Fabrication of BSA@AuNC-Based Nanostructures for Cell Fluoresce

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Fabrication of BSA@AuNCs Based Nanostructures for Cell Fluoresce Imaging and Target Drug Delivery Caifeng Ding, Yujuan Xu, Yanan Zhao, Hua Zhong, and Xiliang Luo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18493 • Publication Date (Web): 19 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

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ACS Applied Materials & Interfaces

Fabrication of BSA@AuNCs Based Nanostructures for Cell Fluoresce Imaging and Target Drug Delivery Caifeng Ding*, Yujuan Xu, Yanan Zhao, Hua Zhong, Xiliang Luo* Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China S Supporting Information

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ABSTRACT: Drug delivery which can offer efficient and localized drug transportation together with imaging capabilities is highly demanded in the development of cancer theranostic approaches. Herein, we report the construction of bovine serum albumin (BSA) gold nanoclusters (BSA@AuNCs) for cell fluoresce imaging and target drug delivery. BSA@AuNCs were modified with cyclic arginine-glycine-aspartate (cRGD) with the product RGD-BSA@AuNCs to enhance the cell internalization of the nanoclusters. Furthermore, doxorubicin (DOX), a widely used a chemotherapy drug, was also used to modify RGD-BSA@AuNCs. The final design of DOX/RGD-BSA@AuNCs system was constructed through the disulfide bond. Physical microstructure and biological characterization of the BSA@AuNCs were realized through the high resolution transmission electronmicroscopy (HRTEM) and confocal laser fluorescence microscope. As the disulfide bonds were cleaved by glutathione (GSH) in cancer cells, DOX-SH molecules were released from the nano-system to inhibit the growth of cancer cells. The as-prepared DOX/RGD-BSA@AuNCs system cannot only be used to deliver drug but also to achieve the antitumor effect by in vivo imaging, demonstrating its promising applications in cancer treatment. KEYWORDS: BSA@AuNCs, doxorubicin, cRGD, GSH, DOX/RGD-BSA@AuNCs system, cell imaging, drugs delivery

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■ INTRODUCTION Accurate cancer detection and target therapy is essential for human health, as cancer is one of the most deadly diseases in the world today. As an effective chemotherapeutic agent, doxorubicin is often used to treat various tumor diseases, such as soft tissue sarcoma,1 breast cancer,2 malignant lymphomas,3 etc. However, some side effects are found during and after doxorubicin treatment, such as the injury to the non-targeted tissues and irreversible cardiotoxicity.4 To enhance the chemotherapy efficiency and the quality of patients’ life, targeted drug delivery has attracted great attention.5,6 Recently, various types of nanosystems, including liposomes,7-9 polymeric nanoparticles,10-12 micelles,13-15 and nanogels16-18 have been tested for delivering doxorubicin in vitro and in vivo to meet the pharmaceutical requirements. In order to minimize immune and fibrotic responses, surfaces of the nano-systerms need to be modified with biologic elements, or alternatively, the substrates are prepared directly from biologic molecules.19 Noble metal nanomaterials, especially gold nanoparticles, have been designed and utilized in many fields due to their unique optoelectronic properties,

excellent

biocompatibility,

high

catalytic

activity,

and

potential

noncytotoxicity.20-23 Compared to gold nanoparticles, gold nanoclusters (AuNCs) demonstrate unique luminescence and charging properties with smaller dimensions. Besides, AuNCs with ultrafine size and nontoxicity24-25 are highly attractive in the field of bio-labeling and bio-imaging.

