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Multifunctional Aptamer-Silver Conjugates as Theragnostic Agents for Specific Cancer Cell Therapy and Fluorescence-Enhanced Cell Imaging Hui Li, Hongting Hu, Yaju Zhao, Xiang Chen, Wei Li, Weibing Qiang, and Danke Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504230j • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Analytical Chemistry

Multifunctional Aptamer-Silver Conjugates as Theragnostic Agents

for

Specific

Cancer

Cell

Therapy

and

Fluorescence-Enhanced Cell Imaging Hui Li, Hongting Hu, Yaju Zhao, Xiang Chen, Wei Li, Weibing Qiang, Danke Xu† †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, China Corresponding Author Tel/Fax (+) 00862583595835 E-mail: [email protected]

1

ACS Paragon Plus Environment


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Abstract: We fabricated a multifunctional theragnostic agent Ag-Sgc8-FAM for apoptosis-based cancer therapy and fluorescence-enhanced cell imaging. For cancer therapy, aptamers Sgc8 and TDO5 acted as recognizing molecules to bind CCRF-CEM and Ramos cells specifically. It was found that aptamer-silver conjugates (Ag-Sgc8, Ag-TDO5) could be internalized into cells by receptor-mediated endocytosis, inducing specific apoptosis of CCRF-CEM and Ramos cells. The apoptosis of cells depended on the concentration of aptamer-silver conjugates, as well as the incubation time between cells and aptamer-silver conjugates. The apoptotic effects on CCRF-CEM and Ramos cells were different. Annexin V/PI staining, AO/PI staining, MTT assays and ROS (Reactive oxygen species) detection demonstrated the specific apoptosis of CCRF-CEM and Ramos cells. For fluorescence-enhanced cell imaging, Ag-Sgc8-FAM was prepared. Compared to Sgc8-FAM molecules, Ag-Sgc8-FAM was an excellent imaging agent as numerous Sgc8-FAM molecules were enriched on the surface of AgNPs for multiple binding with CCRF-CEM cells and signal amplification. Moreover, AgNPs could increase the fluorescence intensity of FAM by metal-enhanced fluorescence (MEF) effect. Therefore, aptamer-silver conjugates can be potential theragnostic agents for inducing specific apoptosis of cells and achieving cells imaging in real time.

Keywords: Silver nanoparticles; Aptamers; CCRF-CEM; Ramos; Apoptosis; Fluorescence imaging

Introduction Nanoparticles (NPs) are used in the biomedicine and biological systems because of their potential functions in diagnostic and therapeutic applications[1]. Their excellent optical property has been used in biological imaging and cancer diagnostic applications; additionally, the ability to perform both imaging and therapy is a newly emerging concept[2]. Theragnostic nanoparticles usually contain imaging agent and therapeutic agent, enabling diagnosis, therapy, and monitoring of therapeutic effect[2], the nanoparticles themselves have no effect on diagnosis or therapy. Silver nanoparticles (AgNPs) have received considerable attention as potential cancer therapeutic agents and extensive researches have been done on their cytotoxic effects in recent researches[3-5]. Among all nanomaterials, AgNPs are most frequently reported for inducing programmed cell death 2


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(Apoptosis). Apoptosis is a highly recognized cellular process that distinct from necrosis, and also plays a significant role in physiological growth and tissue homeostasis[6, 7]. Compared to the detrimental effect of necrosis that can cause inflammation, the apoptosis is a natural and well-controlled process that benifit cells[8]. Apoptosis-based cancer cell therapy has the potential to avoid the side effects of currently applied immunotherapy, chemo-and radiotherapy that are often harmful to normal cells[9, 10]. The proposed reason for apoptosis in AgNPs-treated cells is the generation of reactive oxygen species (ROS), which is known to cause irreversible DNA damage [3, 11]

. AgNPs-induced apoptosis have great potential for cancer cell therapy when AgNPs have

suitable size, micrograph and surface modification. Some reports have shown that mammalian cells undergo increased apoptosis after incubation with unconjugated and conjugated AgNPs [11, 12]. However, AgNPs are never reported as theragnostic agents for both specific cancer therapy and fluorescence-enhanced cell imaging. We aim to design a specific and sensitive theragnostic agent based on AgNPs for cancer therapy and fluorescence-enhanced cell imaging. For cancer therapy, target recognition is extremely important, since targeted therapy will largely improve cancer treatment with more efficiency and fewer side effects[13]. The molecules, such as folic acid[14], peptides[11,

15]

