Indocyanine Green Ternary Hybrid for

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Pea Protein/Gold Nanocluster/Indocyanine Green Ternary Hybrid for Near-Infrared Fluorescence/Computed Tomography Dual-Modal Imaging and Synergistic Photodynamic/Photothermal Therapy Mi Wu, Zhao Li, Jinrong Yao, Zhengzhong Shao, and Xin Chen ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00794 • Publication Date (Web): 26 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019

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ACS Biomaterials Science & Engineering

Pea Protein/Gold Nanocluster/Indocyanine Green Ternary Hybrid for Near-Infrared Fluorescence/Computed Tomography DualModal Imaging and Synergistic Photodynamic/Photothermal Therapy Mi Wu, Zhao Li, Jinrong Yao, Zhengzhong Shao, Xin Chen* State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai 200433, P. R. China. *Corresponding author. Email: [email protected]

ABSTRACT: Developments in technology such as theranostic nanoplatforms, which combine complementary imaging modalities and synergistic therapies, have brought new hope to the diagnosis and treatment of cancer, one of the most deadly diseases. In this article, a nanoplatform (AuNCs/PPI-ICG nanohybrid) with theranostic effect was prepared using indocyanine green (ICG), a cyanine dye with bright near-infrared fluorescence (NIRF) and excellent photodynamic and photothermal performance, and loading it onto the gold nanoclusters/pea protein isolates hybrid nanoparticles (AuNCs/PPI NPs). There was a linear relationship between ICG loading ratio and the concentration, within a concentration range of 05 mg/mL, making it possible to 1

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precisely control the concentration of loaded ICG. In addition, AuNCs/PPI-ICG nanohybrid displayed outstanding loading stability and enhanced aqueous stability of ICG, owing to the protectivity of AuNCs/PPI NPs. In vitro cell experiments showed that this biocompatible nanohybrid could be taken up by A549 cells and used as a fluorescent bioimaging probe. Moreover, because of Au, AuNCs/PPI NPs could also act as a potential computed tomographic (CT) contrast agent, and so the AuNCs/PPIICG nanohybrid had the potential to function as a dual-modal imaging agent. Finally, the as-prepared hybrid, with excellent photodynamic property and exceptional photothermal capability, showed satisfactory therapeutic effect in ablating A549 cancer cells. These results convincingly demonstrate that the AuNCs/PPI-ICG nanohybrid is a promising theranostic nanoplatform for NIRF/CT dual-modal imaging and for synergistic photodynamic therapy/photothermal therapy (PDT/PTT).

KEYWORDS: pea protein isolate, gold nanomaterials, dye, hybrid materials, theranostic nanoplatform

INTRODUCTION Cancer is one of the most deadly diseases, which is a serious threat to human health.1-4 In the past few decades, many researchers have dedicated their work to the conquest of cancer by developing effective diagnostic and treatment methods. It is gratifying that many advances have been made towards precise diagnosis and treatment of tumors, on the basis of various theranostic nanoplatforms, which integrate multi-modal imaging techniques and therapeutic methods.5,6 2


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In recent years, exciting advances in medical imaging techniques have resulted in an upsurge of variety of imaging methods, such as near-infrared fluorescence (NIRF) imaging,7,8 computed tomography (CT) imaging,9 positron emission tomography (PET) imaging,10 single-photon emission computed tomography (SPECT) imaging,11 magnetic resonance imaging (MRI),12 photoacoustic (PA) imaging,13 Raman imaging,14 ultrasound (US) imaging,15 etc. However, any single imaging mode can hardly meet the ideal imaging requirements, due to its own limitations and drawbacks.16 For instance, NIRF imaging has distinct advantages of real-time imaging, high resolution, and superior sensitivity.17 However, it is not useful for distinguishing anatomical structures and its penetration depth is limited to less than 10 mm.18 On the other hand, CT imaging provides high temporal resolution ratio and three-dimensional (3D) anatomic details and has no limitation in tissue penetration depth.19 It is however unsuitable for detecting soft tissues or certain kinds of tumors.20 Moreover, the use of currently available CT contrast agents is severely affected by its fast renal clearance, which not only shortens the blood circulation time, but also causes potential renal toxicity.21,22 Therefore, integration of NIRF imaging and CT imaging techniques can provide a platform with high sensitivity and precise anatomical structure location, which is useful for locating, imaging, and subsequent treatment of different types of tumors.18