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Bovine serum albumin (BSA), as the major soluble protein constituents in the circulatory system with many physiological functions,29 has been extensively used to prepare protein-based nanoparticles. Gold nanoclusters (AuNCs) prepared by BSA (BSA@AuNCs), which can emit red photoluminescence under ultraviolet light, has also been used to detect metal ions,30,31 small biomolecules,32 proteins,33 as well as in biological imaging.34 Arginine-glycine-aspartate (RGD), a peptide, consisting of tri-amino acid sequence, arginine-glycine-aspartate, is an important integrin binding site present in a variety of integrin ligands. An exhaustive literature has established that RGD is highly effective at promoting the attachment of various materials to different types of cell.35 In this study, we constructed a novel BSA@AuNCs-based nanoprobe, DOX/RGD-BSA@AuNCs system for in situ cell fluoresce imaging and drug delivery using RGD as cell penetrating peptide and doxorubicin hydrochloride (DOX) as chemotherapy drug.

■ EXPERIMENTAL SECTION Reagents

and

HAuCl4·3H2O,

Instruments.

N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide

hydrochloride

(EDC),

N-Hydroxysuccinimide (NHS), adenosine triphosphate (ATP), bovine serum albumin (BSA) were purchased from Aladdin reagent (Shanghai) co., LTD. Peptides cyclic (Arg-Gly-Asp)

(cRGD),

doxorubicin

hydrochloride

(DOX),

2-iminothiolane,

N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), alkaline phosphatase (ALP), lysozyme, tyrosine were synthesized and purified by Shanghai Sangon Biotechnology. 4


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Telomerase was purchased from shanghai Fu life Industry Co., Ltd. (Shanghai, China). Cervix carcinoma (Hela cells), breast cancer cells (MCF-7), glioma cell (U251), tongue squamous cancer cells (CAL-27) were obtained from FDCC (Shanghai China). RPMI 1640 cell culture medium and fetal bovine serum (FBS) were purchased from Invitrogen (Gibco). Annexin V-FITC apoptosis detection kit was purchased from Shanghai Sangon Biotechnology. UV-vis spectra were recorded with UH5300 spectrometer. Fluorescence measurements were performed on Hitachi F-4600 spectrofluorimeter (Tokyo, Japan) with a scan rate of 1200 nm/min. Cellular imaging analysis Laser scanning confocal microscopy was performed using Leica SP8 Point Scanning Confocal (Heidelberg, Germany). High resolution transmission electronmicroscopy (HRTEM) images were carried out with Philips CM200UT transmission electron microscope (Eindhoven, Netherlands). The mean particle size and Zeta potential were determined by using Malvern Zetasizer NanoZS (Malvern, United Kingdom). Cell apoptosis analysis was performed by using BECKMAN flow cytometric (Miami, America). Preparation of BSA@AuNCs. BSA@AuNCs was prepared according to the previously reported protocols.36 Briefly, an amount of 5 mL freshly prepared aqueous solution of HAuCl4 (10 mM) was added to 5 mL of aqueous solution of BSA (50.0 mg/mL) under vigorous stir at 37 oC. Two minutes later, 0.5 mL NaOH (1 M) solution was introduced to adjust the pH, and the mixture solution was incubated at 37 oC for 12 h with constant stirring. The light yellow solution turned to light brown and finally turned 5



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to dark brown. Then the solution was dialyzed with 10 kDa cutoff dialysis bag extensively against doubly distilled water to remove all unrelated small molecules. Finally, the BSA@AuNCs was obtained and stored at 4 oC. Preparation of RGD-BSA@AuNCs. An amount of 20 mg EDC was added into 4 mL prepared BSA@AuNCs solution, and the mixed solution was incubated under light protection for 20 min at room temperature with slow tilt rotation. And then 16 mg NHS was added into the mixture solution, then the mixture solution was left incubated in dark for another 1 h. Then 100 µL of c(RGD)fk (10 mg/mL) was added into the mixture solution, the mixture solution was incubated for 24 h at room temperature with slow tilt rotation. After that, the resulting solution is dialyzed with 10 kDa cutoff dialysis bag extensively against doubly distilled water to remove all small molecular impurities. Then the resulting solution was put into 30 kDa centrifugal filter unit 3000 g centrifugal for 20 min. At last, the final synthesized RGD-BSA@AuNCs were stored at 4 oC. Preparation of thiolated (-SH) doxorubicin. Thiolated doxorubicin (DOX-SH) was prepared by incubating the following mixtures of 1.5 mg DOX, 0.36 mg 2-iminothiolane in Phosphate Buffer Solution (PBS) and 1 mM EDTAfor 4 h. Preparation of DOX/RGD-BSA@AuNCs. An amount of 1.5 mg bifunctional cross-linker SPDP was added to the RGD-BSA@AuNCs solution with slow rotation. Four hours later, the mixture solution was dialyzed with 10 kDa cutoff dialysis bag extensively

