Rapid tumor bioimaging and photothermal treatment based on GSH-capped red fluorescent gold nanoclusters

Abstract

Gold nanoclusters (Au NCs) possess outstanding physical and chemical attributes that make them excellent scaffolds for the construction of novel chemical sensors and biological imaging probes. In this study, a simple one-pot synthesis method, employing L-glutathione as the stabilizer, was presented for the preparation of red fluorescent Au NCs. The prepared Au NCs have no obvious cell cytotoxic effect on cancerous cells (i.e., HeLa, U87, and MCF-7 cells) and non-cancerous cells (i.e., L02 cells) in a wide concentration range. Then the prepared Au NCs were applied for tumor-targeted imaging in vitro and in vivo due to their good photo-stability, strong fluorescence emission, excellent water solubility and bio-compatibility. The observations indicate that the as-prepared Au NCs exhibited a near infrared fluorescence emission at 710 nm for in vivo bioimaging of tumors. Furthermore, Au NCs combining with porphyrin derivatives were applied for photothermal treatment to effectively inhibit the growth of tumors. This raises the possibility of utilizing Au NCs as a fluorescent probe for tumor-targeted rapid imaging and thus realize the facile fluorescence imaging-guided photothermal therapy of tumors.

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1 Introduction

Fluorescence bioimaging, offering unique advantages over other imaging modalities in sensitivity, real-time detection abilities and equipment cost, is widely used in biomedical sciences for applications ranging from the morphological analysis of anatomical structures to sensitive measurement of intracellular molecular events.^1–3 As an essential tool, fluorescence imaging has received particular attention in the field of tumor research. Tumor in vivo fluorescence imaging requires imaging agents with good biocompatibility, high sensitivity, sufficient tissue penetration and high spatial resolution.^4–7 Imaging agents for signal transduction play the most important roles in the fluorescent imaging. However, current imaging methodologies utilizing fluorescent organic dyes remain problematic. Conventional fluorophores suffer from rapid photobleaching, which are not fit for long-term molecular imaging,⁸ and are restricted in sensitive detection because of the limited number of fluorophores conjugated per ligand.⁹ Furthermore, the in vivo toxicities of the majority of fluorophores are unknown.³ Compared with small molecular probes, nanomaterials as imaging agents for in vivo imaging have obvious advantages, such as passive accumulation to tumor sites which maximizes their performance in imaging or therapy, while the convenient and feasible modification leads to multifunctional nanoprobes, featured in multimodal imaging with combinations of other contrast agents and early theranostics of disease with loading drugs.^1,10

Metal nanoclusters (NCs) possess an attractive set of features, such as ultrasmall size, variable emission wavelength, good biocompatibility, brightness and photo-stability, which renders them attractive alternatives as fluorescent probes for biological labeling and imaging.^1,11–13 Moreover, large Stoke shifts of metal NCs can prevent spectral cross-talk and, thus, enhance the detection signal. These properties make them promising for in vivo bioimaging, including cell labeling^14–16 and tumor imaging based on fluorescent metal NCs.^1,16–18 Nienhaus et al.¹⁹ reported water-soluble fluorescent Au NCs stabilized with the bidentate ligand dihydrolipoic acid with long fluorescence lifetime (>100 ns), which makes them attractive as labels in fluorescence lifetime imaging (FLIM) applications. The results indicated that the internalization of Au NCs by live HeLa cells is visualized by using the FLIM technique. Wang et al.³ reported ultrasmall near-infrared fluorescent Au NCs are not only feasible as a biocompatible fluorescent contrast agent in vivo, but also well-targeted to the MDA-MB-45 and Hela tumors in mice due to the enhanced permeability and retention (EPR) effect. Our previous studies^20,21 demonstrated a novel strategy through Au(III) reduction inside cancer cells cytoplasm, and ultimately concentrated around their nucleoli to form Au NCs applied in fluorescence imaging in vivo and thus affording precise cell imaging.

