Coordinatively Self-Assembled Luminescent Gold Nanoparticles

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Coordinatively Self-Assembled Luminescent Gold Nanoparticles: Fluorescence Turn-On System for High-Efficiency Passive Tumor Imaging Xuandi Lai, Lishan Tan, Xiulong Deng, Jinbin Liu, Aiqing Li, Jianyu Liu, and Jianqiang Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14681 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 23, 2017

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ACS Applied Materials & Interfaces

Coordinatively Self-Assembled Luminescent Gold Nanoparticles: Fluorescence Turn-On System for High-Efficiency Passive Tumor Imaging Xuandi Lai,a,‡ Lishan Tan,b,‡ Xiulong Deng,a Jinbin Liu,a Aiqing Li,*b Jianyu Liu,a and Jianqiang Hu*a a

Department of Chemistry, College of Chemistry and Chemical Engineering, South China

University of Technology, Guangzhou, 510640, China b

State Key Laboratory of Organ Failure Research, Nanfang Hospital, Southern Medical

University, Guangzhou, 510515, China KEYWORDS: self-assembly, nanoparticles, cancer, fluorescence turn-on system, passive tumor imaging

ABSTRACT: A fluorescence turn-on system for highly efficient and prolonged tumor imaging has been established by Co2+-induced coordination self-assembly strategy, in which luminescent glutathione (GSH)-modified gold nanoparticles (LGAuNPs) are assembled into LGAuNPs assemblies (LGAuNPs-Co) through coordination bond between unoccupied orbit of Co2+ and lone pair electrons of GSH on the surface of LGAuNPs. The LGAuNPs-Co is sensitive to

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microenvironment pH and its quenched luminescence will be turned on in tumor tissues (acidic microenvironment), which behaves as a fluorescence turn-on system for passive tumor imaging. The fluorescence turn-on system combines advantages of enhanced permeability and retention (EPR) effect of NPs and pH-induced fluorescence turn-on property in tumor site, which results in larger fluorescence intensity (FI) difference between normal and tumor tissues as compared with that of luminescent Au NPs (LAuNPs, only with EPR effect) (~12-fold). Such large FI difference results in that LGAuNPs-Co has rapid (~1.6 h), persistent (~24 h p.i.) and highly efficient tumor targeting capability in comparison with LGAuNPs. Moreover, the LGAuNPs-Co also has much longer tumor retention, faster renal clearance and lower eticuloendothelial system (RES) uptake than LGAuNPs. Therefore, the fluorescence turn-on system is very promising for cancer diagnosis and therapy.

1. INTRODUCTION Nanoparticles (NPs)-based fluorescent imaging has attracted extensive attention due to its promising applications in early-stage cancer diagnosis and therapy.1-4 Compared to traditional organic contrast agents, inorganic nanomaterials tend to provide a higher imaging efficiency due to their flexible optical properties, easy surface functionalization and superior biocompatibility.1,5 To achieve NPs tumor imaging, active and passive targeting modes have been established.6-8 By mean of active targeting process, NPs can be precise retention and uptake in targeted sites through selecting specific affinity ligand (e.g., proteins, peptides, nucleic acids, etc.).9-11 In contrast to the active targeting which requires specific affinity ligands, the passive mode can largely broaden recognized tumor species in virtue of enhanced permeability and retention (EPR)


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effect of NPs in tumor site.12,13 The EPR effect can make NPs easily accumulate in tumor sites at much higher concentrations and for longer time than in normal organs or tissues. To achieve tumor imaging in passive targeting, it is usually required to introduce luminescent element for visualizing NPs EPR effect. This can be achieved through designing fluorescence switch triggered by specific reagents (e.g., enzymes) because only tumor site can make the “off” fluorescence release.14-16 However, the luminescent signal of the fluorescence switch depends largely on foreign luminescent probes (usually small organic molecules), which easily induces unpredictable system toxicity.17,18 Therefore, it is desirable to fabricate fluorescent NPs for passive tumor targeting. Chen and his co-workers successfully synthesized near infrared spectroscopy (NIR) quantum dots (QDs) which easily accumulated in solid tumor.19 The tumor uptake could be up to about 13.5%ID g-1. However, they also showed severe reticuloendothelial system (RES) uptake in liver (~35%ID g-1) and spleen (~12%ID g-1). To escape from RES retention and minimize nonspecific accumulation, NIR-emitting ultrasmall AuNPs have recently been developed.20,21 The NIR-emitting AuNPs have much longer tumor retention time and less accumulation in RES organs compared to small dye molecules. However, it is still a tremendous challenge for the NIR-emitting AuNPs to attain high-efficient passive tumor imaging due to its relatively low fluorescence intensity (FI) difference between normal and tumor sites. In this study, we constructed a fluorescence turn-on system for highefficiency and prolonged passive tumor imaging through Co2+-induced coordinative selfassembly of luminescent glutathione (GSH)-modified AuNPs (LGAuNPs). The fluorescence turn-on system combined the merits of NPs EPR effect and pH-induced fluorescence turn-on property in tumor site. The LGAuNPs assemblies (LGAuNPs-Co) whose fluorescence was partly quenched due to the Co2+-induced self-assembly would be “unlocked” and regain partly the