against

doubly

SPDP/RGD-BSA@AuNCs.

distilled And

water

then,

for

DOX-SH

6


24

h

to

reacted

obtain

the

with

the

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SPDP/RGD-BSA@AuNCs to get the final product DOX/RGD-BSA@AuNCs. The product was purified by dialysis in PBS solution with 1 mM EDTA for 24 h, and stored in a refrigerator at 4 oC. In Vitro Release Study of DOX/RGD-BSA@AuNCs. In order to detect GSH, different

concentrations

of

GSH

were

mixed

with

200

µL

purified

DOX/RGD-BSA@AuNCs solution in a series of 2.0 mL calibrated tubes. Then, the mixture solutions were diluted to 1.0 mL with pH 7.4 PBS buffer (10 mM) solution. The mixture solutions were incubated at 25 oC for 4 h. After that, the mixture solutions were put into 30 kDa centrifugal filter unit and centrifuged (3000 g) for 20 min, and then the fluorescent spectra of the supernatants were recorded. In Vitro Imaging. Hela cells, MCF-7 cells, U251 cells, CAL-27 cells (2 × 104 cells per dish) were respectively seeded into confocal dishes with 10 mm bottom well in 1640 medium for 24 h. After growing the cells overnight in medium containing, the cells were separated from the culture medium and rinsed with PBS solution for three times. Later 500 µL DOX/RGD-BSA@AuNCs solution was dispersed into the culture medium of cells, and incubated with cells in an incubator at 37 oC. The cell imaging and morphology of treated tumor cells were observed under bright light and fluorescence with confocal laser fluorescence microscopy, and images were captured every 5 minutes. Flow Cytometric Analysis of Apoptosis. Hela cells at logarithmic growth phase with a density of 1×105 cells/mL were seeded into a six-well plate and treated with DOX/RGD-BSA@AuNCs and PBS dispersion at 37 oC. The control group was treated 7



with PBS solution only. After incubated with different time, cells were rinsed with ice-cold PBS solution, harvested through trypsin (without EDTA), and centrifuged at 1000 rpm for 5 min. After rinsed with PBS solution twice, the cells were dispersed in 100 µL binding buffer followed with the addition of 5 µL annexin V-FITC and 5 µL 7-Aminoactinomycin D (7-AAD). The above mixture solutions were incubated in the dark for 15 min. After the addition of 1 mL binding buffer, the samples were kept in ice before flow cytometry analysis. Cells which were negative for both 7-AAD and annexin V staining will be live ones, while those that were negative to 7-AAD and positive to annexin V were early apoptotic cells. And cells were positive to both 7-AAD and annexin V staining are those at the later stages of apoptosis and can be seen as dead cells. Antitumor Effect Assay in Vivo. Female Balb/c mice were purchased from Nanjing Ling Chang Biological Technology Co. Ltd.. Hela cells (1×106) suspended in 40 µL PBS solution were subcutaneously injected into the back of each female Balb/c mouse. The mice were randomly grouped (n = 2) when the tumor volume reached 100 mm3 and treated with 100µL PBS and DOX/RGD-BSA@AuNCs solutions. After injection, the tumors in the DOX/RGD-BSA@AuNCs-injected groups were locally treated with a 488 nm laser at photodensity of 2.0 W/cm2 for 5 min, 1, 2, 4, 8, 24 h. Body weight and tumor sizes were monitored every day for 3 weeks. The length and width of the tumors were measured with a digital caliper.