, metabolic glycan[16], hyaluronan oligosaccharide[17], and

aptamers[18] for target cell recognition always recognize corresponding receptors on the membrane of cells. Aptamers that target cell membrane can be derived from a process termed cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) [19]. Compared to other recognizing molecules, aptamers are generally more stable and can be synthesized or modified easily[20]. Aptamers can be more easily bound with nanomaterials to fabricate multifunctional nanomaterials for specific cell therapy and imaging[21-25]. Aptamers Sgc8 and TDO5 were selected by Tan’s group[26-28] to recognize membrane protein tyrosine kinase-7 (PTK-7) on CCRF-CEM (CCL-119, T-cell line, human acute lymphoblastic leukemia) cell membrane and immunoglobulin heavy mu chain on the surface of Ramos (CRL-1596, B-cell line, human Burkitt's lymphoma) cells. The combination of Sgc8 or TDO5 with AgNPs can accomplish the specific apoptosis of target cells. The toxicity of AgNPs was highly related to its size, coating surface and shape. Here, approximately 50 nm spherical AgNPs were synthesized according to previously reported method with some modifications[29]. The synthesized AgNPs were then conjugated with thiol-terminated poly-(ethylene glycol) (mPEG-SH 5000) and aptamers to prepare Sgc8-functionalized AgNPs 3



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(Ag-Sgc8) and TDO5-functionalized AgNPs (Ag-TDO5). Pegylation of nanoparticles was beneficial to maintain the stability of the nanoparticles in cell culture and reduced protein nonspecific absorption on the particles’ surface [11, 30]. Aptamer Sgc8 was reported to be taken up by CCRF-CEM cells and delivered into the endosomes through their interaction with membrane protein tyrosine kinase-7 (PTK 7) on target cells[31]. Therefore, Ag-Sgc8 could also be internalized into target cells by aptamer-mediated endocytosis and then induced apoptosis of CCRF-CEM cells. To ensure Ag-Sgc8 was the most efficient at inducing apoptosis, we conducted Annexin V/PI staining, AO/PI staining, MTT assays on both Ag-Sgc8 and Ag-TDO5. The results demonstrated that aptamer-silver conjugates could induce specific apoptosis of CCRF-CEM and Ramos cells. Developing a sensitive cell imaging technique for monitoring the AgNP-induced apoptotic process is important and challenging. Fluorescence techniques are most frequently used in biomedical imaging due to their simplicity, high sensitivity and high information content[32]. Fluorescent probes such as organic dyes are susceptible to photo-bleaching which greatly affect their use in monitoring cellular processes[33, 34]. AgNPs can result in metal-enhanced fluorescence (MEF) effect on nearby fluorescent dyes that can cause a signiﬁcant enhancement of emission intensity with reduced lifetime, photoblinking and increased photostability[35, 36]. The MEF of AgNPs has been used for increasing sensitivity of protein detections[20, 37, 38] and enhanced cell imaging[35,

36]

. AgNPs-enhanced cell imaging technique is still worth of developing. The

theragnostic agent Ag-Sgc8-FAM was prepared to achieve both imaging and therapy purposes on cancer cells, it did not only induce apoptosis of CCRF-CEM cells, but also produce enhanced fluorescence signal to monitor the apoptotic process (Scheme 1).

Materials and methods Materials and reagents. CCRF-CEM (CCL-119, T-cell line, human acute lymphoblastic leukemia) and Ramos (CRL-1596, B-cell line, human Burkitt's lymphoma) were cultured in RPMI 1640 medium (ATCC) with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and penicillin (80U/mL)-streptomycin (0.08mg/mL ) (KeyGEN Biotech, Nanjing, China) at 37 ℃under a 5% CO2 atmosphere. Cells were washed with 1×PBS (Phosphate Buffered Saline, Gibco®, Scotland, UK ). Binding buffer (BB) was prepared by adding yeast tRNA (0.1 mg/mL) and BSA (1 mg/mL) to the Dulbecco's PBS with calcium chloride and magnesium chloride 4


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(Sigma-Aldrich), Annexin V-FITC Apoptosis Detection Kit, Cell Apoptosis Acridine Orange(AO) and Propidium Iodide (PI) Detection Kit, MTT Cell Proliferation and Cytotoxicity Detection Kit, Reactive Oxygen Species Assay Kit (KeyGEN Biotech, Nanjing, China), Na2S2O3 and K3[Fe(CN)6] (Nanjing Chemical Reagent Co., Ltd), mPEG-SH (MW=5000, Yarebio, Shanghai, China),Glass Bottom Cell Culture Dish (Φ15mm, Nest Biotechnology Co., Ltd, China). DNA library (Lib) and oligoncleotides were purchased from Shanghai Sengon Biotechnology Co. The sequences of used oligonucleotides are as follows: Sgc8: 5’SH-(CH2)6-AAAAAAAAAAATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA TDO5: 5’ SH-(CH2)6-AAAAAAAAAAAACACCGTGGAGGATAGTTCGGTGGCTGTTCAGGGTCTCCTCCCGGTG Sgc8-FAM: 5’SH-(CH2)6-AAAAAAAAAAATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-FAM Sgc8-TAMRA: 5’SH-(CH2)6-AAAAAAAAAAATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-TAMRA lib：5’SH-(CH2)6-AAAAAAAAAAA-random sequence lib-FAM：5’SH-(CH2)6-AAAAAAAAAAA-random sequence-FAM