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quenched fluorescence when reached tumor site (acidic microenvironment). Therefore, as compared with that from luminescent AuNPs only having EPR effect, FI difference between normal and tumor tissues from the fluorescence turn-on system would be greatly enhanced. By conducting a direct comparison of LGAuNPs and LGAuNPs-Co, we were able to demonstrate that the LGAuNPs-Co had rapid, persistent and highly efficient tumor imaging ability and fast renal clearance: the FI was significantly higher in tumor site than surroundings 3 h post injection (p.i.) of LGAuNPs-Co, and this predominance could last long for 24 h. Ex vivo organ imaging results also showed that the tumor FI was much stronger than other organs for LGAuNPs-Co-injected mice within 12 h, while for LGAuNPs, FI difference between tumor and other organs was very low throughout the observation period. Moreover, the LGAuNPs-Co showed relatively faster renal clearance compared with LGAuNPs. This prolonged and highefficiency fluorescence turn-on system triggered by minor pH changes in vivo provides a foundation for the design of a new generation of tumor imaging reagents for clinic applications. 2. EXPERIMENTAL SECTION 2.1. Materials and Instruments. Hydrogen tetrachloroaurate (HAuCl4·3H2O) and reduced glutathione (GSH) was purchased from Sigma-Aldrich (USA). Bovine serum albumin (BSA), 1ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (sulfo-NHS) were obtained from Sangon Biotech Co., Ltd (Shanghai, China). Other chemicals were of analytical grade and used as received without further purification. A549 (human lung adenocarcinoma), HepG2 (human hepatocarcinoma) and 4T1 (human breast adenocarcinoma) cancer cell lines and LO2 (human embryo liver cell strand) normal cell were obtained from


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American Type Culture Collection (Manassas, VA, USA). Milli-Q water (18.2 MΩ·cm) was used to prepare all aqueous solutions. The

luminescence

spectra

were

collected

by

a

Hitachi

F-4500

Fluorescence

Spectrophotometer (Japan). Hydrodynamic size of AuNPs in aqueous solution was analyzed by a Malvern Nano-ZS Laser Particle Size Analyzer (UK). Transmission electron microscopy (TEM) and energy disperse spectroscopy (EDS) measurements were performed with a 200 kV JEOL 2010-F transmission electron microscope (Japan). Ultraviolet-visible (UV-vis) absorption spectra were recorded using a Hitachi U-3010 UV-vis spectrophotometer. Thermo gravimetric analyzer (TGA) curves were obtained from a Q600 SDT DTA-TG thermal analyzer (USA). Pharmacokinetics studies were performed by a Leeman Prodigy XP inductively coupled plasmaatomic emission spectrometry (ICP-AES, USA) and an Agilent 7700S inductively coupled plasma-mass spectrometry (ICP-MS, USA). Fluorescent microscope and hematoxylin and eosin (H&E) staining images of cells and animals were captured through a Zeiss digital camera Axio Imager (Germany). Tumor imaging in vivo and in vitro was collected by Kodak in-Vivo Imaging System F (USA). Cell viability was measured by a Thermo Fisher Multiskan GO UV/visible microplate spectrophotometer (Finland) and cell counting kit-8 (CCK-8, Kumamoto, Japan). Cell optical density (OD) was measured at 450 nm using a microplate reader (F-2500 Fluorescence Spectrophotometer, HITACHI). 2.2. Synthesis of Luminescent AuNPs (LAuNPs). The LAuNPs were prepared according to a previously “green” method.22 In a typical procedure, aqueous HAuCl4 solution (5 mL, 10 mM, 37 °C) was added to BSA solution (5 mL, 50 mg·mL-1, 37 °C) under vigorous stirring. Then, the mixture was bubbled with N2 for 30 min to eliminate O2 dissolved in the solution. After 2 min,


