2017, Vol. 28 
,
Lin-Tao Caia,*
b Department of Nano Science and Technology Institute, University of Science and Technology of China, Suzhou 215123, China;
c Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese of Academy of Sciences, Shenzhen 518055, China;
d Paul-Drude-Institut für Festkörperelektronik, Berlin 10117, Germany;
e Department of Ophthalmology, Shenzhen People's Hospital, 2nd Clinical Medical College of Jinan Univiersity, Shenzhen 518020, China
Very recently, noble metal nanoclusters as a potential promising nanomaterial have gained considerable attention in the field of energy, environment and biology [1-3]. Particularly, for the remarkable optical and chemical properties different from atoms and bulk materials, gold nanoclusters (Au NCs) have been extensively investigated in biomedical applications, like antibacterial [4-6], biosensing [7-11], imaging[12-17] and cancer treatments [18-21]. Previous research reported that Au NCs could be prepared with biological components, for instance, saccharides [22], nucleic acid [23-25], protein [5, 6, 26-34], in vitro [35] and in vivo [36, 37]. The as-prepared Au NCs showed size-dependent fluorescence varying from visible light to near-infrared region, which corresponds to their ultra-small size ( < 1.5 nm) closed to the Fermi wavelength of the conduction electrons (0.7 nm) [38]. Therefore, based on their high biocompatibility, distinctive stability and wavelength-adjustable emission, Au NCs as excellent near-infrared (NIR) fluorescent (FL) probes have been applied for in vitro and in vivo imaging. Folate-modified Au NCs were utilized to improve tumor targeting for imaging in vivo and cancer treatments [18]. Au NCs mixed gadolinium was devoted to increasing the imaging modality [13, 39]. However, the short blood-circulating time and low tumor-targeting efficiency of Au NCs have limited their further applications [40, 41].
In addition, several research reported that Au NCs as sensitizers or vehicles have been employed for cancer treatments, like chemotherapy [18, 42], radiotherapy [20, 43]. However, drug molecules and gamma-rays induced damages for normal tissues. As a clinical modality, phototherapy, especial photodynamic therapy (PDT), has several advantages over conventional chemotherapy and radiotherapy due to their non-invasive nature, high tumor selectivity and low side effects [44]. The biocompatible Au NCs showed excellent NIR FL imaging and high cancer treatment efficiency in vivo [19, 45]. Khlebtsov believed that Au NCs conjugated photosensitizers showed dramatically resistant bacteria [46]. Au NCs incorporated the targeting agents and photosensitizers were employed to improve the tumor targeting and selective PDT [47]. Chiral Au NCs were devoted to treat tumor cells for its chiral-associated reactive oxygen species (ROS) generation [48]. However, Au NCs-induced PDT also were limited for the short excitation wavelength [49] and UV excitation wavelength [50].
Herein, we developed theranostic gold clusters nanoassembly (Au CNA) according to the protein-assisted cross-linking process. This "green" preparation strategy was highly facile and controllable without using any toxic reagent and complicated procedure. The functional self-assemblies had great applications in molecular devices and biological delivery [51], which provided the way to improve the tumor targeting of Au NCs. The as-prepared Au CNA exhibited excellent NIR FL properties and enhanced ROS generation efficiency. It demonstrated that Au CNA could maintain high fluorescence and colloid stability, increase the tumor accumulation for FL imaging and improve PDT efficiency of tumor. The theranostic Au CNA would possess a great potential in future fluorescence diagnosis and position for guided NIR FL-induced cancer PDT.