Tetramethylrhodamine anhydride (TAMRA), Fluorescein isothiocyanate (FAM). Apparatus. BioTek (Synergy H1, USA) was used to take UV-Vis spectra, and transmission electron microscope (TEM) (JEM-200CX, Japan) was used for collecting TEM images. Zeta potential were determined by Malvern Zetasizer (Nano-Z, Worcestershire, UK). Flow cytometry (FC 500, Beckman Coulter, USA) was used to take fluorescence intensity of cells. Confocal scanning laser microscopy (CSLM) (TCS SP5, Leica, Germany) was used to take fluorescence images of cells. Synthesis and characterization of Ag-Sgc8, Ag-TDO5 and Ag-Lib. Silver nitrate (AgNO3) was reduced by sodium borohydride (NaBH4) to make nanosilver seed, and then 1% PVP (polyvinylpyrrolidone), 5 mM L-sodium ascorbate, 50 mM trisodium citrate, 5 mM silver nitrate was successively added to nanosilver seed with equal volume to make large silver nanoparticles (AgNPs). Ag-Sgc8 was prepared by mixing 1 mL AgNPs with mPEG-SH (25 µL, 10 µM), Sgc8 (75 µL, 10 µM), NaCl (58 µL, 2 M) and 1µL Tween for 2 h with strong shaking, Ag-TDO5 was prepared by mixing 1 mL AgNPs with mPEG-SH (25 µL, 10 µM), TDO5 (75 µL, 10 µM), NaCl (58 µL, 2 M) and 1µL Tween for 2 h with strong shaking, Ag-Lib was prepared by mixing 1 mL AgNPs with mPEG-SH (25 µL, 10 µM), Lib (75 µL, 10 µM), NaCl (58 µL, 2 M) and 1µL Tween for 2 h with strong shaking, after standing for 12h, the solution was centrifuged for 3 times, the 5



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precipitate was re-dispersed by binding buffer (BB). Cell apoptosis assay. To assess the fate of CCRF-CEM, Annexin V/PI staining was performed to investigate the apoptosis and necrosis induced by Ag-Sgc8, Ag-TDO5 and Ag-Lib. Cells (2.0×105) were seeded in 48 well plate with Ag-Sgc8 or Ag-TDO5 at various concentrations (0.37 nM, 1.5 nM, 6 nM) for different times (6 h, 12 h, 24 h), after centrifugation and washing, Annexin V-FITC (5 µL) and PI (2 µL) were added for 15 min, then flow cytometry was used to take fluorescence intensity in FL1-H and FL3-H channel. For specificity, the CCRF-CEM Cells (2.0×105) were separately seeded in 48 well plate with BB, 6 nM AgNPs, Ag-Sgc8, Ag-TDO5 and Ag-Lib for 2h, after centrifugation and washing, the cells were cultured for another 24 h, after centrifugation and washing, Annexin V-FITC (5 µL) and PI (2 µL) were added for 15 min, fluorescence intensity was taken by flow cytometry. Acridine orange (AO) and propidium iodide (PI) staining. AO and PI staining were used investigate the cell viability. CCRF-CEM Cells (2.0×105) were seeded in 48 well plate with BB, 6 nM AgNPs, Ag-Sgc8, Ag-TDO5 and Ag-Lib for 2h, Ramos Cells (2.0×105) were seeded in 48 well plate with BB, Ag-Sgc8 (1.5 nM) and Ag-TDO5 (1.5 nM) for 2h, after centrifugation and washing step, the cells were cultured for another 24 h, after centrifugation and washing, AO (5 µL) was added for 15 min and PI (5 µL) for 5min, then fluorescence images were taken by CSLM. In Vitro cytotoxicity assays. CCRF-CEM cells and Ramos cells were seeded in 48 well plate (4 × 105 cells/well). 6 nM Ag-Sgc8, Ag-TDO5 and Ag-Lib were added to CCRF-CEM for 2h, Ag-Sgc8 (1.5 nM) and Ag-TDO5 (1.5 nM) were added to Ramos cells for 2h, after washing with 1×PBS for 2 times, RPMI 1640 medium were added for 24 h, after washing, 50 µL 2×MTT was added for 4 h, the absorbance value was taken by Bioteck. Reactive oxygen species (ROS) Generation. CCRF-CEM cells (3.0×105) were treated with BB, Ag-Sgc8 (6 nM) and Ag-Sgc8 (9 nM) for 12 h, after washing step, DCFH-DA (100 µM, 10 µL) was added for 15 min, after washing step, flow cytometry and CSLM were used to take the fluorescence intensity and images. Ag-Sgc8-FAM probe for cell imaging. Five different Ag-Sgc8-FAM probes were prepared by mixing 1 mL AgNPs with mPEG-SH (0~100 µL, 10 µM) and Sgc8-FAM (0~100 µL, 10 µM) for 2 h, the ratio of Sgc8-FAM and mPEG-SH are 100 µL : 0 µL, 75 µL : 25µL, 50 µL : 50 µL, 25 µL : 75 µL, 0 µL : 100µL, after standing for 12 h, the solution was centrifuged for 3 times, the 6


