Black Phosphorus Nanosheets Immobilizing Ce6 for Imaging-Guided

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Biological and Medical Applications of Materials and Interfaces

Black Phosphorus Nanosheets Immobilizing Ce6 for Imaging Guided Photothermal/Photodynamic Cancer Therapy Xiaoyan Yang, Dongya Wang, Yunhao Shi, Jianhua Zou, Qisen Zhao, Qi Zhang, Wei Huang, Jinjun Shao, Xiaoji Xie, and Xiaochen Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00276 • Publication Date (Web): 22 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

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ACS Applied Materials & Interfaces

Black Phosphorus Nanosheets Immobilizing Ce6 for Imaging Guided Photothermal/Photodynamic Cancer Therapy Xiaoyan Yang,a Dongya Wang,a Yunhao Shi,a Jianhua Zou,a Qisen Zhao,a Qi Zhang,b Wei Huang,a,c Jinjun Shao,a* Xiaoji Xie,a* Xiaochen Donga* a

Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials

(IAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211800, China. E-mail: [email protected]; [email protected]; [email protected] b

School of Pharmaceutical Sciences, Nanjing Tech University (NanjingTech), 30 South

Puzhu Road, Nanjing 211800, China. c

Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University

(NPU), 127 West Youyi Road, Xi'an 710072, China.

Keywords: black phosphorus, Ce6, photothermal therapy, photodynamic therapy, synergistic therapy

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Abstract In preclinical and clinical research, to destroy cancers, particularly those located in deep tissues is still a great challenge. Photodynamic therapy and photothermal therapy are promising alternative approaches for tissue cancer curing. Black phosphorus (BP) based nanomaterials, with broad UV-vis near-infrared (NIR) absorbance and excellent photothermal effect, have shown great potential in biomedical applications. Herein, a biocompatible therapeutic platform, Chlorin e6 (Ce6) decorated BP nanosheets (NSs), has been developed for fluorescence and thermal imaging guided photothermal and photodynamic synergistic cancer treatment. Taking advantage of the relatively high surface area of exfoliated BP NSs, the PEG-NH2 modified BP NSs (BP@PEG) are loaded with Ce6 photosensitizer. The resulted BP@PEG/Ce6 NSs not only have good biocompatibility, physiological stability and tumor-targeting property, but also exhibit enhanced photothermal conversion efficiency (PCE, 43.6%) compared with BP@PEG NSs (28.7%). In addition, BP@PEG/Ce6 NSs could efficiently generate ROS due to the release of Ce6 photosensitizer, which is also verified by in vitro study. In vivo fluorescence imaging suggests that BP@PEG/Ce6 NSs can accumulate in the tumor targetedly through the enhanced permeability and retention (EPR) effect. Both in vitro and in vivo studies suggest that BP@PEG/Ce6 can be a promising nanotheranostic agent for synergetic photothermal/photodynamic cancer therapy.

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Introduction In spite of the remarkable achievements made in the cancer prevention and treatment in the past decades, cancer is still one of the most serious diseases and continues to threaten human health. As a promising alternative and/or supplement to the traditional therapeutic techniques, phototherapy, including photothermal therapy (PTT)1,2 and photodynamic therapy (PDT),3 emerges in the forefront of medical and materials science due to their unique advantages, such as high efficiency, minimal invasiveness, specific tumor treatment, few side effects and low systemic toxicity. PTT is a hyperthermia treatment that is realized through the conversion of light energy into heat via photothermal agents, causing thermal ablation to target cells. Meanwhile, PDT relies on the photosensitizers and converts light to cytotoxic reactive oxygen species (ROS), inducing cell death. However, till now, single modal therapy, either PTT or PDT, still suffers from the difficulty in eradicating tumors, particularly those deep-located, due to the intrinsic drawbacks of phototherapy, such as the inevitable depth-dependent decrease of photon intensity, the non-uniform heat distribution within tumor tissues in PTT, and the severe hypoxia environment in tumors for PDT.4,5 Cancer synergistic therapy is one of promising ways to address these issues, which shows high therapeutic efficiency and low risk of tumor recurrence.6,7 Owing to the excellent NIR absorption, two-dimensional (2D) nanomaterials, such as transition metal dichalcogenides (TMDCs),8 graphene and its derivatives,9-10 boron nitride (BN),11,12 transition metal carbide or carbon nitride (Mxene),13,14 graphitic carbon nitride (g-C3N4),15-17 and black phosphorus (BP),18 have been proved to exhibit great photothermal effect and large surface areas, which render them as photothermal agents and drug delivery 3