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NaOH solution (0.5 mL, 1 M) was introduced. Finally, the LAuNPs were obtained through proceeding the reaction for 12 h under 37 °C and vigorous stirring. 2.3. Synthesis of LGAuNPs. The as-prepared LAuNPs were modified with GSH through cross-linking reaction between –COOH residues of the BSA on the surface LAuNPs and –NH2 residues of GSH. Firstly, GSH (20.0 mg), EDC (18.8 mg) and sulfo-NHS (28.0 mg) were dissolved in 10 mL ultrapure water, which was incubated in dark under 50 °C for 12 h. Then, the activated GSH solution was cooled naturally and added to the freshly prepared AuNPs colloid under vigorous stirring (dark, 0 °C for 12 h) to form LGAuNPs. Finally, the mixture solution was dialyzed (13 KD MWCO) in 2 L ultrapure water for 24 h, and desiccated in the freeze drier and stored for the following experiments. 2.4. Co2+-Directed Coordination Self-Assembly of LGAuNPs. Briefly, 200 μL 10 mM Co2+ aqueous solution was added into 1 mL 50 mg·mL-1 LGAuNPs colloid under ultrasound. Then, the mixture was incubated for 30 min at room temperature. Finally, the self-assembly solution was diluted with ultrapure water, pH 7.4 phosphate buffer saline (PBS) and pH 6.4 PBS to 5 mg·mL-1, named as LGAuNPs-Co, LGAuNPs-Co_7.4 and LGAuNPs-Co_6.4, respectively. 2.5. Cell Culture and Cytotoxicity Assessment. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, high glucose, Gibco) supplemented with 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 mg/mL) (obtained from Sigma Chemical) and conditioned at 37 °C with 5% CO2 in a 95% humidified atmosphere. Then, these cultured cells in log phase were trypsinized and subsequently seeded in 96-well plates. After cultured for 24 h again, these cells were treated with various concentrations of LGAuNPs-Co in the range of 0.1~10 mg·mL-1 for 6, 12 and 24 h, respectively. Finally, each well of the plate was added with


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10 µl of the tetrazolium substrate, and incubated at 37 °C for 2 h. The cell survival ratio was calculated according to the following equation:23 cell survival ratio = (OD experiment - OD blank)/(OD control - OD blank)]×100%. All experiments were done in triplicate and repeated three independent times and a set of samples without adding NPs were considered as the control groups. 2.6. Cell Fluorescence Imaging. Typically, HepG2 and LO2 cells were cleaved using trypsin-EDTA (0.05% at 37 °C), and planted into cover-glass chambers for 24 h with a density of 5×104 cells. Then, 100 μL 50 mg/ml LGAuNPs or LGAuNPs-Co were introduced into each well and incubated for 1, 8 or 24 h, respectively. The cells were washed with PBS for 5 times and then fixed with the mixture of acetone: methanol=1:1 for 10 min at room temperature. Finally, the cells were stained with 4', 6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature in the dark and mounted with mounting media (Sigma-Aldrich, USA). Each cell lines were performed at least three times with three wells per group. To quantify the uptake amount of LGAuNPs or LGAuNPs-Co in each cell line, HepG2 or LO2 cells incubated with LGAuNPs or LGAuNPs-Co for 1, 8 or 24 h, respectively, were collected, digested with aqua regia and diluted with ultrapure water for ICP-AES analysis. 2.7. Toxicity Evaluation in vivo. The animal experiments were performed according to the guidelines of the Animal Research Ethics Board of Southern Medical University. For the system toxicity studies, female Balb/c mice of 6-8 weeks old were weighted and randomly divided into three groups with three mice per group: LGAuNPs (0.5 mg/g body weight), LGAuNPs-Co (0.5 mg/g body weight) or PBS (10 µL/g body weight). Their body weights were monitored every other day for 3 weeks p.i. The control group was injected with equivalent volume of PBS.