2. Results and discussion 2.1. Synthesis and characterization Au CNABSA stabilized Au NCs were synthesized with a wet chemical reaction according to Ying et al. [26]. BSA for the good biocompatibility, nontoxicity and easy accessibility, could be used not only as a reductant, but also a stabilizer for entrapping gold to form Au NCs, which is similar to the natural bio-mineralization approach [52]. In the preparation process, HAuCl4 was reduced by tyrosine residues (Tyr) of BSA to obtaining Au (0) and Au (Ⅰ), which further was stabilized by thiol group from cysteine residues (Cys) to form strong Au-S bonds in Au NCs [53]. To improve the tumor targeting, the assembly was synthesized with the protein linkers, like tea polyphenols [54] and urea [55], which changed the optical properties and the structure of Au NCs, respectively. Glutathione (GSH) also was devoted to prepare the protein nanoparticles by cleaving the disulfide bonds of BSA surface and form the intermolecular disulfide bonds [56, 57]. The constructed Au CNA were made up with many Au NCs and different from the selfassembly on the surface of gold nanoparticles [58]. The Au NCs assembly using GSH as linkers had not changed the optical propertiesand structureof Au NCs.As shownin Fig. 1a, thereduced Au NCs were further assembled by free sulfhydryl to form Au CNA in the presence of ethanol on alkaline conditions. TEM images illustrated the size of Au NCs and Au CNA with 1.46±0.11nm and 12.04±0.36nm, respectively (Fig. 1b-c). HRTEM images indicated that Au CNA were composed of amounts of Au NCs (Fig. 1c, inset). TEM image with negative staining was also demonstrated that Au CNA were assembled by Au NCs(Fig. S1in Supporting information). In comparison with Au NCs, the hydrodynamic size of Au CNA increased from 3.98±0.11nm to 40.97±0.41nm via DLS measurement (Fig. 1d-e), which was larger than corresponding sizes measured by TEM. Generally, TEM was employed to measure the dehydration morphology while DLS measurements with the hydrodynamic size. Additionally, the zeta potential scarcely decreased from -40.7±0.86mV to -42.3±1.56mV (Fig. S2 in Supporting information), suggesting that the as-synthesized Au CNA had good mono-dispersity in aqueous solution.
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| Fig. 1. (a) Schematic illustration for the preparation process of Au CNA. TEM images of Au NCs (b) and Au CNA (c). The inset shows the typical enlarged TEM images of separated Au NCs and Au CNA. The scale bars indicate 2 nm and 4 nm, respectively. Size distribution photographs of Au NCs (orange) (d) and Au CNA (purple) (e) in aqueous solution. The inset photographs of Au NCs and Au CNA under visible light (i) and UV-365 nm (ii). The fluorescent emission spectra of Au NCs (orange) and Au CNA (purple)(excitation = 476 nm) (f). UV-vis absorbance spectra of Au NCs (orange) and Au CNA (purple) (g). The inset shows the enlarge photograph of UV-vis absorbance spectra in the range of 225 nm-425 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) | |
Afterwards, the optical properties of Au CNA were investigated. Under visible and UV radiation (365nm), Au NCs showed brown light and red luminescence, respectively (Fig. 1d, inset). After the formation of Au CNA, the color of solution became lighter than that of Au NCs (Fig. 1e, inset). Au CNA had negligible changes in the fluorescence and UV-vis spectra in contrast to these of Au NCs (Fig. 1f-g). The slightly declined intensity revealed partly breakage of Au-S bonds stabilized Au NCs and limitedly instead of BSA with GSH on alkaline conditions [57, 59]. Moreover, the mean fluorescence lifetime of Au CNA was closed to that of Au NCs (Fig. S4 in Supporting information). Itwasfurther indicatedthat Au CNAwere assembled by Au NCs. In addition, Au CNA showed excellent colloid and fluorescence stability in aqueous solution (Figs. S4-S5 in Supporting information), suggesting that the self-assembly of Au NCs had hardly impact on inherent fluorescence and stability. Furthermore, the powder of Au CNA obtained by freeze-drying was also stored for a long time without fluorescence quenching and redispersed in aqueous solution with the same fluorescence. It further indicated Au CNA could be used as a contrast agent for imaging applications.