After thorough investigation of cancer using different imaging techniques, the ultimate focus would be to completely cure cancer. At present, surgery,23 radiotherapy,24 and chemotherapy25 are the most common treatments available for cancer treatment. However, these treatment methods also have obvious disadvantages that cannot be 3



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ignored. First, the limitation of resection surgery is that it fails to completely eliminate all the tumor cells.26 Second, the limitation of radiotherapy is that due to the resistance of hypoxic cancer cells to ionizing radiation, its efficiency to destroy certain kinds of tumors is less.27 Third, in case of chemotherapy, non-specificity and poor biodistribution of anticancer drugs usually causes severe systemic side effects and undesirable treatment effects.28 Moreover, repeated use of chemotherapeutic drugs can lead to multidrug resistance, which further reduces the curative effects of chemotherapy.27 Therefore, in the past few decades, researchers have shifted their focus to phototherapy, which has higher tumor therapeutic specificity and less side effects, especially photothermal therapy (PTT) and photodynamic therapy (PDT).29-31 PTT uses NIR-absorbing agents to convert light energy into heat energy, creating local hyperthermia for thermal ablation of cancer cells.27, 29 However, in case of PDT specific wavelength of light is used to produce singlet oxygen (1O2) or reactive oxygen species (ROS) by photosensitizer to induce apoptosis of cancer cells.27,29 PTT and PDT have overwhelming advantages over traditional therapies, such as lower toxicity to normal tissues, less systematic side effects, higher tumor specificity, and better tolerance after repeated treatment.30,32 Compared to individual use of PTT or PDT, the combination of both therapeutic modalities results in synergistic effects. On one hand, PTT induced hyperthermia can increase the concentration of ROS in cancer cells by enhancing the cellular uptake of photosensitizer, thereby increasing the vascular saturated O2 concentration.33,34 On the other hand, the transient vasoconstriction induced by PDT enables the extension of hyperthermia time.35 4


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Ideal PDT/PTT agents are expected to exhibit strong absorption in the NIR region (7001100 nm), due to minimum scattering and absorption from soft tissues and maximum penetration depth.32,36 Indocyanine green (ICG), a cyanine dye approved by the U.S. Food and Drug Administration (FDA) in 1959, has been widely employed as an NIR fluorescent agent for clinical diagnosis.37 ICG is also a prospective photosensitizer and photothermal agent when exposed to NIR light.38-40 However, further applications of ICG have been compromised because of its intrinsic drawbacks, such as its poor photostability in aqueous solution and rapid clearance from the body.41,42 With an aim to overcome these problems, ICG has been loaded into various nanocarriers, such as liposomes,37 cell membranes,38,43 polymeric micelles,44,45 nanoparticles,40,46 proteins,47,48 etc. These formulations have rendered ICG with higher photostability, prolonged blood circulation time, increased fluorescence intensity as well as facile modification for systematic delivery.37, 48-50

Elements with high atomic numbers and proper X-ray attenuation coefficients, such as iodine,51 lanthanide,52,55 tantalum,56 gold48,57-59 and bismuth,60 are potential CT contrast agents.18 Among them, gold stands out owing to its extreme inertness and ease of fabrication into different nanostructures that have excellent biocompatibility if properly stabilized.21,61It is noteworthy that gold nanoparticles of smaller size (under 5 nm) are highly effective in enhancing the efficiency of CT imaging as compared to the larger ones.48,62 In our previous work, biocompatible gold nanoclusters (AuNCs), synthesized using pea protein isolates (PPI) as reducing and stabilizing agents, have been successfully synthesized,7 but they only have been concerned and utilized as a 5



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fluorescent probe for tumor imaging. Herein, AuNCs can be also considered as a potential CT contrast agent. More importantly, till date, bovine serum albumin (BSA) has been the most commonly used protein carrier for encapsulation of ICG.16,48,63 Nevertheless, to the best of our knowledge, there has been no report on loading of ICG onto plant proteins (such as PPI), which are obviously more abundant and less expensive. Therefore, in this study, we report a facile route to fabricate AuNCs/PPIICG nanohybrid by employing simple mixing and dialysis techniques. The NIRF and CT imaging properties of this hybrid were evaluated, and subsequently, PDT and PTT performances of this hybrid were tested and applied to assess their synergistic therapeutic effects on cancer cells in vitro.