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2.8. Construction of the Xenograft Tumor Mouse Model. Three-week-old female nude mice were purchased from Southern Medical University Health Science Center. All experiments involving mice were approved by the National Institute of Biological Science and Animal Care Research Advisory Committee of Southern Medical University and conducted by following the guidelines of the Animal Research Ethics Board of Southern Medical University. All animals were maintained in a specific pathogen free (SPF) house at 24 ± 2 °C with a standard 12-h light/12-h dark cycle. The animals were allowed free access to tap water and food in the form of a standard pellet diet. Subcutaneous tumor models were generated by the subcutaneous inoculation (0.20 mL volume containing 5×106 cells/mL media) of HepG2 cells in the right side of their hind legs using a 1-mL syringe with a 25G needle. Tumor growth was monitored until a palpable size of about 6-8 mm in length was reached in any direction. 2.9. In vivo and ex vivo Tumor Imaging. HepG2 tumor bearing mice used for in vivo tumor imaging were anesthetized using pentobarbital sodium for 5 min prior to intravenous (i.v.) injection. The imaging parameters for LGAuNPs and LGAuNPs-Co were described as follows: Excitation: 535/10 nm; Emission: 600/20 nm; Exposure time: 30 s. After injecting 200 µL 50 mg/mL LGAuNPs or LGAuNPs-Co, the mice were imaged at different time points. The imaging parameters were kept all the same at each time point. The CI was measured according to the formula:24 CI = (FI of tumor region - FI of mouse autofluorescence)/(FI of normal contralateral region - FI of mouse autofluorescence). For ex vivo organ imaging, major organs (heart, liver, spleen, lung and kidney) and tumor of the intervened mice were dissected and collected 1, 8 and 12 h p.i. of LGAuNPs or LGAuNPsCo, respectively, followed by immediately imaging with in vivo imaging system.


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2.10. Pharmacokinetics Studies. After i.v. injection of 100 μL 100 mg/mL LGAuNPs or LGAuNPs-Co, the blood and urine samples of nude mice without tumor were collected at 10 and 30 min and 1, 4, 8, 12 and 24 h. To accurately quantify the accumulation amount of AuNPs in major organs, tumor, heart, liver, spleen, lung and kidneys of mice 1 h after injection with LGAuNPs or LGAuNPs-Co were collected and freeze-dried. The gold concentrations of these samples were measured using ICP-AES and ICP-MS described as follows: every blood or urine sample was weighted and completely lysed for 24 h using 500 μL freshly prepared aqua regia in a centrifugation plastic tube with lid (2 mL). The samples were then heated at 100 °C in a water bath until the aqua regia was completely evaporated. The residues were diluted with deionized water to 5 mL and analyzed by ICP-AES and ICP-MS. 3. RESULTS AND DISCUSSION 3.1. Co2+-Induced Assembly of LGAuNPs and Its Fluorescence Turn-On Mechanism for in vivo Tumor Imaging. Schematic diagrams of synthesis and Co2+-induced assembly of LGAuNPs and its fluorescence turn on mechanism for in vivo tumor imaging are presented in Figure 1. As shown in Figure 1A, LAuNPs were firstly fabricated by the reduction of Au3+ ions with bovine serum albumin. Then, the LAuNPs were modified with GSH to increase unpaired electron groups (i.e., -SH). Thus, the LGAuNPs with plentiful -SH groups could be assembled in the induction of Co2+ ions in neutral condition by coordination interaction, which would result in that fluorescence of LGAuNPs was partly quenched.25 Nevertheless, the assembly would be partly untied in acidic environment probably because of acid effect of -SH groups.26 At this moment, the quenched fluorescence would partly regain. By taking advantages of EPR effect of NPs and different microenvironments (neutral and acidic microenvironments in normal and tumor tissues, respectively) in in vivo tissues, the


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LGAuNPs-Co could serve as a fluorescence turn-on system for in vivo tumor imaging (Figure 1B). After injecting LGAuNPs-Co into tumor bearing mice via tail vein, it would spread throughout the mice along with blood. Because of the EPR effect of NPs and acidic microenvironment of tumor tissues, LGAuNPs-Co could accumulate in tumor site and partly disintegrate due to acid effect. Thus, the fluorescence turn-on system combined advantages of NPs EPR effect and pH-induced fluorescence turn-on property in tumor site, which resulted in larger FI difference between normal and tumor tissues as compared with that of luminescent Au NPs (only with EPR effect). 3.2. Size, Surface Modification and Stability Characterizations of the LAuNPs and LGAuNPs. The synthesized LAuNPs had the sizes of 2.7 ± 0.3 nm measured by TEM and 3.6 ± 0.8 nm obtained from DLS (hydrodynamic diameter (HD), Figure 2A,C). Comparatively, the GSH-modified LAuNPs showed nearly same TEM-evaluated size (2.8 ± 0.3 nm, Figure 2B) and larger HD (4.8 ± 0.6 nm, Figure 2C) than those of the LAuNPs, indicating that GSH was successfully modified onto the surface of the LAuNPs. LGAuNPs stock solution yielded bright red fluorescent under UV light (inset of Figure 2D) and the luminescent properties of LGAuNPs with an emission peak at approximately 650 nm and an excitation maximum at around 540 nm (Figure 2D) was nearly identical to those for the AuNPs (Figure S1), thus indicating that GSH modification did not affect its luminescence property.27 Except for the evaporation of water before 100 °C, there was only one weight-loss platform for the LAuNPs, whereas the LGAuNPs had two weight-loss platforms (Figure 2E,F). Relative to fourier transform infrared spectroscopy (FTIR) spectra of LAuNPs, there appeared an obvious characteristic vibration peak at nearly 2520 cm-1 of -SH in the LGAuNPs (Figure S2).28 ICP-AES analysis showed that LGAuNPs contained less gold (2.3%, w/w) in comparison with the AuNPs