■ RESULTS AND DISCUSSION

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Scheme 1 Illustration of the strategy of the DOX/RGD-BSA@AuNCs system for cell fluoresce imaging and drug delivery.

The

principle

of

the

novel

BSA@AuNCs-based

nanoprobe

DOX/RGD-BSA@AuNCs system for in-situ cell fluoresce imaging and drug delivery are shown in Scheme 1. For BSA@AuNCs, the surface which retains BSA’s intrinsic structure and biological functions was synthesized firstly. Considering multiple bio-functionalizations of those clusters, we employed them as vehicles to deliver both c(RGD) and DOX, which also enabled the clusters multi-functions. In detail, the carboxyl groups on BSA were reacted with the amino groups of c(RGD) and led to the formation of nanosystem--a recognizer with tumor cells. In order to attach DOX to the nanosystem, the amino groups of BSA reacted with the bifunctional cross-linker SPDP first, forming pyridyldisulfide groups on BSA. Then DOX-SH was introduced to react with the SPDP-conjugated RGD-BSA@AuNCs to construct DOX/RGD-BSA@AuNCs through a thiol exchange reaction with the formation of a disulfide bond between DOX and RGD-BSA@AuNCs.

Due

to

endocytosis,

the

resulted

nanoclusters

DOX/RGD-BSA@AuNCs enter the cells. With c(RGD) as the tumor-targeting ligand,

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the nanosystem can successfully recognize αvβ3 integrin, which is highly expressed in the tumor cells. As the disulfide bond between the DOX-SH and RGD-BSA@AuNCs can be cleaved with GSH in the cells, DOX-SH can be released. Because the functionalised BSA@AuNCs has good biocompatibility and high fluorescent signal, it should be successfully applied in cell imaging and drug delivery in living cells.

Characterization

of

BSA@AuNCs

and

DOX/RGD-BSA@AuNCs.

The

BSA@AuNCs prepared in our work has an average diameter of approximately 8±0.5 nm measured by HRTEM as shown in Figure 1A. From the result we can see that the crystal lattice fringes are 0.23 nm which corresponds to the spacing of crystal plane of Au. Optical properties of BSA@AuNCs were studied with UV−vis absorption, fluorescent excitation and emission spectroscopy were studied to investigate the optical properties of BSA@AuNCs (Fig. 1B, Fig. 1C). Fig. 1B displays the UV-vis absorption spectrum of the BSA@AuNCs, the peak on the UV-Vis spectrum around 280 nm corresponds to the BSA@AuNCs. The inset in Fig. 1B shows the photographs of the BSA@AuNCs under daylight and UV irradiation. The red photoluminescence under illumination by ultraviolet light demonstrates the successful construction of BSA@AuNCs. The fluorescence excitation and emission spectroscopy of BSA@AuNCs are also presented in Fig. 1C. The maximum excitation wavelength is at 510 nm, and it was selected for the subsequent fluorescence measurements. An emission peak maximum of 650 nm is observed. The loading of DOX to the nanosystem was investigated by UV-Vis characterization as shown in Fig. 1D. From Fig. 1D we can see that the UV-Vis spectrum peak of the 10


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RGD-BSA@AuNCs (curve a) is around 280 nm, while the UV-vis absorption spectrum of DOX (curve c) exhibited a characteristic absorption centered at 480 nm. Two obvious peaks around 280 nm and 480 nm in curve b indicate that the DOX was successfully conjugated to the surface of RGD-BSA@AuNCs. The sizes of BSA@AuNCs, RGD-BSA@AuNCs and DOX/RGD-BSA@AuNCs were also detected by Zetasizer NanoZS as shown in Table S1. There is no obvious difference in the sizes of the three nanoclusters because cRGD and DOX are too small to affect the size of BSA@AuNCs due to the BSA’s three-dimensional structure.The zeta potential of RGD-BSA@AuNCs is increased compared with BSA@AuNCs because the amino groups of cRGD were reacted with the carboxyl groups of BSA@AuNCs. The zeta potential of DOX/RGD-BSA@AuNCs is increased compared with RGD-BSA@AuNCs because positive charge of DOX.

