobial properties have recently been found to inactivate a wide range of cancer cells. However, some peptides degrade quickly in biological systems. Therefore, peptide-based nanotherapeutic approaches for cancer have been widely studied but are still in the early stages of research. The objective of this study is to synthesize a nanocomposite, NRC-03 peptide conjugated to polydopamine (pDA)-modified reduced graphene oxide (rGO), to facilitate the use of near-infrared light-activatable photothermal therapy to destroy breast tumor cells. The results show that immobilizing NRC-03 on the surface of dopaminemodified rGO can increase the stability of the NRC-03 peptide in a biological system. Furthermore, a burst release of the NRC03 from NRC-03−pDA/rGO was observed under photothermal and tumor environment conditions. Overall, the results show that NRC-03−pDA/rGO combines the advantages of NRC-03, dopamine, and rGO, displaying excellent biocompatibility and anticancer properties; thus, it shows strong potential for augmenting photothermal therapy for breast cancer.

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INTRODUCTION

Nanomaterial-based PTT agents have been widely investigated. Gold nanostructures, carbon nanomaterials (carbon nanotubes and graphene oxide (GO)), and various other inorganic and organic nanomaterials have strong NIR absorbance and can effectively convert the light energy into heat to kill cancer cells upon NIR irradiation. Reduced graphene oxide (rGO) is a particularly promising material for PTT. It can be prepared from graphene oxide using dopamine as both a reducing and capping reagent9−11 to improve the stability and dispersity without using substances that damage the environment.12,13 During the reduction process, polydopamine (pDA) is formed on the surface via oxidative polymerization of dopamine, which significantly enhances the NIR optical absorbance of the resulting composite.9−11,14 In a previous study, our group demonstrated that pDA/rGO exhibits excellent biocompatibility, photothermal conversion efficiency, and drug-loading capacity.

Breast cancer is the most common type of cancer in women worldwide and in most countries.1 Current treatments for breast cancer include surgery, radiotherapy, chemotherapy, hormonal therapies, and targeted therapies.2 These therapies may be used alone or in combination depending on the stage of the disease. Photothermal therapy (PTT) is a newly developed and promising therapeutic strategy that involves applying near-infrared (NIR) laser light to photoabsorbers, which generate heat and cause irreversible damage to tumor cells (this process is called thermal ablation).3 NIR light (700− 1350 nm) can penetrate biological tissues more efficiently than visible light and has been successfully used in PPT because it can be easily operated and locally focused on a specific region.4,5 Cancer cells are more susceptible to thermal damage than normal cells under hyperthermic treatment (43−45 °C).6−8 According to Elengoe and Hamda (2013), MDA-MB 231 human breast cancer cells died by apoptosis at exponentially higher rates, whereas normal human WRL-68 cells survived under certain temperatures and durations of heat exposure between 38 and 42 °C.7 Thus, PPT is a precise and minimally invasive alternative cancer treatment. © 2019 American Chemical Society

Received: December 21, 2018 Accepted: February 5, 2019 Published: February 14, 2019 3298

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Scheme 1. Schematic of the Route for Preparing NRC-03−pDA/rGO

Figure 1. Tapping-mode AFM image of (A) GO, (B) pDA/rGO, and (C) NRC-03−pDA/rGO and cross-sectional plots of (D) GO, (E) pDA/ rGO, and (F) NRC-03−pDA/rGO.

cells.20 NRC-03 also substantially reduces the EC50 value of cisplatin, suggesting that is may be used as a chemosensitizing agent.18 However, NRC-03 is readily degraded by the trypsin in serum,18 which limits its potential applications in clinical practice. Therefore, we aim to develop a nanocomposite that makes NRC-03 more resistant to protease degradation and allows it to retain its biological activities. In this study, we developed NRC-03-functionalized pDA/rGO (NRC-03−pDA/rGO) to enhance the stability of NRC-03 in biological systems and facilitate efficient PPT with less NIR irradiation. We characterize the anticancer activity of the composite, including the synergistic effects of AMPs combined with thermal effect against breast cancer cells.