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(3.6%). These further demonstrated that GSH was successfully modified onto the surface of the LGAuNPs. Moreover, the LGAuNPs also had good fluorescence stability in different media and pH solutions (Figure S3). 3.3. Morphology and Luminescent Property Characterization of LGAuNPs-Cos. It is well known that normal physiological pH for human is generally neutral (pH≈7.4) but its microenvironment will change into weakly acidic when human is in tumor or inflammatory diseases.29 To mimic microenvironments of normal and tumor tissues in vitro, PBS buffer solution at pHs 7.4 and 6.4 were used.30,31 The LGAuNPs-Co_7.4 was acquired through forming four-coordination of Co2+ with the GSH on the surface of LAuNPs (Figures S3,4). The average HD of LGAuNPs-Co_7.4 was 12.2 ± 3.6 nm, which could be inferred that the LGAuNPs-Co_7.4 were assembled with several LGAuNPs (Figure 3A,C). Whereas, the average HD of LGAuNPsCo_6.4 had only 7.5 ± 1.8 nm, which was because these assemblies were partly untied in acidic microenvironment (Figure 3B,C). The FI of LGAuNPs-Co_6.4 was ~65% higher than that of LGAuNPs-Co_7.4 (Figure 3D), which was probably attributed to the turn-on of the quenched fluorescence. Moreover, the fluorescence turn-on/off properties of LGAuNPs-Co could switch between acidic and neutral microenvironment due to its GSH acid effect. This will be an attractive characteristic for improving FI difference between tumor and normal tissues and thus showing high tumor imaging efficiency. 3.4. Biocompatibility and Cell Imaging and Uptake Efficiency of LGAuNPs and LGAuNPs-Co. To estimate biocompatibility and cell imaging and uptake efficiency of LGAuNPs and LGAuNPs-Co, three cancer cell lines (human hepatocarcinoma (HepG2), human lung adenocarcinoma (A549), human breast adenocarcinoma (4T1)) and a normal cell line (human embryo liver cell strand (LO2)) were incubated with LGAuNPs or LGAuNPs-Co. Both


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cancer and normal cells had high cell viability (>80%) in wide concentration (0.0-10.0 mg/mL) and time (6-24 h) ranges (Figure 4), suggesting that these LGAuNPs and LGAuNPs-Co had good biocompatibility. As showed in Figure 5A,B, the average FI in the HepG2 cells was nearly twice stronger than that in the LO2 cells after 8 h of incubating with LGAuNPs-Co, whereas no obvious difference was observed between the two cell lines when incubated with LGAuNPs. The FI difference between HepG2 and LO2 cells after incubating with LGAuNPs-Co was about 1.80, while the difference was only about 0.15 for LGAuNPs, in which the FI difference was defined as: (FI of HepG2 - FI of LO2)/FI of LO2. The FI difference between tumor and normal tissues of the fluorescence turn-on system (LGAuNPs-Co) was about 12-fold larger than that of the LGAuNPs and the fluorescence turn-on property could be only triggered in tumor cells (Figure S6), which was very useful for tumor imaging and diagnosis. EDS and ICP-AES were performed to measure cell uptake capability of LGAuNPs and LGAuNPs-Co (Figure 5C and Figure S7). The existence of Au indicated that LGAuNPs or LGAuNPs-Co were easy uptake in cell cytoplasm through endocytosis. Compared to the LGAuNPs, the uptake of LGAuNPs-Co was easier and faster by tumor cells and had longer retention time. Meanwhile, in normal cells, the LGAuNPs-Co was excreted faster than the LGAuNPs. Moreover, Figure S8 further demonstrated that the LGAuNPs-Co had rapid tumor cell recognition (within 1 h), persistent tumor cell accumulation (more than 24 h) and fast normal cell elimination (within 8 h). The rapid recognition and persistent retention in tumor cells and relatively rapid excretion in normal cells greatly improve the targeting ability of LGAuNPs-Co in tumor imaging.