Fig. 1 (A) HRTEM image of BSA@AuNC. (B) Absorption spectrum of BSA@AuNC. The inset displays the photographic images of BSA@AuNC solutions (under broad daylight and UV light ). (C) Fluorescence excitation and emission spectra of BSA@AuNC. (D) UV-Vis spectrum of BSA@AuNCs(curve a), DOX/RGD-BSA@AuNCs (curve b) and DOX(curve c).

The stability of BSA@AuNCs and DOX/RGD-BSA@AuNCs. The emission spectra of as-prepared BSA@AuNCs and DOX/RGD-BSA@AuNCs were obtained in different 11



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times as shown in Fig. S1. From the result we can see that BSA@AuNCs and DOX/RGD-BSA@AuNCs are all very stable even after almost 2 months stored at room temperature. Real-Time Study of Cell Imaging and DOX-SH Release in Living Cells. Confocal microscopy was used to verify the cell imaging and drug delivery capability of DOX/RGD-BSA@AuNCs. Hela cells were incubated with PBS solution, BSA@AuNCs and DOX/RGD-BSA@AuNCs system for 2 h respectively. As shown in Fig. S2, no changes are found on the sample of Hela cells cultured with PBS solution for 2 h. For the sample Hela cells cultured with BSA@AuNCs, red fluorescence is not observed either, suggesting that BSA@AuNCs could not enter the cells with the formation of DOX/RGD-BSA@AuNCs system. Due to the recognition of the RGD and the function of GSH, the fluorescence signal of Hela cells cultured with DOX/RGD-BSA@AuNCs system can be observed in both channels of red and yellow. The results demonstrate that the DOX/RGD-BSA@AuNCs system can not only enter cells, but also can provide efficient cell imaging and drug release, demonstrating a promising nanoplatform for cancer therapy. In

order

to

monitor

the

cell

imaging

and

DOX-SH

release

of

DOX-RGD-BSA@AuNCs system in real time, cell images were recorded at different time intervals via multichannel confocal microscopy. As shown in Fig. 2, the BSA@AuNCs channel and DOX channel of DOX/RGD-BSA@AuNCs system are indicated with the red and yellow fluorescence signal respectively. After 2 h of treatment 12


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with DOX/RGD-BSA@AuNCs system, the red fluorescence (BSA@AuNCs) signal and yellow fluorescence signal (DOX) appeared on the cell membrane and inside the cytoplasm. After treated for 4 h, the intensity of the red and yellow fluorescence all increased. This is highly indicative of the DOX/RGD-BSA@AuNCs system translocation to the cell and the subsequent DOX-SH release as a result of the effect of GSH. After 6 h of incubation, the intensity of the red and yellow fluorescence signals from DOX/RGD-BSA@AuNCs system increased dramatically. These results suggest that the DOX/RGD-BSA@AuNCs system is an excellent candidate to be applied in intracellular detection.

Fig. 2 Multichannel confocal microscopy images of Hela cells treated with DOX/RGD-BSA@AuNCs system at different incubation time (0, 2, 4, 6 h). The channel of BSA@AuNCs is indicated by the red fluorescence, and the DOX-SH is indicated by the yellow fluorescence. The scale bar is 100 µm.