EXPERIMENTAL SECTION

Materials. PPI powder was purchased from Staerkle & Nagler AG, Switzerland. Chloroauric acid was bought from Sigma-Aldrich. Indocyanine green was procured from MesGen Biotechnology. Other chemicals were also purchased from SigmaAldrich and were used without further purification. Water used in all experiments was deionized through Millipore purification apparatus (resistivity >18.2 MΩ·cm).

Preparation of AuNCs/PPI NPs. The AuNCs/PPI NPs were prepared according to the procedure reported in our previous work.7 In brief, 10.0 wt% PPI solution was mixed with the same volume of 10 mmol/L HAuCl4 solution. After adjusting the pH to 13 using 1 mol/L NaOH solution, the mixture was cultured in a 60ºC water bath and reacted for 30 min under stirring. Then the mixture was transferred into a Visking 6


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dialysis tube with a molecular weight cutoff (MWCO) of 100 Da and dialyzed against deionized water for 3 days to obtain the AuNCs/PPI NPs.

Preparation of AuNCs/PPI-ICG Nanohybrid. Desired amount of ICG was first dissolved in deionized water to obtain an ICG solution. Then the ICG solution was added to an equal volume of as-prepared AuNCs/PPI NPs. The reaction mixture was stirred in dark for 2 h and then dialyzed against deionized water in a Visking dialysis tube (MWCO: 3,000 Da) for 3 days to obtain AuNCs/PPI-ICG nanohybrid.

Characterization of AuNCs/PPI NPs, ICG, and AuNCs/PPI-ICG Nanohybrid. The UV-Vis absorption spectra of the AuNCs/PPI NPs, ICG, and AuNCs/PPI-ICG nanohybrid aqueous solutions were recorded on a Hitachi UV 2910 UV-Vis spectrophotometer, in the wavelength range of 325 to 1000 nm. The fluorescence spectra of the AuNCs/PPI NPs, ICG, and AuNCs/PPI-ICG nanohybrid aqueous solutions were obtained using FLS 920 fluorescence spectrometer, with an excitation wavelength of 750 nm and scanning range of emission wavelength from 770 nm to 850 nm. The morphology of AuNCs/PPI-ICG nanohybrid was observed under a FEI Tecnai G2 20 TWIN transmission electron microscope (TEM) at an operating voltage of 200 kV. The hydrodynamic diameters of AuNCs/PPI NPs and AuNCs/PPI-ICG nanohybrid were determined by dynamic light scattering (DLS) on Zetasizer Nano ZS90. The CT tests on AuNCs/PPI NPs solutions (Au concentration: 0.5, 1.0, 1.5, 2.0, and 2.5 mmol/L) were conducted using X-ray computed tomographic system for small animals (Bruker SkyScan 1176).

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Loading and Releasing of ICG. In the loading experiment, ICG was loaded onto AuNCs/PPI NPs in different mass ratios of ICG to PPI: 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3. After mixing and dialyzing, the loading ratio was determined by measuring the absorption at 800 nm using Hitachi UV 2910 UV-Vis spectrophotometer. In the releasing experiment, 1 mL AuNCs/PPI-ICG nanohybrid solution was placed in a Visking dialysis tube (MWCO: 3,000 Da) against 5 mL PBS or cell medium RPMI 1640 containing 10% (v/v) fetal bovine serum and cultured in a 37ºC water bath for 6, 12, 24, 48, and 72 h. The amount of ICG released into PBS or cell medium was quantified by measuring the absorption at 800 nm using Hitachi UV 2910 UV-Vis spectrophotometer. Assessment of Amount of Singlet Oxygen Generation. The amount of 1O2 generated by AuNCs/PPI NPs and AuNCs/PPI-ICG nanohybrid was measured using 1,3-diphenylisobenzofuran (DPBF) as an 1O2 sensor. Three milliliter of deionized water, AuNCs/PPI NPs solution (Au concentration: 2.5, 5.0, 10, 20, and 50 μg/mL), ICG solution (ICG concentration: 10 μg/mL), or AuNCs/PPI-ICG nanohybrid solution (Au concentration: 5 μg/mL, ICG concentration: 10 μg/mL) were mixed with 100 μL of DPBF solution (0.5 mg/mL, freshly dissolved in DMSO), respectively. After irradiation with 808 nm laser (1 W/cm2) for 0, 2, 4, 6, 8, 10, and 15 min, the absorbances of DPBF solutions were measured at 420 nm.