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3.5. In vivo Tumor Imaging and Pharmacokinetics and Renal Clearance Kinetics of LGAuNPs and LGAuNPs-Co. It is requisite to evaluate systemic toxicity of LGAuNPs and LGAuNPs-Co through investigating changes in body weight and histological sections of LGAuNPs or LGAuNPs-Co treated mice before in vivo tumor imaging. For a 3-week observation period, mice in all groups appeared healthy and groomed, and maintained stable body weight (Figure S9). Then mice were sacrificed and their major organs (heart, liver, spleen, lung and kidney) were collected for histological sections. The HE staining results indicated that there was no obvious histological difference of major organs among the three groups (Figure S10). The stable body weight and good histological sections revealed that LGAuNPs and LGAuNPs-Co had no systemic toxicity on living body. To compare in vivo tumor targeting specificity and imaging efficiency of LGAuNPs and LGAuNPs-Co, mice bearing HepG2 tumors were used as a real-time imaging model (Figures S11,S12). After intravenous (i.v.) injection of LGAuNPs or LGAuNPs-Co, the luminescent NPs dispersed into the whole body within 30 min (Figure S13). As time went on, the tumor sites gradually became distinguishable and the difference of the imaging efficiency rapidly increased between LGAuNPs and LGAuNPs-Co (Figure 6A). Tumor area could be defined in the mouse injected with LGAuNPs-Co at almost 1.0 h p.i. and till 24 h p.i., whereas for the mouse injected with LGAuNPs, the tumor was discerned only within a narrow time range (from 3 h to 5 h p.i.). Contrast index (CI) is a general parameter used in tumor imaging to distinguish from tumor and normal tissues, and a CI value of 2.5 is generally considered to be the threshold for substantial tumor targeting.20,21,32 As showed in Figure 6B, the LGAuNPs and LGAuNPs-Co took 2.1 and 1.6 h, respectively, to reach a CI value of 2.5, which was much faster than the renalclearable and NIR-emitting luminescent AuNPs (around 3.1 h), indicating that both LGAuNPs


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and LGAuNPs-Co had rapid tumor imaging capability. However, the LGAuNPs only had a narrow time interval (ca. 2.1-7.7 h) of identifying tumor, which was much narrower than the NIR-emitting luminescent AuNPs (~3.0-24 h).20,21 This was perhaps due to the stronger fluorescence penetration ability of the NIR-emitting AuNPs. But, different from LGAuNPs, the LGAuNPs-Co not only had rapid tumor imaging ability but also could maintain CI value over 2.5 from ~1.6 to ~24 h p.i., whose time span (~22.4 h) was 4-fold as long as that of the LGAuNPs (~5.6 h). This very long time span for discriminating tumor site revealed that the LGAuNPs-Co had significant advantage in tumor diagnosis and even therapy. The CI kinetics difference fundamentally arose from whether the LGAuNPs or LGAuNPs-Co had fluorescence turn-on effect. The time-dependent emission intensities from the tumors also implied the difference in the tumor targeting by the LGAuNPs-Co and LGAuNPs (Figure 6C). The area under the curves (AUC) for LGAuNPs and LGAuNPs-Co were about 310 and 1200, respectively, which reflected that the tumor resolution efficiency for LGAuNPs-Co was around 3 times stronger than that for LGAuNPs. More than 65% of the maximum FI from the LGAuNPs-Co remained in tumor site 24 h p.i. In sharp contrast, only approximately 4.0% FI from the LGAuNPs remained in the tumor 24 h p.i., indicating that the tumor retention of LGAuNPs-Co was much longer than that of LGAuNPs. We also compared pharmacokinetics and renal clearance of LGAuNPs and LGAuNPs-Co in tumor bearing nude mice. The pharmacokinetics in the mice at selected p.i. time points showed that distribution half-lives (t1/2α) for LGAuNPs and LGAuNPs-Co were 213 min and 135 min, respectively (Figure 7A), indicating that both LGAuNPs and LGAuNPs-Co had enough prolonged blood retention for tumor accumulation. At the same time, the integral AUC of the


