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Gold nanospheres-stabilized indocyanine green as a synchronous photodynamic-photothermal therapy platform that inhibits tumor growth and metastasis Wei Li, Hanbo Zhang, Xiaomeng Guo, Zuhua Wang, Fenfen Kong, Lihua Luo, Qingpo Li, Chunqi Zhu, Jie Yang, Yan Lou, Yong-Zhong Du, and Jian You ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13351 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017
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ACS Applied Materials & Interfaces
Gold nanospheres-stabilized indocyanine green as a synchronous photodynamic-photothermal therapy platform that inhibits tumor growth and metastasis
Wei Li1, Hanbo Zhang1, Xiaomeng Guo1, Zuhua Wang1, Fenfen Kong1, Lihua Luo1, Qingpo Li1, Chunqi Zhu1, Jie Yang1, Yan Lou2*, Yongzhong Du1, Jian You1*
1
College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road,
Hangzhou 310058, People's Republic of China 2
Key Laboratory of Precision Diagnosis and Treatment for Hepatobiliary and
Pancreatic Tumor of Zhejiang Province, the First Affiliated Hospital, College of Medicine, Zhejiang University, 79 QingChun Road, Hangzhou 310000, People's Republic of China
*Corresponding Authors: Jian You, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of China; Tel: 086-571-88981651; Fax: 086-571-88208439; E-mail:
[email protected]. Yan Lou, the First Affiliated Hospital, College of Medicine, Zhejiang University, 79 QingChun Road, Hangzhou 310000, People's Republic of China; E-mail:
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ABSTRACT Both photothermal therapy (PTT) and photodynamic therapy (PDT) are phototherapeutic approaches, which have been widely investigated for cancer therapy mediated by external light source. Here, a nanosystem presenting synchronous PTT and PDT effect realized through one-step near infrared (NIR) light irradiation is reported. This system was fabricated by conjugating indocyanine green (ICG) on hollow gold nanospheres (HAuNS) using branched-polyethylenimine (PEI, MW=10 kDa) as optimal linker, which provided a high ICG payload as well as a covering layer with suitable thickness on HAuNS to maintain ICG fluorescence and reactive oxygen species (ROS) productivity. The resulting system (ICG-PEI-HAuNS) had the molar ratio of ICG: PEI: Au=3: 0.33: 5. Compared with free ICG, ICG-PEI-HAuNS exhibited dramatically enhanced stability of ICG molecules and greater intratumoral accumulation. The conjugation of ICG caused significantly higher plasmon absorption of ICG-PEI-HAuNS in the NIR region compared with HAuNS alone, inducing remarkably enhanced photothermal conversion efficiency and synchronous photodynamic effect under NIR light irradiation. Interestingly, compared with PTT or PDT alone, synchronous PTT and PDT produced by ICG-PEI-HAuNS upon NIR light irradiation induced significantly stronger antitumor and metastasis inhibition effects both in vitro and in vivo, which might be a promising strategy for cancer treatment. KEYWORDS: Indocyanine green; Hollow gold nanospheres; Photodynamic therapy; Photothermal therapy; Near-infrared light; Tumor metastasis
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INTRODUCTION Malignant tumor and its metastasis have caused an extraordinary high mortality rate of sufferers, and few of them can be cured by commonly used clinical cancer therapies. 1, 2 Phototherapy has attracted widespread focus in the past decade due to its excellent efficacy in treating local cancer with minimal invasiveness. Photothermal therapy (PTT) and photodynamic therapy (PDT) are two major phototherapeutic approaches that employ various photosensitizers, depending on their light conversion ability to produce localized hyperpyrexia (> 42 °C) or reactive oxygen species (ROS, e.g., singlet oxygen) to kill cancer cells. 3-6 The photosensitizers employed are minimally toxic in the absence of light; thus their accumulation in non-specific or non-irradiated parts of the body causes little systemic toxicity. 7-9 Near-infrared (NIR) light can readily penetrate the skin and go deep into the tissues because its tissue absorption is minimal, 10-13 rendering it highly suitable for in vivo PTT and PDT applications. PTT alone is unlikely to eliminate all tumor cells because the resulting heat distribution is nonuniform, especially in areas near large blood vessels where heat can be rapidly dissipated by circulating blood, which leads to sublethal thermal doses in some areas of the tumor. For PDT alone, the mechanism of which is the generation of reactive oxygen species from tissue oxygen via interactions with the photoactivated agents. 14 However, the oxygen level within tumor tissue can be highly heterogeneous due to dysregulated vascular distribution, leading to severely hypoxic regions where tumor cells could be resistant to PDT, and thus decreasing the efficacy of PDT. 15-19 Recently, combining PTT and PDT to compensate for the drawbacks of PTT or PDT alone in treating cancer has been widely studied. 20-27 Although nanomaterials with surface plasmon resonance (SPR) in the NIR region, such as gold nanospheres 28-30 and gold nanorods, 11, 31 have been frequently investigated for NIR-mediated PTT, few studies have been reported on synchronous PTT and PDT mediated by NIR light. Some researchers have employed up-conversion nanomaterials containing rare earth elements to activate photosensitizers with short-wavelength excitation under NIR laser irradiation. 