Assessment of Photothermal Behavior. One milliliter of AuNCs/PPI-ICG nanohybrid solution (ICG concentration: 12.5, 25, 50, and 100 μg/mL) and ICG solution (50 μg/mL) were irradiated with a 808 nm laser (1 W/cm2) for 5 min. The 8


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changes in temperature were determined by an infrared thermal imager (E40, FLIR) with simultaneous acquisition of infrared thermal curves. All experiments were conducted at room temperature (about 25°C).

Cell Viability. The cytotoxicity of the AuNCs/PPI-ICG nanohybrid was evaluated using Cell Counting Kit-8 (CCK-8) assay on human lung adenocarcinoma (A549) cells. A549 cells were first cultured in RPMI 1640 also containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin in a standard cell incubator (37°C, 5% CO2). Then the cells were seeded into a 96-well tissue culture plate with a density of 1×104 cells/well and cultured for 24 h. They were then treated with a series of AuNCs/PPIICG nanohybrid solutions (ICG concentration: 6.25, 12.5, 25, and 50 μmol/L). After another 24 h of incubation, cells were washed thrice with PBS and the cell metabolic viability was assessed by CCK-8 assay.

Fluorescence Imaging. A549 cells were seeded into a 35 mm glass bottom culture dish with a density of 2×105 cells/well and cultured for 24 h. They were then treated with either AuNCs/PPI NPs solution or AuNCs/PPI-ICG nanohybrid solution. After further 4 h of incubation, the cells were washed thrice with PBS, immobilized with 4% glutaraldehyde solution for 15 min and stained with Hoechst 33342 for another 15 min. Finally, the cells were washed again and kept hydrated with PBS. Fluorescence images were observed using an Olympus Fluoview FV 1000 laser scanning confocal microscope (LSCM).

Detection of Intracellular ROS. Dichlorofluorescein diacetate (DCFH-DA) was 9



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used to determine the amount of intracellular ROS by AuNCs/PPI NPs, ICG, and AuNCs/PPI-ICG nanohybrid. Esterase present in the cells hydrolyzed DCFH-DA to DCFH after it entered the cells. Then, in the presence of ROS, DCFH was rapidly oxidized to highly fluorescent dichlorofluorescein (DCF), which could be detected by FITC channel on an LSCM.38-39, 64

A549 cells were seeded into a 35 mm glass bottom culture dish with a density of 2×105 cells/well and cultured for 24 h. They were then treated with AuNCs/PPI NPs solution (Au concentration: 5 μg/mL), ICG solution (ICG concentration: 10 μg/mL), and AuNCs/PPI-ICG nanohybrid solution (Au concentration: 5 μg/mL, ICG concentration: 10 μmol/L). After another 4 h of incubation, cells were washed thrice with PBS, incubated with DCFH-DA for 1 h and irradiated with 808 nm laser (1 W/cm2

) for 5 min. Finally, the intracellular ROS generation was studied using a LSCM.

Evaluation of Phototherapeutic Effect. A549 cells were seeded into a 96-well tissue culture plate with a density of 1×104 cells/well and cultured for 24 h. They were then treated with a series of AuNCs/PPI-ICG nanohybrid solutions (ICG concentration: 2.5, 5.0, 10, and 20 μmol/L). After another 4 h of incubation, cells were irradiated with 808 nm laser (1 W/cm2) for 5 min at 4ºC or 25ºC. After 24 h, the metabolic viabilities of the cells were assessed by CCK-8 assay.