2.2. ROS generation of Au CNAThe generation of ROS was measured byusing the DCFDA probe, of which fluorescence intensity (494nm excitation and 523nm emission) increased with the enhancement of ROS levels. With the irradiation of 660nm laser (0.2W/cm2), ROS was generated in the presence of Au NCs and Au CNA (Fig. 2a). The fluorescence of DCDFA was not showed without irradiation and scarcely changed exposed to the 660nm laser. With the prolonging of irradiation time, ROS generated from Au NCs and Au CNA were gradually increasing. After 12min irradiation, Au CNA showed the 1.5 times increasingof ROS levels compared to that of AuNCs(Fig. 2b).Dueto the same wavelength of laser and Au NCs emission, it could be speculated that Au CNA exposed to laser could generate photon energy, which was further transferred to molecular oxygen for producing ROS. The enhanced ROS levels indicated that Au CNA could be used as excellent photodynamic agents for tumor phototherapy triggered by NIR laser.
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| Fig. 2. (a) Schematic photograph of ROS generation of Au CNA. (b) The fluorescent emission spectra of DCDFA probe at irradiating 12min under 660nm laser with the power density at 0.2W/cm2 in the presence of Au NCs (orange) and Au CNA (purple) (excitation=494nm), respectively. (c) The ROS generated levels with irradiation timeindependent varying of Au NCs (orange) and Au CNA (purple), respectively. The final concentration of Au NCs and Au CNA are 0.176mg/mL as Au. The data are shown as mean ±SD (n=3). (***) P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) | |
2.3. Cellular uptake and in vitro PDT
Cellular viability was performed by MTT assays with 293T normal cell and 4T1 breast cancer cell, respectively. As shown in Fig. S6 (Supporting information), cells treated materials had not shown cytotoxicity after 24h incubation, suggesting the excellent biocompatibility of Au CNA. The fluorescence emission in 660nm was employed to assess cellular uptake by incubating the suspension of materials in 4T1 cells for 4h. Red emission of Au NCs and blue emission of nuclei were observed by confocal laser scanning microscope (CLSM). Au CNA exhibited higher cellular uptake than that of Au NCs, which also demonstrated Au CNA with significant NIR FL imaging (Fig. 3a). Moreover, to determine the uptake efficiency of Au CNA, the flow cytometry (FCM) analysis was investigated. As shown in Fig. 3b, Au CNA showed 1.9 folds increasing in contrast to that of Au NCs, which further confirmed Au CNA with highly cellular uptake for NIR FL imaging in vitro.
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| Fig. 3. (a) Confocal luminescence imaging of 4T1 cell treated with Au NCs and Au CNA at the same equivalent gold concentration (0.352 mg/mL) for 4 h. Blue and red fluorescence images show nuclear staining with Hoechst 33258 and Au NCs, respectively. (b) Flow cytometry analysis of fluorescence intensity in 4T1 cells treated with media (black), Au NCs (orange) and Au CNA (purple) (0.352 mg/mL) for 4 h. (c) Fluorescence images of 4T1 cells after PDT treatment (660 nm laser irradiated 12 min) (0.2 W/cm2) by incubating PBS, Au NCs and Au CNA (0.176 mg/mL). Green and red images show viable cells with calcein-AM and dead cells with PI, respectively. (d) MTTassays of cell viability with PDT treatment (660 nm laser irradiated at different periods of time) (0.2 W/cm2) in 4T1 cells for 4 h after incubating with PBS (black), Au NCs (orange) and Au CNA (purple) (0.176 mg/mL) for 24 h. The scale bars represent 10 μm and 30 μm of A and C, respectively. The data are shown as mean ±SD (n = 3). (**) P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) | |
Additionally, to explore the photo-toxicity of Au CNA to 4T1 cells, calcein-AM and propidium iodide (PI) staining was conducted for distinguishing the viable or dead cells, respectively. As shown in Fig. 3c, cells treated with PBS showed significant green fluorescence and negligible red fluorescence, suggesting the laser irradiation alone could not result in the cell death. However, after 12 min irradiation of 660 nm laser (0.2 W/cm2), cells treated with materials displayed remarkable red fluorescence. And Au CNA showed more obvious red fluorescence than that of Au NCs (Fig. 3c), suggesting Au CNA with high cellular uptake could produce more ROS for improving PDT. Moreover, PDT efficiency was also performed by MTT assays. Without the laser irradiation, all groups (cells treated with PBS, Au NCs and Au CNA) exhibited non-toxicity to 4T1 cells. However, with the prolonging of irradiation time, Au CNA demonstrated more and more cell death and 1.4 times cell killing efficiency in contrast to that of Au NCs after 12 min irradiation (Fig. 3d). Moreover, cells treated with different concentration of Au CNA also indicated lower cellular viability than that of Au NCs (Fig. S7 in Supporting information). The improved PDT efficacy of Au CNA mainly attributed to the dramatic ROS generation and significant cellular uptake. Therefore, Au CNA as an excellent photosensitizer was employed for NIR FL imaging-guided PDT in vitro.