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urine excretion showed that the excretion amount of LGAuNPs-Co was nearly 2-fold higher than that of LGAuNPs in the initial 4 h (Figure 7B). Moreover, the accumulation amount of Au3+ in major organs collected from mice 1 h after injection with LGAuNPs or LGAuNPs-Co revealed that LGAuNPs were trapped much more than LGAuNPs-Co in liver and lung organs (Figure S14).33 These results indicated that LGAuNPs-Co had faster renal clearance and lower RES uptake compared with LGAuNPs. Ex vivo tumor-imaging efficiency measurement was performed by sacrificing mice postinjected with LGAuNPs or LGAuNPs-Co at 3, 8 and 12 h, and collecting major organs for fluorescence imaging. Consistent with in vivo imaging results, LGAuNPs-Co showed significant advantage over LGAuNPs in tumor detection (Figure 8). Both tumors collected from 3 h p.i. of LGAuNPs and LGAuNPs-Co mice were brighter than other organs, which meant that tumors were distinguishable by these two kinds of NPs at this moment. However, the tumor fluorescence became weaker and weaker and even showed no difference with other organs after 8- and 12-h injection of LGAuNPs (Figure 8A,B). In contrast, the tumors still maintained strong fluorescence till 12 h p.i. of LGAuNPs-Co and tumor fluorescence was much stronger than other organs during this period, especially at 8 h (Figure 8A,C). Remarkably, the tumor fluorescence from 8 h p.i. of LGAuNPs-Co was stronger than those from 3 and 12 h p.i. This was because that there existed a balance between the disassembly of LGAuNPs-Co (fluorescence turn-on system) and the metabolizing of LGAuNPs and LGAuNPs-Co at tumor tissues. The persistent and strong fluorescence at tumor site would be very helpful for tumor diagnosis and therapy. 4. CONCLUSIONS


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In summary, we have established a fluorescence turn-on system for high-efficiency tumor imaging by Co2+-induced coordinatively self-assembling LGAuNPs into LGAuNPs-Co. The LGAuNPs-Co would be turned on in tumor site and release partly the quenched luminescence, thus generating a fluorescence turn-on system for passive tumor imaging. The fluorescence turnon/off properties of LGAuNPs-Co could switch between acidic and neutral microenvironment due to its GSH acid effect. The fluorescence turn-on system combined advantages of NPs EPR effect and pH-induced fluorescence turn-on property in tumor site, which resulted in larger FI difference between normal and tumor tissues as compared with that of luminescent Au NPs (ca. 12-fold). Such large FI difference resulted in that the LGAuNPs-Co had rapid (~1.6 h), persistent (~24 h p.i.) and highly efficient tumor targeting capability in comparison with the LGAuNPs. Moreover, the LGAuNPs-Co also had much longer tumor retention, faster renal clearance and lower RES uptake than those for the LGAuNPs. With these attractive characteristics, we believe that metal ion-directed luminescent AuNPs assemblies would be a promising fluorescent agent for highly efficient tumor imaging, which represents an innovative avenue of designing and developing tumor diagnosis and therapy system.


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Figure captions

Figure 1. Schematic diagrams of (A) synthesis and Co2+-induced assembly of LGAuNPs and (B) fluorescence turn on mechanism for in vivo tumor imaging: the fluorescence of LGAuNPs is partly quenched by adding Co2+ under neutral condition (normal tissues), while the assembled structure can be disintegrated in acidic environment (tumor site) and regain “off” fluorescence.

Figure 2. Characterization of the LAuNPs and LGAuNPs. TEM images of (A) LAuNPs and (B) LGAuNPs, (C) hydrodynamic size distributions of LGAuNPs and LGAuNPs, (D) UV-vis and fluorescence spectra and (E,F) TG analyses of LAuNPs and LGAuNPs. Insets A and B are the particle size distributions counted from the TEM images of LAuNPs and LGAuNPs, respectively; Inset D is digital photos of LGAuNPs under white light and UV light, respectively.

Figure 3. Characterization of LGAuNPs-Cos. (A, B) TEM images, (C) hydrodynamic size distributions and (D) fluorescence spectra of LGAuNPs-Co_7.4 and LGAuNPs-Co_6.4, respectively.

Figure 4. Cell viability of (A) HepG2, (B) LO2, (C) A549 and (D) 4T1 cell lines incubated with 0.5, 1.0, 2.5, 5.0 and 10.0 mg/mL LGAuNPs-Co for 6, 12 and 24 h, respectively.

Figure 5. (A) Fluorescent microscope images and (B) fluorescence intensity statistic plot of HepG2 and LO2 cells incubated with LGAuNPs or LGAuNPs-Co for 8 h. (C) Statistic plot of


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Au3+ concentrations in HepG2 and LO2 cells incubated with LGAuNPs (NPs for here) and LGAuNPs-Co (NPs-Co for here) for 1, 8 and 24 h, respectively.

Figure 6. Comparison of tumor-targeting efficiency of LGAuNPs and LGAuNPs-Co in HepG2 tumor bearing nude mice. (A) Time-dependent in vivo fluorescence images (the white circles and arrows show the tumor location). (B) CI values of LGAuNPs and LGAuNPs-Co at different p.i. time points. (C) Tumor targeting kinetics of LGAuNPs and LGAuNPs-Co. Area under the curves (AUC) were 310 and 1200 for LGAuNPs and LGAuNPs-Co, respectively.