Apart from the in vitro detection of biomolecules for cancer diagnosis, in vivo imaging and therapy is the ultimate goal for cancer diagnosis. Photothermal therapy (PTT) has been well known from last few decades as a minimally invasive alternative to conventional approaches, such as surgery and chemotherapy, for therapeutic intervention of specific biological targets.^22–24 It is necessary for photosensitizers to have an absorption peak at wavelengths ranging from 600 to 900 nm, i.e., the near-infrared window, a wavelength region of low absorptivity by tissue. This permits fluorescence signals relatively free of intrinsic background interference with detectable signal intensities and penetrates deeply into living tissues.^4,25 Porphyrin and derivatives are good candidates as photosensitizers for use in PTT which can absorb light and dissipate the absorbed energy through nonradiative decay, inducing a temperature increase in the local treatment environment and resulting in irreversible cell damage.^26–28 Porphyrins have been conjugated to gold nanoparticles or carbon nanotubes for enhanced light energy conversion and combination of photodynamic and photothermal therapy,^25,28–30 but only recently, porphyrin combined with metal nanoclusters as photosensitizer was reported.³¹ Metal nanoclusters, especially Au NCs, can serve as a promising fluorophore not only for biosensing and in vitro/vivo imaging, but also for cancer therapy.^16,19,32 It is also known that Au NCs can vigorously absorb light and then efficiently convert optical energy into local heat, producing a selective and sensitive photothermal effect.^33,34 In addition, Au NCs have abundant binding sites which can be designed to be multifunctional probe realizing simultaneous targeting therapy and fluorescence imaging.³⁵

In view of the above observations, in this study we have explored the possibility of using ultrasmall Au NCs with red fluorescence for cell imaging and tumor near infrared (NIR) fluorescence imaging in vivo (Scheme 1). Furthermore, Au NCs combining with porphyrin derivatives (meso-tetra(4-sulfonatophenyl)porphine dihydrochloride, TSPP) were applied for photothermal treatment to effectively inhibit the growth of tumors, indicating the possibility of applying Au NCs in tumor-targeted rapid fluorescent bioimaging and then readily realize fluorescence imaging-guided photothermal therapy of tumors.

Scheme 1 Schematic illustration of Au NCs used for fluorescent bioimaging and photothermal treatment through combining with porphyrin derivatives (TSPP).

2 Experimental

2.1 Materials, instruments and cancer cells

L-Glutathione (GSH), thiazolyl blue tetrazolium bromide (MTT), streptomycin, penicillin and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, USA) and were used as received. DMEM (high glucose) medium, RMPI-1640 medium and trypsin were purchased from Hyclone. Auric chloride acid (HAuCl₄·4H₂O), dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co. (China). Carbon monoxide (CO, 99.9%) was obtained from Nanjing industrial gas plant, China. All reagents were of analytical grade purity level, there is no need purification before use. All the solutions were prepared by Milli-Q purified water (18.2 MΩ cm). All glassware were washed with chromic acid lotion, and rinsed with copious amounts of ultrapure water.

Fluorescence measurements were performed on SHIMADZU RF-5301 PC instrument. The slit wavelength for excitation and emission was set as 5 nm. Thermo SCIENTIFIC, BioMate 3S UV-visible spectrophotometer was used for the UV-Vis adsorption measurements. Transmission Electron Microscopy (TEM) images were collected using a JEM-2100 microscope to characterize the size and size distribution. A diluted solution was spotted on carbon coated copper grid (300 meshes) and was dried in laboratory ambience.

Cancer cells like cervical cancer Hela cells, glioblastoma U87 cells and human breast adenocarcinoma cell line MCF-7 cells (purchased from Cell Bank of Chinese Academy of Sciences, Shanghai) and non-cancerous cells like human embryo liver L02 cells (supplied by Third Military Medical University, Chongqing) were applied in our study. Hela, U87 and L02 cells were cultured in DMEM medium replenished with 10% fetal bovine serum, 100 U mL⁻¹ penicillin and 100 U mL⁻¹ streptomycin. MCF-7 cells were cultured in RMPI-1640 medium supplemented with 10% fetal bovine serum, 100 U mL⁻¹ penicillin and 100 U mL⁻¹ streptomycin. All cancer cells were placed at 37 °C in a carbon dioxide cell incubator with 5% CO₂ and 95% relative humidity.