Recently, antimicrobial peptides (AMPs) have attracted increasing attention for cancer therapies.15 AMPs may exert antineoplastic effects mainly by suppressing tumor angiogenesis, inducing tumor cell apoptosis and necrosis and inhibiting kinases, and proteases.16 AMPs are typically relatively short (12−100 amino acids), positively charged (net charge of +2 to +9), and amphiphilic. The smaller size of AMPs facilitates their rapid diffusion into cells. They have been isolated from single-celled microorganisms, insects, and other invertebrates, such as plants, amphibians, birds, fish, and mammals, including humans.17 One such peptide, NRC-03 (GRRKRKWLRRIGKGVKIIGGAALDHL-NH2), has been shown to have cytotoxic effects in breast cancer cells18 due to the peptide binding to negatively charged surface molecules. NRC-03 treatment on HL60 cells immediately results in the loss of microvilli, the formation of membrane pores, and cell swelling.19 Hilchie et al. (2015) previously reported that NRC03 causes lysis in breast cancer cells and multiple myeloma

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RESULTS AND DISCUSSION Characterization of pDA/rGO and NRC-03−pDA/rGO. The synthesis of NRC-03−pDA/rGO is shown schematically

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Figure 2. Physicochemical properties of NRC-03−pDA/rGO. (A) Raman spectra of GO, pDA/rGO, and NRC-03−pDA/rGO. (B) Fouriertransform infrared (FTIR) spectra of GO, pDA/rGO, and NRC-03−pDA/rGO. (C) Photograph of GO, pDA/rGO, and NRC-03−pDA/rGO. (D) UV−vis absorption spectra of GO, pDA/rGO, and NRC-03−pDA/rGO. Absorbance (Abs) is plotted in arbitrary units (au).

in Scheme 1. The GO was generated with a uniform length of 1.53 ± 0.72 μm and a width of 1.11 ± 0.48 μm (Figure S1). The atomic force microscope (AFM) images in Figure 1A,D show that the monolayered GO was approximately 0.8 nm thick. Dopamine was polymerized, and the pDA was coated on the GO surface. The thickness of the resulting monolayer GO−pDA was 2.2 nm (Figure 1B,E). NRC-03 was then coated on the pDA/rGO surface to form a uniform film that was 3.5 nm thick (Figure 1C,F). Raman spectroscopy was conducted to confirm that the GO was successfully reduced (Figure 2A). The Raman spectra of GO shows two strong peaks located at 1333 and 1583 cm−1, which were referred to as D band and G band, respectively. The intensity of the D band (1333 cm−1 in GO and 1350 cm−1 in rGO) is related to the size of the in-plane sp2 domains. The relative intensity ratio of both peaks (ID/IG) is a measure of the degree of disorder and is inversely proportional to the average size of the sp2 clusters.21 The ID/IG ratio decreased from 0.92 in the GO to 0.86 in the pDA/rGO, which confirms that a reduction reaction occurred between the GO and dopamine molecules (Figure 2A). When the peptide NRC-03 was applied to the pDA surface, the ID/IG ratio increased to 0.93 (Figure 2A). Fourier-transform infrared (FTIR) spectroscopy was conducted to investigate the bonding interactions in GO, pDA/ rGO, and NRC-03−pDA/rGO. It is well known that chemically derived GO sheets are heavily oxygenated, bearing hydroxyl, carbonyl, and epoxide functional groups.22 As shown in Figure 2B, GO exhibits strong peaks at 1780 cm−1