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precipitate was re-dispersed by binding buffer (BB). BioTek (Synergy H1, USA) was used to take UV-Vis and fluorescence spectra of Ag-Sgc8-FAM probes, Na2S2O3 and K3[Fe(CN)6] were used to dissolve AgNPs into Ag+. CCRF-CEM and Ramos cells (2.0×105) were cultured with Ag-Sgc8-FAM probes (3 nM) for 1h, CCRF-CEM cells (2.0×105) were cultured with Ag-Sgc8-FAM and Ag-Lib-FAM probes (3 nM) for 1h, flow cytometry was used to take fluorescence intensity of cells. CCRF-CEM cells (2.0×105) were cultured with Ag-Sgc8-FAM (3 nM) for different times (6 h, 12 h, 24 h), after washing step, CSLM was used to take cell images. Overlay

apoptotic

cells

images

with

Ag-Sgc8-TAMRA

and

Annexin-V

FITC.

Ag-Sgc8-TAMRA was prepared by mixing 1 mL AgNPs with mPEG-SH (25 µL, 10 µM) and Sgc8-TAMRA (75 µL, 10 µM). CCRF-CEM cells (2.0×105) were treated with Ag-Sgc8-TAMRA (6 nM, 10 µL) 12 h, after centrifugation, Annexin-V FITC was added for 15 min, CSLM was used to take cell images.

Results and discussion Synthesis and characterization of Ag-Sgc8 and Ag-TDO5. The synthesized silver nanoparticles (AgNPs) had a characteristic absorbance peak at 454 nm (Figure 1A). The micrograph of AgNPs was taken by transmission electron microscope (TEM) as shown in Figure1B, the diameter of AgNPs was calculated as D=51.6 ± 7.3 nm. The concentration of AgNPs was calculated as 0.38 nM based on the calculation method reported previously[38]. To achieve the optimal specific recognition of target cells, Ag-Sgc8 was prepared, in addition, Ag-TDO5 and Ag-Lib were prepared as negtive control for CCRF-CEM. The TEM images were shown in Figure S1. Thiol-terminated poly-(ethylene glycol) (mPEG-SH) was used to decrease nonspecific absorption of aptamer-modified AgNPs with other cells, as well as to prevent aggregation of AgNPs. The probes Ag-Sgc8, Ag-TDO5 and Ag-Lib had similar properties. Firstly, they had the same characteristic absorbance peak around 454 nm. The modification of AgNPs with aptamers (Sgc8, TDO5 and Lib) and mPEG-SH made the absorbance intensity of AgNPs decreased a little (Figure 1A). From the OD changes at 454 nm, the concentrations of Ag-Sgc8, Ag-TDO5 and Ag-Lib were calculated as 0.34 nM, 0.32 nM and 0.33 nM. Secondly, they had high OD ratio at 454 nm (ODAg-Sgc8/ODAgNPs=0.89, ODAg-TDO5/ODAgNPs=0.84 and ODAg-Lib/ODAgNPs=0.87(Figure 1a), the high OD ratio at 454 nm indicated the little loss of AgNPs during the functionalization process. 7



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Because of the protection of PVP and trisodium citrate in the synthesis procedure, the prepared AgNPs were stable in 0.1 M NaCl which facilitated the functionalization process between aptamers and AgNPs. Furthermore, the whole modification process was quick (2 h) and simple. The method for functionalization of AgNPs can be popularized as its high efficiency and simplicity compared to other methods reported

[29, 39, 40].

Thirdly, Ag-Sgc8, Ag-TDO5 and and

Ag-Lib were all negatively charged as their zeta potentials in BB were -13.9±0.4 and -12.9±1.1, -12.9±3.4 respectively. Therefore, Ag-TDO5 and Ag-Lib would be excellent control probes for Ag-Sgc8, and vice versa. Since the binding buffer was used as solvent to store Ag-Sgc8 , Ag-TDO5 and Ag-Lib, while CCRF-CEM and Ramos cells were treated with probes in RPMI 1640 medium; therefore, the stability of probes in RPMI 1640 medium and binding buffer was investigated. The UV-Vis spectra showed that probes (Ag-Sgc8, Ag-TDO5 and Ag-Lib) were very stable in binding buffer within a 24- hour period (Figure1 C, D, E) and 48-hour period (Figure S2); there was a little change in OD and peak shift when Ag-Sgc8, Ag-TDO5 and Ag-Lib were stored in RPMI 1640 medium during 24 hours (Figure1 C, D, E) and 48 hours (Figure S2 ), but the difference could be ignored. Hence, the stability of Ag-Sgc8, Ag-TDO5 and Ag-Lib in RPMI 1640 medium was acceptable within a 48-hour period. Cell apoptosis assay. AgNPs were mostly reported to enter into cells by passive endocytosis [41, 42], however, the receptor-mediated endocytosis was more important in cancer therapy. As the aptamer Sgc8 could be internalized into CCRF-CEM cells, Ag-Sgc8 was also able to be internalized into cells by the receptor-mediated internalization. To assess the state of the CCRF-CEM cells, ﹣