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The Efficiency of DOX-RGD-BSA@AuNCs with Multifold Cancer Cells. Here we further investigated the capabilities of the DOX/RGD-BSA@AuNCs system for intracellular imaging in other tumor cells. Hela cells, MCF-7, U251, CAL-27 were incubated with the DOX/RGD-BSA@AuNCs system for 2 h. The cellular internalization and intracellular localization of DOX/RGD-BSA@AuNCs system in different cells were studied using confocal laser microscope after treated for 2 h (Fig. 3). Notably, the red (BSA@AuNCs channel) and yellow (DOX channel) fluorescence signal suggests that plenty of DOX/RGD-BSA@AuNCs have entered into the tumor cells. The results implied that DOX/RGD-BSA@AuNCs system not only has an effect on Hela cells, but also can enter into other cancer cells. Thus DOX/RGD-BSA@AuNCs system is membrane permeble and feasible for cell fluoresce imaging and drug delivery.

Fig. 3. Confocal microscopy images of Hela, MCF-7, U251, CAL-27 cells incubated with DOX/RGD-BSA@AuNCs (the figure respectively is blank, BSA@AuNCs, DOX and merge channel fluorescence imaging ) : from top to bottom images represent Hela, MCF-7, U-251, CAL-27 cells incubated for 2 h. The scale bar is 25 µm.

To trace the intracellular distribution in cancer cells, DOX-SH has been monitored by confocal

laser

microscope

after

the

Hela

cells

14


were

treated

with

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DOX/RGD-BSA@AuNCs system for 6 h (Fig. S3). From the result we can see that the DOX/RGD-BSA@AuNCs (orange), which was the merge of fluorescence signal from BSA@AuNCs (red) and DOX-SH (blue), was observed in the cytoplasm and nuclei comparing with DAPI (blue). It demonstrates that intracellular DOX-SH was caused by the efficient delivery of DOX/RGD-BSA@AuNCs system and efficient release from the nanosystem in the presence of GSH in cancer cells. Thanks to cRGD and GSH, DOX-SH can be able to be carried into cells quickly and accurately, transfer, and distributed in cytoplasm and nucleus regions. In vitro release study in presence of glutathione. To demonstrate the distribution of DOX-SH in cancer cells, the responses of DOX/RGD-BSA@AuNCs to GSH (Fig. S4) were studied. The fluorescence signal intensity of supernatant gradually increases with concentrations of GSH (Fig. S4A). Meanwhile, a good linear relationship in the range from 1-10 mM with a correlation coefficient of 0.9946 was obtained (Fig. S4B). Clearly, we can see that more DOX-SH can be released with the increase of GSH concentration. We have to point out that despite the detection limit of this approach, considering the concentrations of GSH in cancer cells which is around 2-10 mM,37 our work is still a practical approach. To demonstrate the release of DOX-SH from DOX/RGD-BSA@AuNCs in presence of GSH, the release behaviors of DOX-SH loaded within DOX/RGD-BSA@AuNCs was further studied in cancer cells. Fluorescence and flow cytometry measurements of DOX in Hela cells which were incubated with DOX/RGD-BSA@AuNCs system at 37 oC were 15



shown in Fig. S5A. DOX fluorescence from Hela cells measured by flow cytometry confirms the increasing cellular uptake of DOX/RGD-BSA@AuNCs system at 37 oC. While all cells incubated with DOX/RGD-BSA@AuNCs system shows increased fluorescence over Hela cells without DOX/RGD-BSA@AuNCs system. After 0.5 h, the fluorescence signal of the DOX in Hela cells shifts to the right, indicating a significant release of drug into the cells. The fluorescence signal of the DOX in Hela cells incubated at 37