2.2 Cell growth inhibition study by MTT assay

To test the cytotoxicity of Au NCs in vitro, the Hela, U87, MCF-7, and L02 cells were seeded in a 96-well plate in cell medium overnight and subsequently incubated with different concentrations of Au NCs (0, 10, 50, 100, 300, and 600 μM) in cell medium for 24 h at 37 °C and 5% CO₂. Afterwards, 20 μL MTT (5 mg mL⁻¹) was added to each well, and the cells incubated for 4 h. Thereafter, the cell medium in each well was removed and 150 μL DMSO was added to each well and gentle shaking in the shaker for 10 min. The absorbance at 492 nm was measured with a microplate reader (MK3, ThermoFisher). Cell viability was expressed as follows: cell viability (%) = [A]_test/[A]_control × 100%, where [A] represents the absorbance.

2.3 Cell imaging

Cancer cell lines like HeLa, U87 and normal cell lines like L02 (10⁵ cells in 1 mL) in DMEM medium containing 10% fetal bovine serum and antibiotics were seeded in 6-well plate with coverslips. After 12 hours, Au NCs decorated with GSH dissolved in PBS were added to each well, which were incubated for 24 hours at 37 °C in a 5% CO₂. The medium was then removed and the cells were washed three times with PBS. Then samples were incubated with 1 mL of 4% paraformaldehyde in PBS for 15 minutes. The supernatant was removed and the samples covered with coverslips were maintained in the dark at 4 °C. Confocal microscopy was performed on a Leica TCS SP5 Leica Microsystems GmbH microscope (Wetzlar, Germany) using the Leica Application Suite advanced fluorescence 2.0.2 software for data acquisition. Images were acquired by using a 63× oil immersion lens with argon laser set at 532 nm to detect cells with Au NCs. The confocal fluorescence images were quantitatively analyzed by using the Image J software. The fluorescence intensity of Au NCs within each cell was obtained by calculating the integrated intensity divided by the cell area.

2.4 In vivo bioimaging study

The BALB/c female athymic nude mice harboring MCF-7 cells and U87 cells (female, weighed 18–22 g, aged 4–5 weeks) were initially prepared. All in vivo animal experiments involved in mice have been approved by the National Institute of Biological Science and Animal Care Research Advisory Committee of Southeast University, and experiments were conducted following the guidelines of the Animal Research Ethics Board of Southeast University. For in vivo bio-imaging of Au NCs (5 mM, 100 μL) in the tumor location complex solution was administered into the solid tumor mouse model through local injection. The xenografted tumor nude mice of none injection were used as self-control. All mice were fully anesthetized by gaseous 5% isoflurane anesthesia. The in vivo bio-images were acquired on Perkin Elmer in vivo imaging system at different time points after the active fluorescent probes were injected, with the excitation and emission wavelengths of 520 nm and 710 nm, respectively. The ex vivo fluorescence images were obtained immediately after the major organs/tumors were excised from the tested nude mice.

2.5 Au NCs combined with porphyrin for photothermal therapy of tumors

The nude mice harboring U87 cells were divided into three groups with three mice in each group. Two control groups were only injected with 0.1 mL of 5 mM Au NCs and 0.1 mL of 0.1 mM TSPP, respectively. The experimental group deal with 0.1 mL of TSPP modified Au NCs complex (TSPP–Au NCs), which was made through mixing TSPP and Au NCs and completely shaking for self-assembly combination. When the tumor volume became around 120 mm³, each group was injected for treatment. Solutions was administered into the solid tumor mouse model through local injection and all the groups were exposed for 30 min per day in the infrared irradiation. The tumor volume of nude mice were measured for the last 16 days and calculated by the formula V = 1/2 × a × b², where a is the largest and b is the smallest diameter of the tumor.