2.4. In vivo imaging and bio-distribution of Au CNATumor imaging in vivo was performed by the BALB/C athymic nude mice bearing 4T1 tumors. When tumor attached to 150 mm3, four 4T1 tumor-bearing female mice of each group were intravenously injected Au NCs and Au CNA suspensions (13.2 mg/kg) for NIR FL imaging in vivo, which was acquired on IVIS Spectrum Imaging System. Mice without administration were studied as a control. As shown in Fig. 4a, the fluorescence signal of Au CNA in tumor location showed after 7 h post-injection and obviously increased after 24 h post-injection. On the contrary, mice treated with Au NCs had not observed the fluorescence signals in the whole range of 24 h post-injection. It indicated that Au CNA was largely accumulated into tumor tissue with the prolonging of injection time interval. Moreover, the fluorescence intensity on tumor location was further qualified with region of interest (ROI) about 2.2 folds increasing of Au CNA compared to that of Au NCs at 24 h post-injection (Fig. 4b). It revealed Au CNA could keep longer blood-circulation time than Au NCs for improving tumor targeting.
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| Fig. 4. (a) In vivo NIR fluorescence images of 4T1-bearing mice treated with Au NCs and Au CNA (13.2 mg/kg) at different time points. The green cycle indicates the tumor location. (b) The fluorescence intensity is illustrated with ROI. (c) Ex vivo NIR fluorescence images of major organs and tumors after 24 h post-injection. (d) Gold concentration is measured with ICP-MS. (**) P < 0.01, (***) P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) | |
In addition, to determine the fluorescence intensity on tumor location, the bio-distribution of Au CNA was measured by NIR FL imaging ex vivo. After 24 h post-injection, two mice of each group were sacrificed for detecting the fluorescence signals of major organs and tumor tissues. As shown in Fig. 4c, Au CNA showed higher intensity on liver and kidney than these of Au NCs, suggesting that the liver accumulation by reticuloendothelial system (RES) failed to avoid in this scale of size and the renal clearance came from free Au NCs or some Au NCs disaggregated from Au CNA. However, Au CNA had more notable intensity than that of Au NCs on tumor location, which further demonstrated the enhanced tumor targeting of Au CNA. The average fluorescence intensity ex vivo was further qualified with ROI about 2.4 folds increasing of Au CNA at 24 h post-injection in contrast to that of Au NCs (Fig. S8). Furthermore, the other mice were euthanatized for measuring the gold contents of tissues by ICP-MS. In comparison to Au NCs, the gold contents of Au CNA in tumor tissues exhibited about 3.1 folds increasing, which further confirmed the improved tumor targeting of Au CNA (Fig. 4d). The difference of ROI and ICP results was contributed to the decrease of Au NCs intensity due to some gold released from Au NCs, suggesting that the formation of Au CNA reduced the release of gold from clusters. Although Au CNA exhibited exactly signals on liver and kidney, the precise tumor targeting remain displayed excellent NIR FL imaging in vivo for the next therapeutic investigation.