Trypan blue staining was also employed to intuitively observe the phototherapeutic performance of the AuNCs/PPI-ICG nanohybrid (ICG concentration: 20 μmol/L) on A549 cells. Trypan blue solution (0.4%) was added to A549 cells in presence or absence 10


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of 808 nm laser irradiation and the microscopic images of cells were obtained using a microscope (IX71, Olympus). RESULTS and DISCUSSION Preparation and Characterization of AuNCs/PPI-ICG Nanohybrid. ICG, an FDA-approved clinical NIR imaging agent, has in recent years been applied for photodynamic and photothermal treatment of cancer.65-67 According to our previous work, AuNCs/PPI-ICG nanohybrid was prepared by simply adding fresh ICG aqueous solution into an equal volume of AuNCs/PPI NPs solution. The mixture was then stirred in dark for 2 h and dialyzed against deionized water to remove unloaded ICG. After the successful loading of ICG, the hybrid solution turned from yellow to green (Figure S1).

The loading capacity of AuNCs/PPI NPs as an ICG carrier was first investigated. As shown in Figure 1a, in contrast to AuNCs/PPI NPs, which had no obvious absorption between 700900 nm, ICG showed a characteristic peak at 800 nm in UV-Vis absorption spectrum of AuNCs/PPI-ICG nanohybrid, indicating the successful loading of ICG. It is noteworthy that, due to changes in local environment, AuNCs/PPI-ICG nanohybrid showed a small red shift in its absorption maxima, as compared to that of free ICG.68 Moreover, the appearance of an emission peak at 822 nm in the fluorescence spectrum also confirmed the loading of ICG. Importantly, the loading process had hardly made any influence on the fluorescence emission peak of ICG (Figure 1b), which retained its NIR imaging property. Figure S2 shows the TEM image of AuNCs/PPIICG nanohybrid. Compared to AuNCs/PPI NPs,7 the morphology of AuNCs/PPI-ICG nanohybrid was irregular and loose. The reason for such a phenomenon may be 11



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assumed to the fact that both PPI and ICG were negative charged. Therefore, the repulsion of PPI and ICG caused the structure of AuNCs/PPI NPs to be loose, thus showed an irregular morphology. Result of DLS showed a slight increase in the hydrodynamic diameter of AuNCs/PPI NPs after ICG loading (from 262 ±8 to 277±2 nm, Figure 1c), which proved such an assumption. This phenomenon could be another evidence for the successful loading of ICG onto AuNCs/PPI NPs.

Figure 1. (a) UV-Vis-NIR spectra and (b) fluorescence emission spectra of pure ICG, AuNCs/PPI NPs, and AuNCs/PPI-ICG nanohybrid; (c) hydrodynamic diameters of AuNCs/PPI NPs and AuNCs/PPI-ICG nanohybrid.

After confirming the successful loading of ICG onto AuNCs/PPI NPs, the relation between loading ratio and loading concentration was investigated. It can be seen from Figure 2a that the maximum loading ratio was 10.7% for highest ICG loading concentration of 6 mg/mL. However, further increase in ICG loading concentration led to aggregation of proteins and the formation of a precipitate. If the loading concentration was maintained between 05 mg/mL, a linear relation between loading concentration and loading ratio could be established (R2 = 0.999). In other words, the loading amount of ICG could be well controlled by simply changing the loading 12


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concentration within a certain range.

The purpose of this work is to establish a theranostic nanoplatform, which is applicable to a complex in vivo environment, so the stability of AuNCs/PPI-ICG nanohybrid is of vital significance. Hence, the loading stability of ICG in AuNCs/PPIICG nanohybrid was first evaluated. Only about 3% of loaded ICG was released from AuNCs/PPI-ICG nanohybrid when cultured in PBS and RPMI 1640 containing 10% (v/v) fetal bovine serum for 72 h (Figure 2b). This result demonstrated that AuNCs/PPI NPs were excellent carriers of ICG. ICG is highly unstable in aqueous solution and when exposed to light, which can further accelerate its degradation,69,70 Hence, the stability of ICG in aqueous solution before and after loading onto AuNCs/PPI NPs was also determined by storing under normal laboratory conditions (20C and 50% relative humidity). As shown in Figure 2c, without the protection of AuNCs/PPI NPs, the absorption of free ICG was quickly reduced to 25.4% after storage for one week. Contrastingly, in case of AuNCs/PPI-ICG nanohybrid solution, more than 70% of loaded ICG was preserved, indicating remarkably improved aqueous stability. Both experiments proved that AuNCs/PPI NPs were ideal carriers for ICG, which favored the subsequent delivery of ICG to tumor sites.