3 Results and discussion

3.1 Synthesis and characterization of Au NCs

The ultrasmall fluorescent Au NCs were firstly prepared by using a simple one-pot synthesis method stabilized by L-glutathione as reported by Xie et al. (for details see ESI†).³⁶ The size of the prepared Au NCs was about 2 nm (Fig. S1†), which was small enough to be effectively excreted from the body. When imaging by using IVIS Lumina XRMS in vivo optical imaging system, the brown solution of Au NCs emitted an intense red fluorescence (Fig. 1). In contrast, there was no red fluorescence signal for the control GSH, HAuCl₄ and mixture GSH and HAuCl₄. In addition, the Au NCs exhibited high photostability as quantum dots. Fig. 1B demonstrated the relative fluorescence intensity variation of Au NCs with different excitation and emission in vivo. Under the 520 nm excitation, the NIR fluorescence emitted at 710 nm attained the maximum.

Fig. 1 (A) The photograph of Au NCs (a), GSH solution (b), and the mixture of GSH and HAuCl₄ (c) imaging using IVIS Lumina XRMS in vivo optical imaging system. (B) The relative fluorescence intensity of Au NCs under different excitation and emission in vivo.

3.2 Application of Au NCs for cancer cell imaging

Based on the above observations, the cytotoxic effect and bio-labeling of Au NCs on cancer cells was further explored, where U87, HeLa and MCF-7 cancer cells were chosen as relevant experimental models while non-cancerous ones L02 cells were chosen as the control model. Fig. 2 illustrates the cytotoxic effect of Au NCs on different cell lines including HeLa, U87, MCF-7, and L02 cells. It is evident that upon incubation with Au NCs for 24 h, no significant differences in the proliferation of the relevant cells were observed. The viabilities of both cancer cells and normal cells maintained above 85% even at relatively high concentrations. MTT assay revealed that the as prepared Au NCs is of low toxicity and good biocompatibility.

Fig. 2 Cytotoxicity results of different concentration Au NCs on HeLa, U87, MCF-7 and L02 cells determined by MTT assay.

Confocal fluorescence microscopy study was explored to evaluate the relevant bio-imaging efficiency for different cell lines including normal cells (i.e., non-cancerous cells) and cancer cells when cultured with DMEM (high glucose) contained Au NCs. As shown in Fig. 3A–E, the effect of Au NCs in different concentrations to Hela cells was initially explored in details. It is evident that with the increase of Au NCs concentration, the intracellular fluorescence of target cells apparently increased. When the added Au NCs was 300 μM (Fig. 3F), the Au NCs were well distributed in the cells and the relevant edges and morphologies of the cells were neatly delineated. Furthermore, the difference of Au NCs imaging in normal cells and cancer cells were also observed. As shown in Fig. 4, when incubated with the Au NCs for 24 h, the intracellular fluorescence of target cancer cells could be observed with many bright red spots inside the cells. In contrast, when non-cancerous ones (L02 model, Fig. S2†) incubated with the Au NCs, no apparent intracellular fluorescence could be observed. The above studies performed on cell cultures have evidenced that incubation of cancerous cells (i.e., U87 and HeLa cell models) and non-cancerous ones (i.e., L02 model) with Au NCs led to drastic differences in relevant cell interactions. This could be attributed to the microenvironment and some specific biochemical characteristics of cancer cells differentiate them from cells under normal homeostasis.^21,31 The intracellular fluorescence indicates that the Au NCs could readily enter inside the target cancer cells, indicating the possibility for the promising use of these fluorescent Au NCs for bioimaging or bio-marking of cancer cells and potential monitoring of the relevant treatment process.

Fig. 3 Laser confocal fluorescence micrographs of HeLa cells. HeLa cells incubated in the absence of Au NCs (A) and in the presence of 50 (B), 150 (C), 300 (D), and 600 μM (E) Au NCs incubated for 24 h. (F) Relative fluorescence intensity variations of A–E. Fluorescence micrographs collected by using 532 nm as fluorescence excitation wavelength.

Fig. 4 Laser confocal fluorescence micrographs of Hela and U87 cells after incubation for 24 h in the absence of Au NCs (A, B) and in the presence of 300 μM Au NCs (C, D). The micrographs of cells were acquired by 40 × IR coated objective. The excitation wavelength was at 532 nm.