﹣

﹢

Annexin V-FITC and PI was performed to differentiate intact cell (FITC PI ), apoptotic (FITC ﹣

﹢

﹢

PI ) and necrotic (FITC PI ) cells population. Annexin V binds to phosphatidylserine of the plasma membrane at an early stage of apoptosis, and PI enters into late apoptotic or necrotic cells[5]. It was found that the apoptotic and necrotic cells population was highly related with Ag-Sgc8 concentrations and the incubation time between Ag-Sgc8 and CCRF-CEM cells. Different concentrations of Ag-Sgc8 (0.37 nM, 1.5 nM, 6 nM) and incubation time (6 h, 12 h, 24 h) were investigated; and apoptosis of CCFR-CEM cells induced by 0.37 nM and 1.5 nM Ag-Sgc8 could be ignored as 90% cells remained alive after the 24 h treatment (Figure S3). 6 nM Ag-Sgc8 could induce obvious apoptosis of CCRF-CEM cells (Figure 2); the apoptotic cells population 8


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(13.0%) is larger than that of necrotic cell population (3.3%) at a 6 h incubation period; the apoptotic and necrotic cells population increased to 34.9% and 8.6% respectively after a 12 h treatment. After a 24 h treatment, the apoptotic cells had decreased to 18.3% and necrotic cells population had increased to 19.9%. From these results, we concluded that the Ag-Sgc8 could induce apoptosis of CCRF-CEM cells and then the cells went through necrotic process; in addition, only higher concentrations of Ag-Sgc8 could trigger and induce the apoptosis of CCRF-CEM cells. Thus, the Ag-Sgc8 induced apoptosis can be highly controlled and has great potential for cancer therapy. To further prove that the apoptosis was induced by AgNPs, Ag-TDO5 was prepared to specifically bind with Ramos cells to induce apoptosis. Ag-TDO5 could also be internalized into cells by receptor-mediated internalization. Different concentrations of Ag-TDO5 (0.37 nM, 1.5 nM, 6 nM ) and incubation time (6 h, 12 h, 24 h) were investigated. It was found that Ramos cells were more easily going into the apoptotic process in low concentration of Ag-TDO5 (0.37 nM), and the apoptotic and necrotic cells population also increased with the incubation time between Ag-TDO5 and Ramos cells. The apoptotic cells population of Ramos cells increased from 17.3% to 60.1% with 0.37 nM Ag-TDO5 treatment; the necrotic cells population increased from 2.4% to 33.2% (Figure 3), after a 24 h treatment. There was few living Ramos cell observed. The higher concentration of Ag-TDO5 resulted in higher rate of apoptosis; the apoptotic cells population of Ramos cells increased from 15.6% (6 h) to 57.2% (12 h), and then deceased to 37.2% (24 h). The necrotic cells population increased from 2.4% (6 h) to 12.1% (12 h) and then reached 57.2% (24 h) at 1.5 nM Ag-TDO5 (Figure S4 A). For 6 nM Ag-TDO5, the rate of apoptosis was much more quicker, the apoptotic cells population increased from 41.2% (6 h) to 47.6% (12 h), then deceased to 13.0% (24 h) and the necrotic cells population increased from 4.5% (6 h) to 29.0% (12 h) and then 81.8% (24 h) (Figure S4 B). Ramos cells are more sensitive to AgNPs, and AgNPs can easily induce the apoptosis of Ramos cells. It was the same as apoptotic process of CCRF-CEM cells that Ramos cells had firstly undergone apoptosis and then went through necrotic process; however, even lower concentrations of Ag-TDO5 could trigger and induce apoptosis of Ramos cells. The mechanism of AgNPs-induced apoptosis for CCRF-CEM and Ramos was due to the generation of reactive oxygen species (ROS), but degree of apoptosis for two cells was different. Therefore, we can conclude that AgNPs-induced apoptosis is different for various cells and the rule is still 9