(corresponding to the deformation of CO), 3000−3700 cm−1 (O−H stretching), and 2800−3000 cm−1 (vibration of C−H). After dopamine reduction, the intensities of the peaks at 1780, 3000−3700, and 900−1500 cm−1 decreased significantly, as seen in the spectra of pDA/rGO and NRC03−pDA/rGO (Figure 2B). Moreover, the absorbance over the entire visible range increased in pDA/rGO and NRC-03− pDA/rGO compared to that in GO because they had deeper colors (Figure 2C). In addition, the absorbance of visible/NIR light of 808 nm increased in pDA/rGO and NRC-03−pDA/ rGO compared to that in GO (Figure 2D). Together, these results indicate the successful reduction of GO by dopamine. Photothermal Properties and Biocompatibility. To examine the photothermal heating capabilities of GO, pDA/ rGO, and NRC-03−pDA/rGO, the nanocomposite suspensions were exposed to NIR irradiation and the temperature was measured in real time using a thermocouple. As shown in Figure 3A, the temperature of the control (medium only) increased slightly, whereas those of GO, pDA/rGO, and NRC03−pDA/rGO suspended in Dulbecco’s modified Eagle’s medium (DMEM) increased with time after 300 s of continuous NIR irradiation. Although the GO exhibited outstanding photothermal conversion performance, that of pDA/rGO and NRC-03−pDA/rGO was even higher (resulting in a solution temperature of 52 °C after 3 min of NIR illumination) because of their strong light absorption. The effects of the NIR laser on the photothermal conversion efficiency (the ratio of the absorption cross section to the extinction cross section23) were studied by measuring the 3300

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Figure 3. Photothermal effects and biocompatibility of NRC-03−pDA/rGO. (A) Photothermal heating of GO, pDA/rGO, and NRC-03−pDA/ rGO under NIR illumination (880 nm, 1.5 W/cm2). (B) Photothermal conversion efficiency of GO, pDA/rGO, and NRC-03−pDA/rGO under NIR illumination. (C) Photothermal stability (four consecutively repeated cycles) of NRC-03−pDA/rGO under NIR illumination. (D) Cytotoxicity of GO, pDA/rGO, and NRC-03−pDA/rGO in human normal BEAS-2B cells was measured by thiazolyl blue tetrazolium bromide (MTT) assay (*P < 0.05).

Figure 4. Stability of NRC-03 peptide over NRC-03−pDA/rGO. (A) NRC-03 and (B) NRC-03−pDA/rGO were incubated with/without trypsin at 37 °C for 4 and 24 h. Intact and fragmented peptides were detected by MALDI-TOF mass spectrometry.

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temperature increase of each PTT agent compared to that of the medium. The experimental setup had a 10 mm path-length quartz cuvette and an 808 nm laser that was used to irradiate the sample with a power density of 1.5 W/cm2 for 20 min. Between experiments, the laser was turned off and allowed to cool to its initial temperature. The photothermal conversion efficiency of NRC-03−pDA/rGO was estimated to be 41.10%, which was higher than that of GO and rGO (11.61 and 35.03%, respectively) (Figure 3B). Furthermore, to test the stability of the photothermal heating, the temperature increase of the NRC-03−pDA/rGO suspension under NIR irradiation was measured over four cycles of irradiation and cooling to room temperature. The thermal curves shown in Figure 2C indicate that the heating effect was extremely repeatable, demonstrating highly stable photothermal performance (Figure 3C). The biocompatibility of NRC-03−pDA/rGO is an important factor for materials intended for biomedical applications. The cytotoxicities of GO, pDA/rGO, and NRC-03−pDA/rGO to normal human BEAS-2B cells were tested using an MTT assay. Previous data showed that GO exposure significantly decreases cell viability.24 In this study, GO was shown to be cytotoxic to BEAS-2B cells only at a concentration of 160 μg/ mL (Figure 3D). Moreover, the viability of BEAS-2B cells was about 100% when exposed to pDA/rGO and NRC-03−pDA/ rGO even at the highest concentration tested (160 μg/mL) after 24 h of incubation. This finding implies that the dopamine on the surface of GO can greatly enhance its biocompatibility.25 Thus, NRC-03−pDA/rGO can be considered a biocompatible material for PTT. NRC-03 Peptide Stability. Trypsin will cleave NRC-03 peptides from the C-terminus at lysine and arginine amino acid residues into shorter fragments.18 To verify that conjugation to pDA/rGO improves the peptide’s stability, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) was used to assess the NRC-03 degradation by trypsin. NRC-03 alone and NRC-03−pDA/rGO were incubated in the presence of trypsin (2 μg) at 37 °C for 4 and 24 h. Figure 4A shows that only cleaved fragments of NRC-03 were present following trypsin treatment for 4 h with complete degradation within 24 h. In contrast, no cleaved NRC-03 fragments were observed in the NRC-03−pDA/rGO samples with trypsin (Figure 4B), which indicates that pDA/rGO protects the NRC-03 peptides from protease degradation. In addition, no NRC-03 peptides were observed in the trypsin-treated NRC-03−pDA/rGO samples, which confirms that the NRC-03 was strongly conjugated to the pDA/rGO surface and was not removed by trypsin (Figure S2). Release of NRC-03 Peptide from NRC-03−pDA/rGO. In this composite, the NRC-03 peptide was conjugated to the pDA/rGO surface via a Michael addition and/or Schiff base reaction.26 It has been demonstrated that Schiff bases are a promising candidate for the development of acid-sensitive linkages and their stability decreases as the surrounding pH decreases.27 Under biological conditions, the interaction between the amine groups in NRC-03 and the pDA/rGO surface can be broken by either proteases or acidic ion to release the NRC-03. Thus, the release profile of NRC-03 from pDA/rGO was evaluated by UV spectrophotometry under different pHs (3−7) and temperatures (37 and 42 °C). The results shown in Table 1 indicate that, at pH 7, 4.8−35.4% of the peptide was released at 37 °C and 16.8−35.7% was released at 42 °C in 24−48 h (Table 1). This slow release of