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platforms for cancer treatment. Among 2D nanomaterials, BP has been widely investigated in

electronic,

photoelectronic

applications

and

energy

storage

due

to

its

thickness-dependent bandgap (0.3-2.0 eV) and relatively large interlayer distance.19,20 Since 2015, BP based nanomaterials have attracted enormous interest in biomedicine due to its high

photothermal

conversion

efficiency

(PCE),

large

surface

area,

sufficient

biocompatibility and good biodegradability. What’s more, phosphorus is a vital element in the organism, and its metabolism will not induce certain immune responses. For example, BP quantum dots as well as nanoparticles have been utilized as photothermal agents, demonstrating their inappreciable cytotoxicity and excellent therapeutic effects of tumor.21,22 Ultrathin BP NSs have been shown to be efficient photosensitizers for ROS production.23 Furthermore, the corrugated plane configuration of BP NSs offers large surface area, which is in favor of drug loading. For instance, BP based nanomaterials have been

demonstrated

as

a

multifunctional

theranostic

delivery

platform

for

PTT/chemotherapy when loading with DOX, for PTT/bio-imaging system while loading with Cy7, for PTT/targeting delivery system by modifying with FA-PEG-NH2, and for MRI guided PTT system via loading with Fe3O4 nanoparticles.24-26 Recently, BP reinforced 3D-printed bio-glass scaffold is fabricated for photothermal treatment of osteosarcoma and subsequent bone regeneration.27 Despite these intriguing advances, thus far, no synergetic PTT/PDT theranostic delivery platform, based on BP NSs and photosensitizers has been reported. It is anticipated that combining photosensitizers with BP NSs should be a promising approach for cancer treatment.7,28 Cholrin e6 (Ce6) is a commercial photosensitizer with NIR light absorption, which has 4


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been widely used for PDT and NIR fluorescence imaging in living animals.29,30 Herein, we have prepared a nano-system by immobilizing Ce6 on BP@PEG NSs, which can be used for fluorescence and thermal imaging guided synergistic PTT/PDT (Figure 1). BP NSs prepared by liquid exfoliation of bulk BP tend to aggregate and precipitate in the presence of salts due to the electron screening effect (Figure S1). Thus, BP NSs are modified with PEG-NH2 by electrostatic adsorption to enhance their biocompatibility. BP@PEG NSs are further decorated with Ce6 to construct BP@PEG/Ce6 NSs. BP@PEG/Ce6 NSs dispersed into phosphate buffered saline (PBS) exhibit a higher PCE (η = 43.6%) than BP@PEG NSs (η = 28.7%) due to the synergistic effect of BP@PEG and Ce6. Besides, BP@PEG/Ce6 NSs can generate ROS efficiently due to the release of Ce6. Both in vitro and in vivo studies show that BP@PEG/Ce6 is a more promising nanotheranostic agent for dual-modal imaging-guided PTT/PDT. Results and Discussion The chemical composition of BP NSs was analyzed by energy-dispersive X-ray spectroscopy (EDX). The EDX pattern shows that the nanosheets mainly consist of phosphorous, as well as oxide with weak signal due to the minor degradation of NSs (Figure S2) 24. From X-ray diffractometry (XRD), BP NSs show orthorhombic crystal structure, which is in consistency with the standard JCPDS card No. 73-1358 (Figure S3).24,25 Raman spectral analysis (Figure S4) shows peaks located at around 358.7, 435.3, and 462.7 cm-1, corresponding to Ag1, B2g, and Ag2 modes of BP, respectively, which is in accordance with literature.23 The morphology of BP NSs was characterized by transmission electron microscopy (TEM) 5



and atomic force microscopy (AFM). The TEM image shows that BP NSs are thin and separated with a lateral size about 90 nm (Figure 2a). The average thickness of BP NSs is determined to be about 14.3 nm by AFM images (Figure 2b, 2c). After modified with PEG-NH2, the average lateral size almost remains constant (about 100 nm, Figure 2d) and the average height is about 15.4 nm (Figure 2e, 2f)), which can be attributed to PEG coated on the surface of NSs. Dynamic light scattering (DLS) results display that the diameter of BP NSs and BP@PEG NSs is 89.8 ± 24.2 nm and 90.5 ± 24.7 nm, respectively (Figure 2g, 2h). After loading Ce6, the diameter increases to 157.5 ± 41.9 nm for BP@PEG/Ce6 NSs (Figure 2i), indicating the successful loading of Ce6. In addition, the photographs of BP@PEG/Ce6 NSs in PBS before and after centrifugation intuitively demonstrate the loading of Ce6 (Figure 2i (I, II)). The successful loading of Ce6 onto BP@PEG NSs was further confirmed by UV-vis-NIR and fluorescence spectra. BP@PEG/Ce6 NSs in PBS exhibit two new absorption peaks at 404 and 703 nm, compared to BP@PEG NSs, corresponding to the characteristic Soret-band and Q-band for Ce6 (Figure 3a). According to the fluorescence spectra (Figure 3b), BP@PEG/Ce6 NSs show a fluorescence peak at 696 nm, with an 18-nm red-shift compared with that of Ce6 (678 nm). Furthermore, the fluorescence intensity of BP@PEG/Ce6 NSs is lower than that of free Ce6 in PBS at the same concentration, resulting from the quenching effect of BP@PEG NSs. In addition, BP@PEG NSs, as well as BP@PEG/Ce6 NSs dispersions show good physiological stability, which is validated through the typical Tyndall effect (Figure 3a (I, II)) The loading behavior of BP@PEG NSs with Ce6 was further investigated. Different 6