Figure 7. (A) Pharmacokinetics and (B) renal clearance kinetics of LGAuNPs and LGAuNPs-Co measured by ICP-AES, respectively.

Figure 8. (A) ex vivo fluorescence images and relative fluorescence intensity of major organs (1-8: heart, liver, spleen, lung, left kidney, right kidney, intestine and tumor) collected from (B) LGAuNPs-treated or (C) LGAuNPs-Co-treated mice at 3, 8 and 12 h. The fluorescence intensity of organs was normalized by setting the fluorescence intensities of tumors in different time points as 1.


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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Figure 5.


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Figure 6.


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Figure 7.


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Figure 8.


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ASSOCIATED CONTENT Supporting Information. Electronic Supplementary Information (ESI) available: UV-vis, FTIR, EDS and fluorescence spectra, fluorescence stability analysis, fluorescent microscope images, body weight change plots, histological section images, digital photograph and in vivo fluorescence images of LGAuNPs or LGAuNPs-Co. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel & Fax: 86-20-222-36670; E–mail: [email protected]. Tel: 86-20-62787973. Fax: 86-20-87281713. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡Xuandi Lai and Lishan Tan contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The work is supported by National Natural Science Foundation of China (Grants 51273070, 81270825, 21173087, 21673081) and Natural Science Foundation of Guangdong Province (Grants 2014A030313232).


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BRIEFS: Fluorescence turn-on property and passive tumor imaging of

Au nanoassemblies.


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Figure 1. Schematic diagrams of (A) synthesis and Co2+-induced assembly of LGAuNPs and (B) fluorescence turn on mechanism for in vivo tumor imaging: the fluorescence of LGAuNPs is partly quenched by adding Co2+ under neutral condition (normal tissues), while the assembled structure can be disintegrated in acidic environment (tumor site) and regain “off” fluorescence. 101x119mm (300 x 300 DPI)



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Figure 2. Characterization of the LAuNPs and LGAuNPs. TEM images of (A) LAuNPs and (B) LGAuNPs, (C) hydrodynamic size distributions of LGAuNPs and LGAuNPs, (D) UV-vis and fluorescence spectra and (E,F) TG analyses of LAuNPs and LGAuNPs. Insets A and B are the particle size distributions counted from the TEM images of LAuNPs and LGAuNPs, respectively; Inset D is digital photos of LGAuNPs under white light and UV light, respectively. 103x119mm (300 x 300 DPI)


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Figure 3. Characterization of LGAuNPs-Cos. (A, B) TEM images, (C) hydrodynamic size distributions and (D) fluorescence spectra of LGAuNPs-Co_7.4 and LGAuNPs-Co_6.4, respectively. 151x119mm (300 x 300 DPI)



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Figure 4. Cell viability of (A) HepG2, (B) LO2, (C) A549 and (D) 4T1 cell lines incubated with 0.5, 1.0, 2.5, 5.0 and 10.0 mg/mL LGAuNPs-Co for 6, 12 and 24 h, respectively. 154x119mm (300 x 300 DPI)


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Figure 5. (A) Fluorescent microscope images and (B) fluorescence intensity statistic plot of HepG2 and LO2 cells incubated with LGAuNPs or LGAuNPs-Co for 8 h. (C) Statistic plot of Au3+ concentrations in HepG2 and LO2 cells incubated with LGAuNPs (NPs for here) and LGAuNPs-Co (NPs-Co for here) for 1, 8 and 24 h, respectively. 165x119mm (300 x 300 DPI)



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Figure 6. Comparison of tumor-targeting efficiency of LGAuNPs and LGAuNPs-Co in HepG2 tumor bearing nude mice. (A) Time-dependent in vivo fluorescence images (the white circles and arrows show the tumor location). (B) CI values of LGAuNPs and LGAuNPs-Co at different p.i. time points. (C) Tumor targeting kinetics of LGAuNPs and LGAuNPs-Co. Area under the curves (AUC) were 310 and 1200 for LGAuNPs and LGAuNPs-Co, respectively. 127x119mm (300 x 300 DPI)


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Figure 7. (A) Pharmacokinetics and (B) renal clearance kinetics of LGAuNPs and LGAuNPs-Co measured by ICP-AES, respectively. 303x119mm (300 x 300 DPI)




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TOC 80x83mm (300 x 300 DPI)


Coordinatively Self-Assembled Luminescent Gold Nanoparticles

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