3.3 Application of Au NCs for tumor imaging

On the basis of aforementioned in vitro results, the feasibility of in vivo fluorescence bioimaging of tumors based on fluorescent Au NCs was further explored in this study. The fluorescent gold nanoclusters were subcutaneously injected into the two kinds of mice models, i.e., with MCF-7 and U87 xenograft tumors, respectively, as shown in Fig. 5 and S3.† It is evident that upon injecting the fluorescent Au NCs into the tumor-bearing mice, the substantially enhanced NIR fluorescence signal could be detected about 1 h after the Au NCs injection, and then the NIR fluorescence signal in tumors was becoming weaker gradually. After 8 h, the fluorescence signal was almost disappeared. In addition, no obvious toxic effects were observed during the whole experimental trail in the two different tumor models, suggesting that the biocompatible Au NCs can be administered for rapid and high-sensitive in vivo fluorescence cancer imaging.

Fig. 5 Representative xenograft tumor nude mice models bearing MCF-7 tumor by in vivo NIR fluorescence imaging. (A) In vivo fluorescence imaging pre injection and after a subcutaneous injection 100 μL of 5 mM Au NCs for 1, 2, 4.5, and 8 h. The excitation wavelength was 520 nm. (B) Statistical analysis of the fluorescence intensity corresponded to the fluorescence images of bearing MCF-7 tumor mice.

The ex vivo fluorescence images of various organs/tissues were also obtained via thoracotomy, which were obtained after 24 h post-injection of the Au NCs. Representative organs/tissues including heart, liver, spleen, lung, kidney and tumor were excised, washed with PBS buffer, and then used for fluorescence imaging. From Fig. 6, we can find that the ex vivo MCF-7 tumors showed much stronger fluorescence, while almost no fluorescence was observed in other organs. It is noted that fluorescent Au NCs are well-targeted to the tumors in mice mainly due to the EPR effect.³ Moreover, the tumor microenvironments are different with normal tissues that make the GSH capped Au NCs easy to fluorescence imaging in the tumor.²¹ The fluorescence on the liver could be pertaining to the relevant accumulation in liver through normal biological metabolism of Au NCs in the mice. However, our previous studies on histopathologic analyses have demonstrated the biosafety of Au NCs in normal tissues.²¹

Fig. 6 Ex vivo fluorescence images of the tumor tissue and major organs of the mice bearing MCF-7 tumor, which were sacrificed 24 h after the injection of fluorescent Au NCs: (a) spleen, (b) lung, (c) kidney, (d) heart, (e) tumor, (f) liver. The excitation wavelength was 520 nm.

3.4 Au NCs combining with porphyrin derivatives for photothermal treatment

Based on the above study, we have further explored the synergistic effect on photothermal therapy of tumors by using gold nanoclusters complex (i.e., TSPP–Au NCs) through combination with a porphyrin derivative TSPP, a potential PTT agent, where the self-assembly of TSPP–Au NCs complex was made through mixing TSPP and Au NCs with completely shaking. After subcutaneous injection of TSPP–Au NCs complex solution near the tumor, time-dependent fluorescence imaging was obtained by using the IVIS Lumina XRMS in vivo imaging system. As shown in Fig. 7, as soon as 1 h the NIR fluorescence emitted at 710 nm could be readily detected under the excitation wavelength of 520 nm. Compared with the control group (i.e., injection of Au NCs alone or TSPP alone), the fluorescence intensity in TSPP–Au NCs complex significantly stronger. The mean fluorescence intensity of the tumor region obtained by the IVIS Lumina XRMS imaging software through statistical analysis indicates that after 3 h the fluorescence was best both experiment and control group. This result indicated that the TSPP–Au NCs could be used for fast tumor fluorescence imaging in vivo and simultaneously they have synergistic effect. The synergistic effect on photothermal therapy of tumors by TSPP and Au NCs could readily be assessed by comparison of the tumor growth and the survival period between the relevant experimental mice exposed to infrared (IR) irradiation by using TSPP–Au NCs complex as well as other control mice. As shown in Fig. 8, the tumors xenografted in mice treated by the TSPP–Au NCs complex had been reduced to a smaller size than those of control, suggesting that TSPP readily interacting with the Au NCs in tumors could further enhance the relevant treatment efficacy. Furthermore, all treated mice were in good health condition, while those from the control groups were very emaciated or had passed away during this period. These results demonstrated that the combination of gold nanocluster with TSPP for in vivo fluorescence imaging and photothermal treatment of tumors can readily provide a synergistic effect and thus lead to a fluorescence imaging-guided photothermal therapy of tumors.