Table 1. NRC-03 Release Profiles from NRC-03−pDA-rGO in a Variety of pH over 24 and 48 h at 37 and 42 °C percentage of NRC-03 release from NRC-03−pDA/rGO 24 h pH 2 3 6 6.5 7

37 °C 21.1 40.9 18.4 21.6 4.8

± ± ± ± ±

1.1% 0.6% 0.5% 0.6% 0.4%

48 h 42 °C 30.4 47.1 25.1 23.0 16.8

± ± ± ± ±

0.5% 1.2% 0.7% 0.5% 0.3%

37 °C 36.5 49.3 54.1 42.3 35.4

± ± ± ± ±

0.5% 0.6% 1.1% 0.6% 0.4%

42 °C 58.6 55.3 61.2 52.6 35.7

± ± ± ± ±

1.2% 0.5% 1.3% 1.0% 0.4%

the NRC-03 peptide may reduce its degradation in the biological system. Furthermore, pHs of 6.5 and 6.0 resulted in the burst release of NRC-03 peptide (18.4−61.2%). This indicates that the bonds between the NRC-03 peptides and pDA/rGO are cleaved under acidic conditions, which are often present in tumors, thus releasing the peptides in a targeted manner. Moreover, upon coming in contact with cancer cell membranes, NRC-03−pDA/rGO may be internalized and localized in acidic intracellular compartments (cytoplasm, nucleus, or lysosomes). Table 1 shows that, under very acidic conditions (pHs of 2 and 3), significant release of NRC-03 peptide (21.1−58.6%) occurs; such release of large numbers of NRC-03 peptides will further enhance the therapeutic efficacy of NRC-03−pDA/rGO on breast cancer cells. Anticancer Efficiency of PTT with NRC-03−pDA/rGO. Next, the effect of NRC-03−pDA/rGO on the efficacy of PTT was evaluated. MCF-7 cells were cultured on GO, pDA/rGO, or NRC-03−pDA/rGO, and NIR irradiation (1.5 W/cm2) was applied for 5 min. The cell viability was measured after 0, 24, and 48 h incubations at 37 °C using an MTT assay. The results (Figure 5) show that the MCF-7 viability decreased after prolonged incubation times of 24 and 48 h on all three PTT agents (Figure 5). This result suggests that all three materials

Figure 5. Photothermal therapy effect of NRC-03−pDA/rGO. Cell viability of MCF-7 cells was measured by MTT assay 24 and 48 h after incubation with GO, pDA/rGO, NRC-03−pDA/rGO, and cell culture medium as a control under NIR illumination. (*P < 0.05, **P < 0.01). 3302

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Figure 6. Cellular uptake of NRC-03−pDA/rGO in MCF-7 cells. (A) TEM images of MCF-7 cells exposed to NRC-03−pDA/rGO for 24 h. White arrows denote Apt-AuNPs−GO. (B) Higher magnification of MCF-7 cells.