2.5. In vivo PDTPDT in vivo was also carried out by the BALB/C athymic nude mice bearing 4T1 tumors. Eight female 4T1-bearing mice of each group were intravenously injected Au NCs and Au CNA suspensions (17.6 mg/kg) for in vivo PDT when tumor attached 100 mm3. Mice treated with PBS were studied as the control group. Five mice and three mice of each group were employed to evaluate PDT effects and tissue toxicity. With the increase of time interval, the body weight of mice had not showed significant changes, the nontoxicity in tissues of materials (Fig. S9). After that, three mice of each group were exposed to the 660 nm laser for 10 min, 20 min and 30 min at a power density of 0.2 W/cm2 after 24 h postinjection. After further maintaining 24 h, mice were scarified to obtain the tumor tissues for preparing the freezing sections, which were utilized to evaluate PDT efficiency of tumor. H&E stained tumor slices revealed the tissue toxicity under the NIR laser irradiation. As shown in Fig. 5, mice treated all materials (PBS, Au NCs and Au CNA) had not showed the tissue toxicity without laser irradiation. After 30 min irradiation, Au CNA treated mice displayed significant cell apoptosis and karyopyknosis but slightly cell death compared to these of Au NCs. It suggested that Au CNA with the enhancement of PDT effects were contributed to the improved tumor targeting and enhanced ROS generation in contrast to these of Au NCs. Given limitedly ROS generation efficiency and passive tumor targeting of Au CNA, the completely destroy of tumor need to further explore by improving the targeting routes and optimizing the PDT approaches.
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| Fig. 5. Tumor histological sections stained with H&E from mice maintained 24 h after irradiating different points under 660 nm laser (0.2 W/cm2) with different periods (10 min, 20 min, 30 min) after mice intravenously injected Au NCs and Au CNA suspensions (17.6 mg/kg) for 24 h. The arrows indicate cell apoptosis and karyopyknosis. The scale bar is 100 μm for all the photos. | |
3. Conclusion
BSA as a template was employed to prepare Au NCs, which further formed the reduced Au NCs by cleaving the disulfide bonds of protein surface with the reductant, GSH. The reduced Au NCs were utilized to synthesize Au CNA with the self-assembly process. In this approach, ethanol as one of controlled and protecting agents was utilized to obtain the spherical structure of Au CNA and reduce the aggregation of protein. The as-synthesized Au CNA exhibited obviously increasing of size but scarcely variation in optical properties. In the prepared approach, there was not completely avoided from fluorescence quenching due to partly breakage of Au--S bonds in Au NCs and GSH instead of BSA for forming GSH stabilized Au NCs. Additionally, two of materials demonstrated non-toxicity for 293T normal cell and 4T1 tumor cell. Au CNA also showed higher cellular uptake than that of Au NCs for NIR FL imaging in vitro. Compared to the low tumor targeting of Au NCs, Au CNA for EPR effects were largely concentrated on tumor after 24 h post-injection. Moreover, under the NIR laser irradiation, Au CNA had higher ROS generation than that of Au NCs. When exposed to 660 nm laser, Au CNA displayed highly cell killing efficiency in contrast to that of Au NCs. Furthermore, after 30 min irradiation, tumor sections from Au CNA treated mice indicated the cell apoptosis and karyopyknosis in comparison to these of Au NCs. It suggested that Au CNA as an excellent photosensitizer had dramatically PDT efficiency for the inhibition of tumor growth. In summary, the well-defined Au CNA exhibited the improved tumor targeting and enhanced ROS generation, which revealed the novel system of NIR FL imaging-guided PDT for further realizing the integration of diagnosis and therapy.