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Figure 2. (a) The loading ratio of ICG onto AuNCs/PPI NPs versus its loading concentration; (b) the loading stability of AuNCs/PPI-ICG nanohybrid in PBS and RPMI 1640 containing 10% (v/v) fetal bovine serum; (c) time-dependent decrease in absorbance of ICG and AuNCs/PPI-ICG nanohybrid under the normal laboratory conditions over a period of one week.

NIRF Imaging and CT Imaging in vitro. After comprehensive understanding of loading properties and stability, the biocompatibility and imaging performance of AuNCs/PPI-ICG nanohybrid were assessed. Firstly, the cytotoxicity of AuNCs/PPIICG nanohybrid towards A549 cells was evaluated by CCK-8 assay for 24 h (Figure 3a). Due to the inherent biocompatibility of PPI, all groups of A549 cells, even those cultured with ICG concentration up to 50 μg/mL retained relatively high viability. The cytotoxicity results strongly demonstrated the excellent biocompatibility of AuNCs/PPI-ICG nanohybrid, qualifying it for in vivo application.

Since the loading process had no noticeable effect on the fluorescent imaging performance of ICG (Figure 1b), the in vitro intakes of AuNCs/PPI NPs and AuNCs/PPI-ICG nanohybrid by A549 cells were compared. In Figure 3b, after 4 h of incubation, fluorescent signal of AuNCs could be clearly seen in both groups and that 14


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of ICG was distinctly visible in AuNCs/PPI-ICG nanohybrid group, indicating a weak effect of ICG loading on the endocytosis of AuNCs/PPI NPs by A549 cells. The Zstack confocal fluorescence images also demonstrated the internal localization of AuNCs/PPI-ICG nanohybrid in A549 cells (Figure S3). All these results sufficiently conclude that AuNCs/PPI-ICG nanohybrid could be well internalized by A549 cells and thus can be applied as a bioimaging probe in vivo.

Figure 3. (a) Viabilities of A549 cells cultured with different concentrations of AuNCs/PPI-ICG nanohybrid for 24 h; (b) fluorescence images of A549 cells incubated with AuNCs/PPI NPs and AuNCs/PPI-ICG nanohybrid (ICG concentration: 20 μg/mL) for 4 h; (c) X-ray attenuation intensity (HU) of AuNCs/PPI NPs solution at different Au concentrations. 15



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Due to high X-ray attenuation coefficient of Au, the property of AuNCs/PPI NPs as CT contrast agent was determined and demonstrated in Figure 3c. The quantitatively calculated CT value showed a linear relationship with Au concentration, proving it to be a potential CT contrast agent.

In vitro Photodynamic Performance. According to previous researches, BSAsynthesized AuNCs could produce singlet oxygen under UV or NIR irradiation.39,48 Hence, the photodynamic capability of AuNCs/PPI NPs was determined first. DPBF was chosen as the acceptor to verify the production of 1O2.71,72 Under irradiation with 808 nm, the absorption of DPBF at 420 nm decreased obviously with time (Figure 4a). Interestingly, rather than a linear correlation, the amount of 1O2 increased at first and then decreased with attenuation of Au concentration. This phenomenon was attributed to the embedding of AuNCs by PPI. Since the mass ratio of Au to PPI was constant, higher Au concentration implied larger PPI content. In other words, the entanglement of PPI chain was intensified, resulting in deeper and tighter wrapping of AuNCs and thus less exposure of reaction sites. Moreover, higher the concentration of PPI was, more was the absorption of NIR light by PPI instead of AuNCs, leading to lesser generation of 1O2. However, when the AuNCs/PPI NPs solution was adequately diluted, increase in the number of reaction sites and absorbed NIR light compensated for the decrease in AuNCs concentration, consequently displaying an upward trend of 1O2 production. Nevertheless, further dilution of AuNCs/PPI NPs solution caused a drop in 1

O2 generation, due to insufficient number of reaction sites added and absorbed NIR

light in comparison to the decrease in AuNCs concentration. 16


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The effect of ICG on enhancing the photodynamic property of AuNCs/PPI NPs was also studied. As shown in Figure 4b, decrease in DPBF absorption was most evident in the AuNCs/PPI-ICG nanohybrid group, which was a reduction to mere 25% after 15 min of irradiation, as compared to pristine ICG and AuNCs/PPI NPs groups, which showed 49% and 76% reduction, respectively. The loading of ICG onto AuNCs/PPI NPs endowed them with superior photodynamic capability to generate considerable number of 1O2 species under NIR irradiation. This suggested that AuNCs/PPI-ICG nanohybrid was a promising photosensitizer in photodynamic therapy.