Aldrich (Milwaukee). NRC-03 peptide (GRRKRKWLRRIGKGVKIIGGAALDHL-NH2) was purchased from KareBayTM Biochem, Inc. (Taipei, Taiwan). All other reagents were purchased from Sigma-Aldrich. All chemicals were used without further purification. Preparation of NRC-03−pDA/rGO. First, pDA/rGO (20 mg) was dissolved in 10 mL of deionized (DI) water. Then, aqueous peptide solution (20 μM) and Tris−HCl (80 mM, pH 8.5) were added to the solution and stirred for 12 h at room temperature. The product was centrifuged for 1 h and washed with DI water. The supernatants from each centrifugation were collected. The final product was dissolved in 10 mL of DI water and stored at 4 °C until further use. Characterization of GO, pDA/rGO, and NRC-03−pDA/ rGO. The morphologies of the synthesized materials were characterized via Bruker dimension icon@AFM (Cambridge, U.K.). UV−visible−NIR spectra were acquired using a spectrophotometer (Lambda 750; PerkinElmer). Raman spectra were acquired using a spectrometer (LabRam-HR; Jobin Yvon, France). The amount of peptide was quantified using a UV−visible spectrophotometer (SP-UV500DB; Spectrum, Victoria, Australia). The temperature of the synthesized materials suspended in the cell culture medium under NIR irradiation (PSU-H-LED, Taiwan) was measured using a thermocouple. The quality of each sample was evaluated by FTIR measurements. Stability of NRC-03 in Biological Conditions. The stability of the NRC-03 was assessed by MALDI-TOF. NRC03 and NRC-03−pDA/rGO were incubated in the presence of trypsin (50:1) overnight at 37 °C. Samples were dried in trifluoroacetic acid (0.1% [w/v]) and diluted in a matrix solution at a 1:1 ratio. Samples were then spotted on a MALDI plate, dried, and analyzed on a MALDI-TOF mass spectrometer. Release of NRC-03 from the Composite. NRC-03− pDA/rGO samples were incubated in solutions with different pHs (acidic and neutral condition) for 24 or 48 h at 37 or 42 °C. The amount of peptide in solution was analyzed by a Branford protein assay. Cell Culture. MCF-7 human breast cancer cells were maintained in the DMEM cell culture media containing 10% fetal bovine serum, 1% (v/v) penicillin, and 1% (v/v) streptomycin. BEAS-2B human bronchial epithelium (normal) cells were maintained in LH9 media. All cells were cultured at

facilitated serious damage to MCF-7 cells via PTT, leading to irreversible cell death. Next, the synergistic effects of PTT (pDA/rGO) and peptide therapy (NRC-03) were evaluated in terms of cancer cell viability. pDA/rGO with NIR illumination reduced the MCF-7 cell viability to 43.7% after 48 h. However, the NRC03−pDA/rGO exhibited a stronger anticancer effect, reducing the MCF-7 cell viability to 33.8% after 48 h (Figure 5). This shows the combination of pDA/rGO and NRC-03 is more efficient for the treatment of breast cancer than PTT with pDA/rGO alone. This might be due to the accumulation of NRC-03−pDA/rGO in MCF-7 cells, which would prolong the circulation of NRC-03−pDA/rGO, leading to a massive release of NRC-03 to the MCF-7 cells and thereby increase in the therapeutic efficiency of the peptide.28 To determine the cellular uptake of NRC-03−pDA/rGO, the slice of NRC-03− pDA/rGO-treated MCF-7 cells were analyzed by transmission electron microscopy (TEM). The NRC-03−pDA/rGO attached on the cell surface was then internalized through endocytosis by MCF-7 cells. Most of the NRC-03−pDA/rGO was confined to cytoplasm and did not enter the nucleus (Figure 6). Thus, the use of NRC-03−pDA/rGO has a strong potential for highly effective cancer treatment.