32-34 Nevertheless, the inherent biotoxicity of these materials limits their future clinical applications. Alternatively, researchers have applied NIR photosensitizers, such as indocyanine green (ICG), for adsorption onto the nanocarriers to obtain the combined effects of PTT and PDT mediated by NIR light. 35-39 However, the physical interaction between ICG and nanoparticles resulted in a high risk of ICG molecules dissociating from the nanoparticles during circulation before arriving at the target site. The leaked ICG molecules were extremely unstable, and prone to be degraded and eliminated from the body, leading to the low efficacy of PDT. 6, 40 In this work, we have developed a combined photothermal and photodynamic therapy system based on ICG and hollow gold nanospheres (HAuNS) for enhanced antitumor efficacy via one-step NIR irradiation. HAuNS have plasmon absorption in the NIR region and display a strong photothermal conducting property. In our previous studies, HAuNS have also been used as a photothermal agent for PTT and a vehicle for delivering biomolecules or triggering drug release under NIR irradiation. 41-44 3 ACS Paragon Plus Environment
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Herein, we grafted branched-polyethylenimine (PEI, MW=10 kDa) onto HAuNS to expand the surface area and provide a covering layer of suitable thickness, which could decrease the fluorescence quenching of ICG caused by HAuNS and increase the stereo stability of HAuNS. Then, ICG molecules were covalently conjugated with PEI to obtain a high payload on HAuNS. The physicochemical properties, biodistribution, antitumor effect and metastasis inhibition property of this nanosystem mediated by NIR light were evaluated. Encouragingly, we found that covalently conjugating ICG on HAuNS could induce improved stability of ICG molecules and increased intratumoral accumulation, presenting synchronous PTT and PDT to achieve remarkably enhanced antitumor and metastasis inhibition effect under NIR light irradiation. RESULTS Characterization of ICG-PEI-HAuNS The fabrication procedures of ICG-PEI-HAuNS were presented in Fig. 1a. The 1 H-NMR spectra showed multiple amine group peaks at approximately 2.5~2.7 ppm in PEI and an increase of sulfhydryl peaks at approximately 2.1 ppm in PEI-HAuNS. The amine peak in PEI-HAuNS completely disappeared after loading ICG while the sulfhydryl peak remained (Fig. s1a). Furthermore, the FTIR spectra showed that the double peaks of amino groups in PEI-HAuNS were greatly weakened after covalent ICG conjugation, while the characteristic peaks of ICG at approximately 800 cm-1~1,500 cm-1 emerged in ICG-PEI-HAuNS (Fig. s1b). These results demonstrated the successful conjugation of ICG and PEI on HAuNS. A polymeric layer was found on the surface of HAuNS for PEI-HAuNS (~8 nm) which became clearer and expanded after the conjugation of ICG to form ICG-PEI-HAuNS (~15 nm) (Fig. 1b and 1c). The average diameters of HAuNS, PEI-HAuNS and ICG-PEI-HAuNS were 50.6±2.4, 106.4±7.2, and 122.5±13.5 nm, respectively, as determined by DLS (Table s1). The zeta potential of HAuNS was changed from -22.40±0.61 mV to 10.98±0.41 mV after PEI conjugation and returned to -4.58±0.78 mV after loading ICG (Fig. 1e and Table s1). The elemental mappings of Au and S in each material were detected by a HR-TEM equipped with an electron energy spectroscope (JEM-2010, JEOL, Japan), which further showed that an increased and more widespread amount of S element was in ICG-PEI-HAuNS because of the hydrosulfide groups in PEI and the sulfonic acid groups in ICG molecules (Fig. 1c and 1d). The ratios of Au and S in each material were calculated according to the peak areas in the energy spectrum (Fig. s2). ICG-PEI-HAuNS was found to have a final molar ratio of ICG:PEI:Au=3:0.33: 5. The ICG content in ICG-PEI-HAuNS was verified to be 35.8% (w/w), which perfectly matched the results acquired from the energy spectrum analysis (Fig. 1d and s2), and the conjugation efficiency was 83.5% (Table s1). The spectra shown in Fig. 1f demonstrated that the absorption peak of ICG-PEI-HAuNS in the NIR region was significantly enhanced and showed an obvious red shift (~880 nm) compared with ICG@HAuNS as a result of the chemical structure change in ICG. Interestingly, PEI linkers with different molecular weights induced significant 4 ACS Paragon Plus Environment
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differences in the ICG fluorescence signal and the stereo stability of our nanosystem. ICG-PEI25k-HAuNS was more prone to aggregating and crosslinking during the synthesis procedure due to the longer branched chain of PEI25k (Fig. s3a), which was confirmed by TEM (Fig. s3d). The long-term storage study showed no significant size change of ICG-PEI-HAuNS over 2 months (Fig. s3b), while ICG-PEI2k-HAuNS exhibited serious aggregation within two days due to the thin covering layer on HAuNS (Fig. s3c). Compared with an equivalent amount of free ICG, ICG fluorescence on the nanospheres was quenched by approximately 35%, 90% and 95% when PEI, PEI2k and PEI25k were used as the linker, respectively (Fig. s4a). The reasons of the stronger fluorescence quenching in ICG-PEI2k-HAuNS and ICG-PEI25k-HAuNS might be the close proximity (