Based on the above findings, biocompatibility and endocytosis properties of AuNCs/PPI-ICG nanohybrid were evaluated. Therefore, generation of ROS inside A549 cells was exploited. For this DCFH-DA was chosen as the detective fluorescent probe. In presence of ROS, non-fluorescent DCFH-DA could be oxidized into DCF with green fluorescence.40 Figure 4c shows A549 cells, cultured with AuNCs/PPI NPs, ICG, and AuNCs/PPI-ICG nanohybrid in absence of NIR irradiation, with negligible green fluorescence. In sharp contrast to this, after the exposure to 808 nm laser, a bright green fluorescence was visible in cells incubated with AuNCs/PPI-ICG nanohybrid, indicating the generation of a large number of 1O2 species (Figure 4d). The other two groups also exhibited green fluorescence, but much weaker in intensity, because the photodynamic capability of AuNCs/PPI NPs was unsatisfactory while the uptake of free ICG by cells was poor.40,73 This result was consistent with the above DPBF experiments, confirming the exceptional photodynamic performance of AuNCs/PPIICG nanohybrid. 17



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In vitro Photothermal Performance. ICG is considered to be a good photothermal agent, and so the photothermal property of the as-prepared AuNCs/PPI-ICG nanohybrid was explored. The changes in temperature of ICG solution and a series of AuNCs/PPI-ICG nanohybrid solutions when exposed to 808 nm laser irradiation (1 W/cm2) for 5 min were measured, as shown in Figure 5a. For the same ICG concentration of 50 μg/mL, the temperatures of pure ICG and AuNCs/PPI-ICG nanohybrid solutions were increased by 23.5 and 22.8ºC, respectively. This meant that the photothermal property of ICG was well preserved in the AuNCs/PPI-ICG nanohybrid, suggesting it to be a promising photothermal therapeutic agent. Particularly, such a slight decrease in temperature increment of AuNCs/PPI-ICG nanohybrid compared to pure ICG could be ascribed to two reasons. Firstly, a portion of NIR light absorbed by the AuNCs/PPI-ICG nanohybrid, which was used to generate 1O2, was not completely transformed into heat. The loss of light energy was responsible for decreased photothermal effect. Secondly, the generated 1O2, in turn, accelerated the degradation of ICG, further reducing the photothermal performance.48 To prove the second assumption, decrease in maximum absorbance of ICG under continuous NIR irradiation for 10 min was measured (Figure S4). As expected, the degradation of loaded ICG in AuNCs/PPI-ICG nanohybrid was faster than that of free ICG when exposed to 808 nm laser irradiation. However, despite the slightly accelerated degradation, photothermal performance of AuNCs/PPI-ICG nanohybrid was hardly affected within 5 min, which meant the AuNCs/PPI-ICG nanohybrid was still fully qualified to be a photothermal agent for subsequent cancer therapy. 18


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Figure 4. (a) Decrease in DPBF absorbance at 420 nm in AuNCs/PPI NP solutions with different Au concentrations under 808 nm laser irradiation (1.0 W/cm2) for 15 min; (b) decrease in DPBF absorbance at 420 nm in AuNCs/PPI NP (5 μg/mL), pure ICG (10 μg/mL), and AuNCs/PPI-ICG nanohybrid (Au concentration: 5 μg/mL, ICG concentration: 10 μg/mL) solutions under 808 nm laser irradiations (1.0 W/cm2) for 15 min; (c and d) confocal images of A549 cells cultured with AuNCs/PPI NPs, ICG, and AuNCs/PPI-ICG nanohybrid for 4 h, followed by without (c) and with (d) NIR laser irradiation (1.0 W/cm2).