4. Experimental 4.1. Materials and reagentsBovine serum albumin (BSA) and L-glutathione reduced (GSH) were obtained from BIOSHARP. 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), propidium iodide (PI), phosphotungstic acid hydrate, Hematoxylin, Hoechst 33258 and 2', 7'-dichlorofluorescin diacetate (DCFDA) were purchased from Sigma-Aldrich. Pentobarbital sodium was bought from Merck (Germany). Chloroauric acid hydrated (HAuCl4), dimethyl sulfoxide (DMSO), sucrose, xylene, sodium hydroxide (NaOH) and ethanol were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Phosphate-buffered saline (PBS, pH 7.4), fetal bovine serum (FBS), DMEM, Penicillin-streptomycin and trypsinEDTA were purchased from Gibco Life Technologies (Switzerland). Isoflurane was obtained from RWD Life Science (Shenzhen, China). 7000 MW dialysis bag was bought from Huashi Science (Shenzhen, China). 4% parafoemaldehyde (4% PFA) was obtained from Boster (Wuhan, China). Neutral balsam was purchased from Solarbio life science (Beijing, China). Ultrathin Carbon Film on Lacey Formvar/ Carbon on 200 Mesh (Copper) was obtained from Beijing Zhongjingkeyi Technology Co., Ltd (Beijing, China). Tissue OCTfreeze medium was bought from Leica (Germany). Amicon ultra-4 centrifugal filter with a molecular weight cutoff of 10 KDa was bought from Millipore (USA). All other chemicals used in this study were of analytical reagent grade and used without further purification. Ultrapure water (18.25 MΩ cm, 25 ℃) was used to prepare all solutions.
4.2. Synthesis of Au CNABSA-assisted preparation of gold nanoclusters (Au NCs) had been reported firstly [26]. The preparation steps were as follows: BSA (2.5 mL 100 mg/mL) was mixed to 6.5 mL ultrapure water. After shaking for 1 min, 1 mL HAuCl4 (50 mmol/L) was added into the solution, followed by vigorously stirring 2 min at room temperature. Then, 0.5 mL NaOH (1 mmol/L) was added into this system for stirring 1 min. The obtained mixture with lightproof package was put into the shaking table at 37 ℃ for reacting 12 h. After transferring it to room temperature for a while, the final suspension would be preserved at 4 ℃ for the following use.
Au CNA was constructed by using the self-assemble approach. GSH as a protein linker was employed to synthesize albumin nanoparticles with the formation of intermolecular disulfide bonds [57]. GSH was dissolved in ultrapure water at concentrations of 60 mmol/L. Considering the self-assemble process of albumin, Au CNA was prepared by following steps: 1 mL GSH (60 mmol/L) solution was added to 1 mL Au NCs suspension with vigorously stirring 1 h at room temperature. Afterwards, 4 mL ethanol was added to the mixture for shaking 1 h. The excessive ethanol solution partly would be abandoned under the vacuum condition. The obtained suspension was dialyzed against ultrapure water using dialysis membrane (7000 MW) under alkaline condition for 12 h. The solution was further filtered with 10 KDa cutoff to 1 mL. The final solution would be preserved at 4 ℃.
To obtained theranostic materials, Au NCs and Au CNA suspensions could be re-dispersed with ultrapure water after freeze-drying. Gold contents were employed to normalize the concentration of Au NCs and Au CNA, which was measured by Pekin Elmer OPTIMA 7000 DV inductively coupled plasma mass spectrometry (ICP-MS). As revealed by ICP-MS, the gold concentration of Au NCs and Au CNA were 1.98 mg/mL and 1.54 mg/mL, respectively.
4.3. Characterization of Au NCs and Au CNAThe size and morphology of materials were measured with Tecnai G2 F20 transmission electron microscope (TEM). For the protein scaffolds of Au NCs and Au CNA, TEM samples were prepared with the regular dropwise, which further negatively stained with 2% (W/V) phosphotungstic acid hydrate. Dynamic light scattering (DLS) was performed by Mastersizer Zetasizer Nano ZS to measure the hydrodynamic diameter and zeta potentials. Au NCs and Au CNA solutions could be observed under visible light and UV light (365 nm) which obtained by a digital camera under Spectronics CX-20 UV observation box (365 nm). The FL spectra and the mean fluorescence lifetime were measured by Edinburgh Instruments F900 (excitation 476 nm). The UV-vis absorption spectra were acquired by Pekin Elmer Lambda25. All optical measurements were performed at room temperature.