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worth researching. To investigate the specificity of Ag-Sgc8 to CCRF-CEM cells, AgNPs, Ag-TDO5, Ag-Lib were chosen as control. To avoid the nonspecific absorption of Ag-TDO5, Ag-Lib to CCRF-CEM cells, AgNPs, Ag-TDO5, Ag-Lib and Ag-Sgc8 were cultured with CCRF-CEM cells for 2 hours, after washing, CCRF-CEM cells were cultured for another 24 hours. Annexin V-PI staining was used to monitor the apoptosis of CCRF-CEM cells. It was found that the apoptosis of CCRF-CEM cells induced by AgNPs, Ag-TDO5, Ag-Lib was smaller than that induced by Ag-Sgc8 in Figure 4A and B. The survival rate of CCRF-CEM cells was decreased from 90.4% to 88.0%, 83.5%, 77.0% after treatment of AgNPs, Ag-TDO5, Ag-Lib; however, the survival rate of CCRF-CEM cells decreased to 42.2% after treatment of Ag-Sgc8 in Figure 4A. In addition, AO and PI staining were used to label living and dead cells. The living cells were stained with green fluorescence and the dead cells were stained with red fluorescence. It was found that the survival rate of CCRF-CEM cells treated with Ag-Sgc8 was much smaller than that of with AgNPs, Ag-TDO5, Ag-Lib in Figure 4B. Therefore, Ag-Sgc8 is specific for apoptosis of CCRF-CEM cells. Ramos cells were also used to further investigate the specificity of probes to cells, the survival rate of Ramos cells with Ag-TDO5 was smaller that of with Ag-Sgc8 in Figure S5, indicating that Ag-TDO5 was specific for Ramos cells. In Vitro cytotoxicity assays. The in vitro cytotoxicity of Ag-Sgc8, Ag-TDO5 and Ag-Lib on cells were also investigated, MTT assay was used assess the viability of cells. Living cells reduce MTT to produce a blue formazan dyes with purple color, and absorbance of formazan dyes at 550 nm indicated the viability of the cells. For CCRF-CEM cells, the mortality with Ag-Sgc8 was higher than that with Ag-TDO5 and Ag-Lib (Figure 5A). The mortality with Ag-TDO5 on CCRF-CEM cells could be ignored indicating the specificity of Sgc-8 for CCRF-CEM cells’ apoptosis. The mortality with Ag-Sgc8 was higher than that with Ag-Lib. For Ramos cells, both Ag-Sgc8 and Ag-TDO5 could result in obvious cells death, but the mortality with Ag-TDO5 was higher than that with Ag-Sgc8 (Figure 5B). It was obvious that Ag-Sgc8 could also enter Ramos cells by passive endocytosis and induced apoptosis of Ramos cells. We attributed the passive endocytosis of Ag-Sgc8 into Ramos cells to the different electronegativity of cells. CCRF-CEM and Ramos cells are negatively charged, the zeta potentials of CCRF-CEM and Ramos cells are -12.9 ± 0.9 and -1.6 ± 1.0. Ag-Sgc8 (-13.9±0.4) and Ag-TDO5 (-12.9±1.1) are also negative. Based on 10


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electrostatic adherence principle, the negative nanoparticles are more easily absorbed on Ramos cells and then enter into cells by passive endocytosis. Therefore, Ag-Sgc8 could enter into Ramos cells and induce vitro cytotoxicity on Ramos cells. Reactive oxygen species (ROS) Generation. It was reported that AgNPs-induced apoptosis increased the ROS production. We monitored the ROS production by flow cytometry and CSLM. ROS can oxidize non-fluorescent DCFH-DA into fluorescent DCF, and the fluorescence intensity of DCF indicates the ROS amount. The DCF intensity increased with concentration of Ag-Sgc8 in Figure 6A, when 6 nM Ag-Sgc8 was treated with CCRF-CEM cells, the DCF increased a little compared with the control. In order to attain obvious increased fluorescence intensity, 9 nM Ag-Sgc8 were used to treat CCRF-CEM cells, and the results showed that the increased concentration could induce increased DCF intensity. Therefore, ROS production would increase with Ag-Sgc8 concentration. The fluorescence images collected by CSLM in Figure 6 B also showed that the fluorescence intensity of cells with 6 nM Ag-Sgc8 was higher than that of cells without Ag-Sgc8. Therefore, CCRF-CEM cells with Ag-Sgc8 treatment will increase the ROS production and induce cell apoptosis. Cell imaging. Apart from inducing apoptosis, AgNPs can also be an excellent material to be adapted in fluorescence-enhanced imaging of apoptotic cells. Sgc8-FAM and mPEG-SH were used to bind with AgNPs to prepare Ag-Sgc8-FAM for real-time cell imaging. The ratio between Sgc8-FAM and mPEG-SH was optimized, the UV-Vis spectra of AgNPs with different ratios of Sgc8-FAM and mPEG-SH had little difference (Figure7 A); however, the fluorescence intensity increased with the ratio and changed a little when the ratio was over 3 (Figure 7 B). In order to prove the fluorescence enhancement of AgNPs, Na2S2O3 and K3[Fe(CN)6] were used to dissolve ﹢