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CONCLUSIONS Here, we proposed a novel nanocomposite, NRC-03−pDA/ rGO, to increase the stability of NRC-03 peptides in biological conditions and augment the therapeutic efficiency of PTT. The addition of pDA to GO strongly enhances its stability, biocompatibility, and NIR absorption. The NRC-03−pDA/ rGO exhibits excellent NIR photothermal energy conversion and can induce photothermal effects under NIR irradiation. In addition, conjugation to pDA/rGO enhances the degradation resistance of the NRC-03 peptide in the biological environment and allows it to be slowly released over time, thereby enhancing its therapeutic effects. Moreover, the increasing temperature due to PTT both kills cancer cells and facilitates more efficient release of NRC-03 from the NRC-03−pDA/ rGO composite, thus improving its therapeutic efficiency.

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EXPERIMENTAL SECTION Chemicals. Graphite (7−11 nm), dopamine hydrochloride, potassium permanganate, hydrogen peroxide, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma3303

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ACS Omega 37 °C in an incubator with a humidified mixture of 5% CO2 and 95% ambient air. The culture media were changed twice per week, and the cells were passaged with trypsin every week. Cytotoxicity Measurements. MCF-7 and BEAS-2B cells were treated with a gradient concentration of GO, NRC-03, pDA/rGO, and NRC-03−pDA/rGO diluted with the prepared culture media for 24 h. The trypan-blue and MTT assays were conducted to measure cell viability. Before measuring the absorbance, the GO, NRC-03, pDA/rGO, and NRC-03− pDA/rGO were removed from the resultant solution by centrifugation. Temperature Monitoring during NIR Irradiation. GO, pDA/rGO, and NRC-03−pDA/rGO samples suspended in cell culture medium were irradiated by an 808 nm NIR laser (PSU-H-LED, Taiwan) at 1.5 W/cm2 for 3 min. The temperature was measured in real time using a thermocouple. Cellular Uptake of NRC-03−pDA/rGO. MCF-7 cells were exposed to NRC-03−pDA/rGO for 24 h and analyzed by TEM (Hitachi HT7700, Japan). NRC-03−pDA/rGO-treated MCF-7 cells were washed with phosphate-buffered saline and fixed in 1% OsO4. The cells were dehydrated with ethanol and immersed in LR white resin. The cells were subsequently embedded in epoxy resin, and then the thin sections were sliced using a diamond microtome. Each slice was imaged under TEM. In Vitro Efficacy of PTT. MCF-7 cells were cultured on a cell culture dish with GO, pDA/rGO, NRC-03−pDA/rGO as cell culture medium. Each sample was irradiated by the NIR 808 nm laser at 1.5 W/cm2 for 5 min. The cell viability was measured using an MTT assay immediately after irradiation and after 24 and 48 h of further incubation. Before measuring the absorbance, the GO, NRC-03, pDA/rGO, and NRC-03− pDA/rGO were removed from the resultant solution by centrifugation. The visible light absorbance of the MTT in each solution was analyzed using a PerkinElmer Victor X4 light plate reader (Victor X4, 2030 Multilabel Reader) at a wavelength of 490 nm, and the results were compared to those of the controls. Statistical Analysis. The results for the different treatments and their corresponding controls were compared using a one-way analysis of variance, followed by Dunnett’s multiple comparison tests. Differences with P < 0.05 or 0.01 were considered statistically significant.

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ACKNOWLEDGMENTS

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REFERENCES

We are grateful to the Ministry of Science and Technology (MOST, Taiwan) for providing financial support for this study under contracts 105-2221-E-150-002 and 106-2314-B-150001.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03604. Preparation and characterization procedures of GO nanosheets and pDA/rGO, quantification of NRC-03, photothermal conversion efficiency of NRC-03-pDA/ rGO and TEM image of small and large GOs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 886-5-6315558. Fax: 8865-6315502. ORCID

Chia-Hua Lin: 0000-0003-4103-9617 Notes

The authors declare no competing financial interest. 3304

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DOI: 10.1021/acsomega.8b03604 ACS Omega 2019, 4, 3298−3305