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amounts of Ce6 were added into BP@PEG NSs solution (Mass ratio of Ce6 and NSs: 1, 2, 4, 6, 8, respectively). After removing excessive free Ce6, the obtained BP@PEG/Ce6 NSs were measured by UV-vis spectra, and the Ce6 loading capacities were calculated (Figure 3c). As shown in Figure 3d, with Ce6/NSs mass ratio increasing, the Ce6 loading capacity increases linearly and reaches 12.8% when feed ratio is 8.0 (Figure S5b and S6), which is probably due to the relatively large surface area of BP NSs. The photothermal effect of BP@PEG/Ce6 NSs was carefully evaluated by taking 660 nm laser as light source. Figure 3e shows the temperature elevation (∆T) of BP@PEG/Ce6 NSs at different concentrations under laser irradiation (660 nm, 0.65 W/cm2,10 min). The temperature of BP@PEG/Ce6 NSs dispersion increased by about 21.6 °C and 17.9 °C at BP@PEG concentration of 200 ppm and 100 ppm, respectively. As shown in Figure 3f, with the laser power density increasing, the temperature elevation increases. For example, the temperature of BP@PEG/Ce6 NSs dispersion (BP@PEG concentration of 100 ppm) increased by 20.2 °C and 24.9 °C at the power density of 0.75 W/cm2 and 0.90 W/cm2, respectively. The photothermal effect of Ce6 on BP@PEG NSs was then investigated. As shown in Figure 4a, the temperature increases are 12.6, 9.9, 6.5, and 2.6 °C for BP@PEG/Ce6 NSs (in PBS, BP@PEG concentration of 70 ppm), BP@PEG NSs (70 ppm), Ce6 (9 ppm) and PBS, respectively. To calculate the PCE, the time-dependent temperature evolutions of the suspensions were recorded under irradiation, followed by naturally cooling to room temperature. The calculated photothermal conversion efficiencies (η) are 43.6%, 28.7% and 35.4% for BP@PEG/Ce6 NSs, BP@PEG NSs and Ce6, respectively (Figure S10a and 7



S10b).31 It can be concluded that the BP@PEG/Ce6 NSs present the best photothermal conversion efficiency, demonstrating the excellent photothermal synergistic effect of BP@PEG NSs and Ce6. ROS are the key factors to assess PDT effect. 9,10-Anthracenediylbis(methylene)dimalonic acid (ABDA) was taken as the probe to estimate the photodynamic effect of BP@PEG/Ce6 NSs, judging from the absorbance decay (300-400 nm) in the presence of ROS (Figure 4b).3 Noteworthy, considering the excellent ROS yield of Ce6, the measurement was performed at low power density (0.15 W/cm2). When BP@PEG NSs is irradiated under 660 nm laser, no absorbance changes can be observed, even under a high laser power density of 0.45 W/cm2 (Figure S11a and S11b). This phenomenon may be attributed to the relatively large thickness of BP NSs.23,32,33 As for free Ce6, the absorbance at 380 nm drops quickly, suggesting a good yield of ROS (Figure S11c). When BP@PEG/Ce6 NSs are irradiated under the same condition, the absorbance intensity gradually decreases, with a relative slow rate, indicating the photodynamic effect of BP@PEG/Ce6 NSs deriving from the slow release of Ce6 in BP@PEG/Ce6 NSs (Figure S11e). It can be concluded that the BP@PEG/Ce6 nano-system could make Ce6 release slowly and prolong the PDT therapeutic time, which is preferred in hypoxic tumors. With the laser power density increasing, the absorption intensity at 380 nm of BP@PEG/Ce6 NSs and ABDA mixture decreases more significantly (Figure 4c, S11f, S11g). Nanomaterials in biomedical application must be of good biocompatibility, and the hemolysis in the blood is one of the key factors of biocompatibility. As shown in Figure 4d, there is little hemolysis (

Black Phosphorus Nanosheets Immobilizing Ce6 for Imaging-Guided

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