o

C for 2h shifts to the most right side. These results confirm the

DOX/RGD-BSA@AuNCs system can enter cells easily in 2 h, and the drug release from DOX/RGD-BSA@AuNCs system could be accelerated after interacting with GSH in the Hela cells. In Fig. S5B, Confocal microscope images in Hela cells incubated with DOX/RGD-BSA@AuNCs system after various incubation times (0, 0.5, 1, 1.5, 2.0, 2.5 h), and the fluorescence signal intensity of supernatant was gradually increased with time. These results clearly demonstrate that GSH are potentially useful for delivering thiolated anticancer drugs into cancer cells. Therefore, a short incubation time could extend the chances of interaction between the carrier and GSH, resulting in enhanced drug release. The selectivity of the DOX/RGD-BSA@AuNCs systems. Selective recognition capability is a very significant character to investigate the performance of the DOX/RGD-BSA@AuNCs system. Therefore, in order to verify the specificity of the DOX/RGD-BSA@AuNCs system for the recognition of GSH, the influence of some common substance including inorganic salts (K+, Ca2+, Mg2+ and Na+), ATP, BSA, enzyme (ALP, Telomerase, Lysozyme) and amino acids (tyrosine) was respectively 16


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studied in aqueous solutions. The results are shown in Fig. S6. With the addition of the solution of interfering substance (1 mM), the fluorescence intensity of the supernatant is very low (fluorescence intensity change less than 20 a.u.), the enhanced intensity is found with the addition of GSH. The results indicate that the DOX/RGD-BSA@AuNCs system shows the high selectivity to GSH. To make it simple, after DOX/RGD-BSA@AuNCs system enters the cells, the DOX-SH can be successfully released from the nanosystem in the presence of GSH. Thus, the DOX/RGD-BSA@AuNCs system is suitable for the drug delivery. Flow Cytometric Analysis of Apoptosis. Apoptosis, a kind of gene-mediated programmed cell death and a key phenomenon, can be induced by drugs in antitumor treatment. In this study, the annexin V-FITC/7-AAD double staining assay with flow cytometry detection was implemented to evaluate the extent of cell apoptosis caused by as-prepared DOX/RGD-BSA@AuNCs system. The existence of apoptosis cells after cultured with DOX/RGD-BSA@AuNCs system was observed as shown in Fig. 4, the results of flow cytometry indicate that there is an increase of apoptosis rate with the treatment of the DOX/RGD-BSA@AuNCs system and the apoptosis rates increase with time. Around 9.05% of cells are in apoptotic stage following treatment with DOX/RGD-BSA@AuNCs system for 1 h, and 58.02% of apoptosis when treated with DOX/RGD-BSA@AuNCs system for 12 h.(Fig. 4) The results of flow cytometry suggesting that DOX/RGD-BSA@AuNCs system can implement drug transport and release, at last leading cell to death. 17



Fig. 4. Cell apoptosis analysis. Annexin V-FITC/7-AAD double staining flow cytometric analysis of HeLa cells A, B, C, D, E, F incubated with DOX/RGD-BSA@AuNCs system for 1, 2, 3, 6, 12, 24h.

The cytotoxicity assay of different nanoparticles. The cytotoxicity assay of BSA@AuNCs, RGD-BSA@AuNCs, DOX/RGD-BSA@AuNCs and DOX against HeLa cells was studied as shown in Fig. S7. After cultured with PBS solution, BSA@AuNCs solution, RGD-BSA@AuNCs solution, DOX/RGD-BSA@AuNCs solution (equivalent to 2.0 × 10-5 M free DOX according to the intensity of fluorescence), and free DOX solution (2.0 × 10-5 M), respectively, for 2h, the cell apoptosis rate was detected by flow cytometry. From Fig. S7 we can see that the rate of apoptosis cells was 0.2%, 6.39%, 14.19%, 38.56%, and 53.70% according to different agents. The results suggest that DOX/RGD-BSA@AuNCs resulted in a stronger tumor cell inhibition compared with free DOX. However, they all exhibited significantly higher cytotoxicity than BSA@AuNCs and RGD-BSA@AuNCs. In vitro assays demonstrated that the anticancer efficiency is dependent on the enhanced cell uptake mediated by cRGD moieties on the nanoclusters and the function of DOX.