AgNPs to Ag , the mixture of Na2S2O3 and K3[Fe(CN)6] did not affect the fluorescence intensity of FAM (Figure S6). After the addition of Na2S2O3 and K3[Fe(CN)6], the UV-Vis absorbance of ﹢

AgNPs disappeared which indicated the change of AgNPs into Ag (Figure 7 C). In addition, the ﹢

fluorescence intensity of FAM was decreased when the AgNPs were dissolved into Ag (Figure 7 D). Therefore, AgNPs could increase the fluorescence intensity of FAM by MEF effect. In order to prove the specificity of Ag-Sgc8-FAM for CCRF-CEM cells in cell imaging, Ramos was chosen as control cells. The probes Ag-Sgc8-FAM with different ratioes of Sgc8-FAM and mPEG-SH were cultured with CCRF-CEM and Ramos cells in RPMI 1640 medium for 1 h, 11



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and fluorescence intensity was measured by the flow cytometry. Fluorescence intensity increased with the ratio of Sgc8-FAM and mPEG-SH, Ag-Sgc8-FAM can specifically bind to CCRF-CEM cells and result in high fluorescence intensity (Figure 8A). However, the fluorescence intensity of Ramos cells with different Ag-Sgc8-FAM probes and control showed no difference (Figure 8B), changes in fluorescence intensity was negligible. Therefore, the interaction between Ag-Sgc8-FAM and Ramos cells could be ignored and Ag-Sgc8-FAM was specifically to CCRF-CEM cells. In addition, to further demonstrate the enhanced fluorescence intensity of Ag-Sgc8-FAM, different concentrations of Sgc8-FAM (100 nM, 1µM, and 10 µM) were cultured with CCRF-CEM cells in RPMI 1640 medium for 1h. Fluorescence intensity of Sgc8-FAM was obvious much smaller than that of Ag-Sgc8-FAM (Figure S7). The conclusion indicated that Ag-Sgc8-FAM was superior to Sgc8-FAM as an imaging probe. AgNPs-enhanced fluorescence intensity could be attributed for two reasons: AgNPs could result in the metal-enhanced fluorescence on nearby FAM; and many Sgc8-FAM were enriched on the surface of AgNPs for signal amplification and the multiple binding of Sgc8 to CCRF-CEM cells enhance the capture ability of Ag-Sgc8-FAM. The flow cytometry of Ag-Sgc8-FAM and Ag-Lib-FAM with CCRF-CEM was conducted and the results were shown in Figure 8C. The fluorescence intensity of CCRF-CEM with Ag-Sgc8-FAM was much higher than that with Ag-Lib-FAM, which indicated good specificity of Ag-Sgc8-FAM for CCRF-CEM cell image. The fluorescence images of CCRF-CEM cells with Ag-Sgc8-FAM at different treatment periods were collected (Figure 9). At 6 h, most cells were labeled with Ag-Sgc8-FAM and the fluorescence images were in optical rings, and the morphology of cells was good. After 12 h, the higher fluorescence intensity was observed on the atrophic cells that were undergoing the process of apoptosis. CCRF-CEM cells might undergo apoptosis when large amount of Ag-Sgc8-FAM entered into cells with high fluorescence. Then after 24 h incubation, some cells were full of Ag-Sgc8-FAM with high fluorescence intensity, and the morphology was completely altered indicating that the late apoptotic and necrotic process of CCRF-CEM cells. Based on the apoptosis results from Figure 2 and Figure 9, we concluded that Ag-Sgc8-FAM could monitor the apoptotic and necrotic process in real time. To further prove the multi-function of AgNPs in cell apoptosis and imaging, Ag-Sgc8-TAMRA (6 nM, 10µL) were prepared to incubate with CCRF-CEM cells for 12 h to 12


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induce apoptosis and took fluorescence images of CCRF-CEM cells. Annexin-V FITC was used to label apoptotic CCFR-CEM cells. The apoptotic CCRF-CEM cells would be labeled with two kinds of fluorescent dyes that had different emission spectra. The results showed that Ag-Sgc8-TAMRA (red fluorescence) and Annexin-V FITC (green fluorescence) could be found in the same CCFR-CEM cells (Figure 10). Therefore, Ag-Sgc8-TAMRA could be used to induce apoptosis of cells and monitoring the apoptotic and necrotic process in real time simultaneously. The different distribution of TAMRA on cells may due to two reasons. Firstly, cells incubate with Ag-Sgc8-TAMRA under static condition resulting in heterogeneity of distribution of TAMRA on cells. Secondly, the amount of Ag-Sgc8-TAMRA is not enough for all cells to bind with enough TAMRA on their suface, so some cells are labeled more while some are labeled less or even no TAMRA on cell’s surface. The unlabeled cells can not be seen in fluorsecence images.