Fig. 7 In vivo fluorescence imaging of U87 tumor-bearing mice after subcutaneous injection of (A) 0.1 mL, 5 mM Au NCs, (B) 0.1 mL, 0.1 mM TSPP, (C) 0.1 mL TSPP modified Au NCs complex solution near the tumor for different time b–j: 0, 1, 3, 6, 9, 21, 25, 32, 48 h, a is pre-injection as the control. (D) The mean changes of relevant fluorescence intensity vs. time for the xenograft tumor mouse models for A, B, and C. The excitation wavelength was at 520 nm.

Fig. 8 (A) Excised tumors from mice, corresponding to those treated with Au NCs, TSPP, and Au NCs combined with TSPP from left to right; (B) in vivo tumor growth inhibition curves for those of Au NCs, TSPP and TSPP modified Au NCs complex on U87 tumor mice model. Error bars are the standard error of the mean for three times measurement.

The ex vivo fluorescence images of various tissues were also obtained via thoracotomy, which were obtained after photothermal treatment. Representative tissues including tumor, heart, liver, spleen, lung, and kidney were excised, washed with PBS buffer, and then used for fluorescence imaging. The corresponding regions of interest (ROI) analysis on the ex vivo fluorescence images was conducted to semi-quantitatively study the uptake of Au NCs and TSPP in each organ by the IVIS Lumina XRMS software. As shown in Fig. 9, the ex vivo tumor and organs from injected Au NCs mice showed much lower fluorescence. For those injected with TSPP–Au NCs complex, the fluorescence of tumor and kidney was much lower than the control group injected with only TSPP, and its average ROI fluorescence intensity for tumor was at least 2 times lower than the control group. This result indicates that Au NCs can promote the metabolism of TSPP after their combination. In addition, the accumulation in tumor and kidney indicate TSPP–Au NCs complex are promising candidates for non-invasive real-time imaging in vivo and can be readily removed from the body through renal clearance, which is consistent with that reported in the literature.^21,31 The high quality imaging in vivo and effective PTT treatment on tumor could be attributed to the ultrasmall size and red-fluorescent gold nanoclusters, which can also effectively avoid the disturbance of auto-fluorescence background from living body.

Fig. 9 Ex vivo (A–C) fluorescence images and (D) average fluorescence intensity analysis of the tumor tissue and major organs of the mice bearing U87 tumor, which were sacrificed after two weeks treatment: (A) injected with Au NCs; (B) injected with TSPP; (C) injected with TSPP modified Au NCs complex; (a) tumor, (b) lung, (c) liver, (d) spleen, (e) heart, and (f) kidney; (D) statistical analysis of the fluorescence intensity corresponding to the ex vivo fluorescence images of (A–C).

4 Conclusions

In this study, a simple one-pot synthesis method, employing L-glutathione as the stabilizer, was presented for preparation of fluorescent Au NCs. The as-prepared Au NCs exhibited a near infrared fluorescence emission at 710 nm in vivo. The prepared Au NCs have no obvious cell cytotoxicity effect on cancerous cell (i.e., HeLa, U87, and L02 cells) and non-cancerous cell (i.e., L02 cells) in a wide concentration range. The as-prepared Au NCs could be readily applied for tumor-targeted imaging in vitro and in vivo due to its good photo-stability, strong fluorescence emission, excellent water solubility and biocompatibility. Furthermore, Au NCs combining with porphyrin derivatives applied for photothermal treatment could effectively inhibit the growth of tumors. This raises the possibility of utilizing Au NCs as a sensitive fluorescent probe for tumor-targeted rapid imaging, which can further facilitate to realize fluorescence imaging-guided photothermal therapy of tumors.

Acknowledgements

This work is supported by National High Technology Research & Development Program of China (2015AA020502), the National Natural Science Foundation of China (81325011, 21327902 and 21175020) and Suzhou Science & Technology Major Project (ZXY2012028).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra10409f

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