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Antitumor Effect Assay in Vivo. To further improve the efficacy of our combination therapy based on DOX/RGD-BSA@AuNCs system, PBS and DOX/RGD-BSA@AuNCs solutions (the same as mentioned previously) were administered by a single intratumoral injection into Hela-tumor-bearing mice. After injection of DOX/RGD-BSA@AuNCs solution into the subcutaneous tumor-bearing mice, the fluorescence images of cancer site could be distinguished from the surrounding normal tissue at 5 min postinjection (p.i.). The fluorescence images of cancer site increased gradually and reached a maximum at 2 h p.i., which was maintained even after 4 h p.i. After 8 h p.i., the fluorescence images of cancer site reduce gradually, and reached the minimum at 24 h p.i. (Fig. 5A). The result demonstrates the targeted delivery and long retention of DOX/RGD-BSA@AuNCs system in cancer tissue. The tumor size and body weight after treated with PBS solution and DOX/RGD-BSA@AuNCs system were then monitored (Fig. S8). Compared with the mice treated with PBS solution, there is no significant weight loss observed in the mice which was treated with DOX/RGD-BSA@AuNCs system, suggesting that the treatment with DOX/RGD-BSA@AuNCs system was nearly nontoxic despite the pronounced tumoricidal effects (Fig. S8A). At the same time, the tumor development was monitored by measuring the tumor size at regular intervals for 19 days (Fig. S8B). The tumor size of the mice which was treated with DOX/RGD-BSA@AuNCs system was smaller than the mice that treated with PBS solution, indicating that the DOX/RGD-BSA@AuNCs system can delay tumor growth effectively as shown in Fig. 5B. This result further strengthened our in vitro observation and signified that the tumor growth could be greatly inhibited by 19



the effect of DOX/RGD-BSA@AuNCs system in vivo.

Fig. 5. (A) In vivo targeted cancer fluorescence images of Hela tumor-bearing mice exposed to the laser (488 nm, 425 mW cm2, 25 min) after it injection of the DOX/RGD-BSA@AuNCs system. (B) The images of tumor excised from mice after injection of PBS solution or DOX/RGD-BSA@AuNCs solutions at 19 day.

■ CONCLUSIONS In conclusion, a multifunctionalized nanostructure has been successfully developed through sequential assembly of BSA@AuNC-based nanoprobe for cell fluoresce imaging and drug delivery in living cell. As intrinsic structure of BSA@AuNCs surface and biological functions of BSA, the reported nanocarrier in our work shows high biocompatibility and stability. The DOX/RGD-BSA@AuNCs system can significantly enhance cell internalization due to the integrin targeting of cRGD. Moreover, the anticancer drug (DOX-SH) can be released quickly and efficiently to inhibit tumor cell growth. The as-prepared DOX/RGD-BSA@AuNCs system not only delivers the anticancer drug (DOX) into cells and enables multi-channel imaging of various cancer

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cells but also signifies the important role of targeting units for improving antitumor efficiency in vivo. This DOX/RGD-BSA@AuNCs system represents an important strategy for efficiently cell fluoresce imaging and drug delivery, indicating its great potential to be used in disease diagnosis.

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■ ASSOCIATED CONTENT * S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: The detection of GSH, confocal fluorescence microscope images of DOX/RGD-BSA@AuNCs system with Hela cells, and the Selection of DOX/RGD-BSA@AuNCs system.

■ AUTHOR INFORMATION *Corresponding Authors E-mail: [email protected] E-mail: [email protected] Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS The authors greatly acknowledge support from the National Natural Science Foundation of China (21375070, Grants 21422504), the Taishan Scholar Program of Shandong Province of China (Grant ts20110829), and the Fund Project for Shangdong Key R&D Program (2017GGX20121).

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Fabrication of BSA@AuNC-Based Nanostructures for Cell Fluoresce

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