4.4. Detection of ROSROS generation was detected by using DCFDA probe. 660 nm laser (LWRL660 nm, Laserwave, Beijing) was used to trigger photochemical reaction for ROS generation. The power density was tested by an optical power meter (LI-P20W, Laserwave, Beijing) to be 0.2 W/cm2 for irradiation. DCFDA (10μmol/L) was employed to monitor ROS levels generated from Au CNA with 660 nm laser irradiation. DCFDA in aqueous solution was set as the control group. The generated ROS was determined by detecting the increase of DCFDA fluorescence with 494 nm excitation from Pekin Elmer LS55.
4.5. Cellular viabilityThe mouse cell lines 4T1 (breast tumor) and the human cell lines 293T (embryonic kidney) were purchased from Cellbank (Shanghai, China). All cells were cultured at 37 ℃ 5% carbon dioxide (CO2) cell incubator with DMEM supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100μg/mL streptomycin (10% FBS DMEM). All cells were utilized to assess the cytotoxicity of Au NCs and Au CNA in vitro. And 4T1 cells were employed to investigate cellular uptake of materials. Cellular viability was measured by MTT assays. 5×103 cells per well were inoculated into 96 well plates for incubating overnight. The suspension of materials was diluted for different concentration with 10% (v/v) FBS DMEM and filtrated with 0.45 μm membrane for further incubating 24 h. After washing 2 times with PBS (PH = 7.4), 20 μL MTT (5 mg/mL) solution was added into well for incubating above 4 h. After that, 200 μL DMSO was further mixed into well to measure the value of OD490 nm with ELISA Reader after stirring the plates about 10 min. Cells alone and PBS treated cells were studied as the black group and control group, respectively.
4.6. Cellular uptake5×103 4T1 cells per well were inoculated into the confocal dishes for incubating overnight. 200 μL 10% FBS DMEM containing 35.2μg/mL materials was added into well for incubating 4 h. After washing with PBS (pH 7.4) for 2-3 times, 4T1 cells were fixed by 4% PFA about 10 min at 37 ℃. Cell nuclei were stained with Hoechst 33258 (10 μg/mL) about 5 min at 37 ℃ after washing 2 times with PBS. After that, cellular uptake was assessed by FL imaging acquired by Leica TCS SP5-Ⅱ confocal laser scanning microscope (CLSM). The channel of nuclei and materials were chosen with 405/ 476 nm excitation and 461/660 nm emission, respectively.
Moreover, BDAccuriC6flowcytometry(FCM)wasalsoemployed to quantitatively determine cellular uptake. As described previously, 5×104 4T1 cells per well were seeded into 48 well plates, which further were incubated the suspension of materials as experimental groups and PBS as control group. After that, 4T1 cells were collected with the centrifuge under 1, 000 rpmabout4 minandwashed2times with PBS. The obtained cells were dispersed with 1 mL PBS for measuring FL intensity with FCM (FL-3A).
4.7. In vitro PDTFor in vitro PDT experiments, 1×104 cells per well were seed into 48 well plates with incubating the suspension of materials at 176 μg/mL as gold for 24 h. Subsequently, cells were exposed to the 660 nm laser with the energy density of 0.2 W/cm2 for 12 min. After incubating 4 h and washing with PBS, cells were stained with calcein-AM/PI dyes (2 μg/mL Calcein-AM, 3 μg/mL PI) for 8-10 min at 37 ℃. After rinsing again with PBS, cells were observed with fluorescent microscope (FM). As the control, cells treated with PBS were irradiated by the 660 nm laser also at 0.2 W/cm2 for 12 min.
PDT efficiency of materials was also confirmed by cellular viability for using MTT assays. As described previously for MTT assay, after 24 h incubation with different concentration of materials, cells were irradiated by the 660 nm laser under a power density of 0.2 W/cm2 at various time points. PBS treated cells under the 660 nm irradiation were studied as the control group. After that, cells were incubated 4 h for further measuring the value of OD490 nm with ELISA Reader after adding MTT and DMSO.