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In vitro Phototherapeutic Property. Considering the fact that the biocompatibility and PDT/PTT properties of AuNCs/PPI-ICG nanohybrid were good, its in vitro phototherapeutic property against A549 cells was assessed using CCK-8 assay. Figure 5b shows the effects of PDT at 4ºC and synergistic effects of PDT/PTT at 25ºC. When PDT was used alone, the effect of treatment was not significant, especially at lower ICG concentration. For instance, the cell viability remained at 76% with ICG concentration of 10 μg/mL. In contrast, the implementation of PDT/PTT treatment showed greater advantage in ablating cancer cells with remarkable decrease in cell viability up to 41% and 12% at ICG concentrations of 10 and 20 μg/mL, respectively.

The effect of AuNCs/PPI-ICG nanohybrid in cell destruction could also be intuitively observed after staining with trypan blue, in which dead or injured cells with incomplete cell membranes were stained and appeared as blue spots, whereas live cells remained colorless.74 Figure 5c shows that only cells cultured with AuNCs/PPI-ICG nanohybrid and irradiated by 808 nm laser simultaneously were stained by trypan blue, which is consistent with the results of CCK-8 assay. Therefore, the AuNCs/PPI-ICG nanohybrid could efficiently ablate cancer cells, indicating outstanding synergistic PDT/PTT effect.

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Figure 5. (a) Photothermal effect of AuNCs/PPI-ICG nanohybrid (ICG concentration: 12.5, 25, 50, and 100 μg/mL), pure ICG (concentration: 50 μg/mL), AuNCs/PPI NP, and PBS solution using 808 nm NIR laser irradiation for 5 min; (b) effects of PDT and PTT of AuNCs/PPI-ICG nanohybrid against A549 cells using 808 nm NIR laser irradiation for 5 min; (c) optical microscopic images of trypan blue stained A549 cells in the presence of AuNCs/PPI-ICG nanohybrid. The intensity of irradiation energy of the 808 nm laser was 1 W/cm2.

CONCLUSIONS In this work, a facile route for the preparation of AuNCs/PPI-ICG nanohybrid using AuNCs/PPI NPs as ICG carriers was presented. For an ICG loading concentration within the range of 05 mg/mL, the loading ratio of ICG in nanohybrid had a linear relation with the loading concentration (R2 = 0.999). The as-prepared AuNCs/PPI-ICG nanohybrid displayed superior ICG loading stability, with only about 3% of ICG 21



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releasing into PBS or RPMI 1640 after 72 h of incubation. In addition, the aqueous stability of ICG was also remarkably improved due to the protectivity of AuNCs/PPI NPs. Due to outstanding biocompatibility of PPI, AuNCs/PPI-ICG nanohybrid could be successfully used as a fluorescent bioimaging probe in vitro. It showed that the loading of ICG almost did not affect the fluorescence of AuNCs/PPI NPs. Moreover, Au in AuNCs/PPI NPs acted as a potential CT contrast agent, endowing AuNCs/PPIICG nanohybrid with NIRF/CT dual-modal imaging property. Subsequent assessments of therapeutic effects of nanohybrid showed exceptional photodynamic performance and excellent photothermal property, with AuNCs/PPI-ICG nanohybrid showing an effective ablation of A549 cells at low ICG concentration. Therefore, such a highly biocompatible theranostic nanoplatform combining NIRF/CT dual-modal imaging with PDT/PTT synergistic therapeutic effects has a bright future in the field of cancer diagnosis and treatment. ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/***.

Images of AuNCs/PPI NPs and AuNCs/PPI-ICG nanohybrid solutions; TEM image of AuNCs/PPI-ICG nanohybrid; a series of 9-step z-stack confocal fluorescence images of A549 cells incubated with AuNCs/PPI-ICG nanohybrid (ICG concentration: 20 μg/mL) for 4 h (step = 1 μm, ICG channel only); the decrease of 22


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ICG absorbance of AuNCs/PPI-ICG nanohybrid and pure ICG using 808 nm NIR laser irradiation (1 W/cm2) (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

M. W. and Z. L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (No. 21574023 and 21574024). We thank Dr. Yuhong Yang for her valuable suggestions and discussions.

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For Table of Contents Use Only

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Mi Wu, Zhao Li, Jinrong Yao, Zhengzhong Shao, Xin Chen*

35


Indocyanine Green Ternary Hybrid for

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