4. Conclusion A multifunctional probe Ag-Sgc8-FAM was developed for apoptosis-based cell therapy and fluorescence-enhanced cell imaging. Aptamer-functionalized AgNPs could induce apoptosis of target cells with high specificity and the target-apoptosis had great potential in cancer therapy. The AgNP-induced apoptotic degree was different on different cell types. Besides the apoptosis, AgNPs are also good materials for fluorescence-enhanced imaging. The probe Ag-Sgc8-FAM could induce apoptosis of CCRF-CEM cells for targeted cancer therapy as well as fluorescence-enhanced cell imaging. The probe was highly specific and sensitive, it open a new way for targeted cancer therapy and imaging. Multifunctional AgNPs for simultaneous cancer therapy and cell imaging have great potential in future theragnosis and drug discovery.

Conflict of interest: The authors declare no competing financial interest.

Acknowledgment We thank Prof. Weihong Tan in Hunan University for providing CCRF-CEM and Ramos cells. We acknowledge the financial support of the National Basic Research Program of China (973 Program, 2011CB911003) and National Natural Foundation of China (Grant Nos. 21405077, 13



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21227009, 21175066 and 21328504), Natural Science Foundation of Jiangsu Province (BK20140591), Fundamental Research Funds for the Central Universities, Jiangsu Province Science and Technology Support Program (No. BE2011773) and Research Foundation of Jiangsu Province Environmental Monitoring (No. 1116), the National Science Funds for Creative Research Groups (NO. 21121091).

Supporting Information Available Additional information as noted in text. This information is available free of charge via the Internet at http://pubs.acs.org/.

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Scheme 1. Aptamer-silver conjugates induced apoptosis for specific cancer therapy and fluorescence-enhanced cell imaging.

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Figure 1. (A) UV-Vis spectra, (B) TEM image of AgNPs, UV-Vis spectra of (C) Ag-Sgc8, (D) Ag-TDO5 and (E)Ag-Lib in RPMI 1640 medium and binding buffer (BB) for 24 h.

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Figure 2. CCRF-CEM cells were treated with BB (A) and Ag-Sgc8 (6 nM) for (B) for 6 h, (C) for 12 h, (D) for 24 h, then Annexin V-FITC (5 µL) and PI (2 µL) were used to label cells, the fluorescence intensity were taken by flow cytometry.

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Figure 3. Ramos cells were treated with BB (A) and Ag-TDO5 (0.37 nM) for (B) for 6 h, (C) for 12 h, (D) for 24 h, then Annexin V-FITC (5 µL) and PI (2 µL) were used to label cells, the fluorescence intensity were taken by flow cytometry.

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Figure 4. CCRF-CEM cells were separately treated with BB, AgNPs, Ag-TDO5, Ag-Lib and Ag-Sgc8 for 2h, after washing, the cells were cultured for another 24 h, then staining was used to label cells, (A) fluorescence intensity of Annexin V-PI staining, (B) fluorescence images of Acridine Orange (AO) and Propidium iodide (PI) staining.

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Figure 5. In vitro cytotoxicity of (A) CCRF-CEM cells with BB, aptamer-silver conjugates (Ag-Sgc8, Ag-TDO5, Ag-Lib), (B) Ramos cells with BB, Ag-Sgc8, Ag-TDO5.

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Figure 6. (A) ROS level of CCRF-CEM cells by flow cytometry, (B) ROS level of CCRF-CEM cells by confocal scanning laser microscopy (CSLM).

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Figure 7. (A,C) UV-Vis spectra, (B) Fluorescence spectra of Ag-Sgc8-FAM with different ratio of SH-Sgc8-FAM and mPEG-SH, (D) Fluorescence spectra of Ag-Sgc8-FAM with and without 100 mM Na2S2O3 and 10 mM K3[Fe(CN)6].

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Figure 8. Fluorescence intensity, (A) CCRF-CEM (2.0×105) and (B) Ramos (2.0×105) cells were cultured with five kinds of Ag-Sgc8-FAM for 1h, (C) CCRF-CEM (2.0×105) cells were cultured with Ag-Sgc8-FAM and Ag-Lib-FAM for 1h.

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Figure 9. Bright, fluorescence and merged images of CCRF-CEM cells, the cells were cultrued with Ag-Sgc8-FAM for 0 h, 6 h, 12 h, 24 h, then CSLM was used to taken the imges.

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Figure 10. CCRF-CEM cells were simutaneously labeled by Ag-Sgc8-TAMRA and Annexin-V FITC. Bright image of CCRF-CEM cells (A), fluorescence images of CCRF-CEM cells with (B) Ag-Sgc8-TAMRA and Annexin-V FITC, (C) Annexin-V FITC, (D) Ag-Sgc8-TAMRA.

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Table of Contents (for TOC only) AgNPs

PTK-7 Mediated Endocytosis

AgNPs

ROS AgNPs

Apoptosis Ag-Sgc8-FAM DNA f ragmentation

AgNPs

Enhanced Cell Imaging

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