4.8. Mouse modalBALB/c athymic nude mice weighting 18-20 g were obtained from Charles River Laboratories (Beijing, China) and maintained under specific pathogen free (SPF) conditions in a small animal isolator. All food, water, bedding and cages were autoclaved before use. Tumor imaging and therapeutic effects were performed by 4T1 tumor-bearing nude mice. 4T1 cells (about 1×106 cells in 100 μL of PBS) were collected and further subcutaneously injected onto the right backside of female mice. The tumor volume was estimated using the formula, volume = length× (width)2/2.
4.9. NIR FL imaging and bio-distributionWhen tumor approached 150 mm3, 4T1-tumor bearing mice were employed for NIR FL imaging in vivo. Four mice of each group were injected intravenously with Au NCs and Au CNA suspensions (13.2 mg/kg) after anaesthetizing mice with 1% (W/V) pentobarbital sodium (5-7 μL/g). Mice without administration were studied as control. NIR FL imaging in vivo was acquired on IVIS Spectrum Imaging System using a 465 nm excitation wavelength and a 660 nm filter at different time points.
All mice were sacrificed after 24 h post-injection. Major organs and tumors were collected and subjected to ex vivo imaging from two mice treated with Au NCs and Au CNA suspensions, respectively. The FL intensity was qualified with region of interest (ROI). For the rest of mice, major organs and tumors were weighted with Mettler Toledo AB265-S analytical balance and transferred into 1-2 mL nitric acid (HNO3) solution. The dissolved mixture was heated at 100 ℃ as much possible as removal of HNO3 which further neutralized with 30% hydrogen peroxide (H2O2). The final solution was diluted to 10 mL with ultrapure water which further was employed to measure the gold concentration by ICP-MS.
4.10. In vivo PDTThe PDT imaging studies were performed when tumors reached 100 mm3. Eight mice of each group were utilized to assess the tissue toxicity and PDT efficiency. PBS treated mice were studied as control and materials (17.6 mg/kg) as experiment groups. The body weight of five mice in each group was measured every two or three days with Mettler Toledo AB265-S analytical balance. The other mice were employed to evaluate PDT efficiency when tumor exposed to the 660 nm laser (0.2 W/cm2) for irradiating different time. After 24 h post-treatment, Mice were euthanized for obtaining tumor tissues, which further were utilized to prepare the tissue frozen sections. The obtained sections were observed by optical microscope after hematoxylin and eosin (H&E) staining. The steps of H&E staining were as follows: Tissue by fixing with 4% PFA (overnight) and dehydrating with 10%-30% (w/v) Sucrose (disperse in PBS) was embedded into tissue OCT-freeze medium, which further was stored at -80 ℃. The frozen tissues were further cut as 8-10 μm for preparing sections with Leica CM 1950. The sections were covered with 100%-75% (v/v) ethanol for H&E staining and further re-dehydrated with 75%-100% (v/v) ethanol. Furthermore, the prepared sections were covered with 50%-100% (v/v) xylene (disperse in ethanol) and then sealed with neutral balsam for putting blank condition overnight.
AcknowledgmentsThis work was supported by the Major State Basic Research Development Program of China (973 Program) (No. 2015CB755500), National Natural Science Foundation of China (Nos. 31571013, 21375141, 81501580, 81401521, 81301272 and 81571745), Shenzhen Science and Technology Program (Nos. KQCX20140521115045447, JCYJ20150403091443298, JCYJ20130402092657771, JCYJ201504-01145529015 and JCYJ20160229200902680). Instrument Developing Project of the CAS (No. YZ201439), Key International S&T Cooperation Project (No. 2015DFH50230), Guangdong Natural Science Foundation of Research Team (2016A030312006).
Appendix A. Supplementary dataSupplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.12.038.
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