âSense-and-Treatâ DNA Nanodevice for Synergetic Destruction of

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A “sense-and-treat” DNA Nanodevice for synergetic destruction of circulating tumor cells Nandi Chen, Shiya Qin, Xiaohai Yang, Qing Wang, Jin Huang, and Kemin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08695 • Publication Date (Web): 21 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016

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ACS Applied Materials & Interfaces

A “sense-and-treat” DNA Nanodevice for Synergetic Destruction of Circulating Tumor cells Nandi Chen, Shiya Qin, Xiaohai Yang*, Qing Wang, Jin Huang and Kemin Wang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China.

ABSTRACT: DNA nanostrucures are promising materials for biomedical applications. Herein, we have established a “sense-and-treat” localized drug delivery system based on DNA Nanodevice to specifically destroy circulating tumor cells (CTCs) by synergetic chemotherapy and photodynamic therapy. The DNA Nanodevices could sense the existence of CTCs and treat CTCs with anticancer agents. Typically, the presence of target cell promoted the formation of hairpin structure of aptamer, and then the aptamer accompanied DNA tetrahedron would release from the supporter. The chemotherapy drugs (doxorubicin, Dox) loaded in DNA tetrahedron would destroy the CTCs specifically. Moreover, the photosensitizer labelled on DNA tetrahedron would be activated by lights and generated toxic 1O2, once DNA Nanodevices bound CTCs flow through the superficial capillary. Unlike the aptamer only labelled with photosensitizer, the DNA Nanodevice have showed the capability to promote cellular internalization of anticancer agents,

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increase drug loading capacity and realize synergetic therapy, which enhanced the destructive ability of anticancer agents. As proof of concept, this DNA Nanodevice has the potential to inhibit metastasis by synergetic destruction of CTCs.

KEYWORDS: DNA Nanodevice, Circulating tumor cell, Aptamer, Photodynamic therapy, Chemotherapy, Metastasis.


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INTRODUCTION DNA has been used as a powerful unit to construct molecularly precise nanodevices. Nanostructures established by DNA have become promising materials in biomedical application owing to their significant advantages, such as precise interactions between base paires, outstanding biocompatibility, programmability and automated synthesis.1-2 Especially, scientists have paid great attention to DNA tetrahedrons about their biological and biomedical application.3-4 The DNA tetrahedron has six double-stranded edges and four triangular faces, which could be rapidly assembled by annealing four designed single-stranded DNA. Vertexes and dsDNA edges of DNA tetrahedron can be readily functionalized with different chemical moieties and biomolecules.5 Up to now, DNA tetrahedrons have been regarded as a kind of multifunctional, biocompatible and stable nanostructures for intracellular sensing6-7, molecular imaging5 and tumor treatment8-10. In recent years, medical scientists tried to integrate diagnosis and therapy in a single system, resulting the “sense-and-treat” concept has attracted much attention. 11-12 Owing to the facile design feature of DNA-based nanostructures, they have the capability to establish “sense-andtreat” drug delivery systems, especially for targeting objectives in circulatory system. Moreover, this concept could be used in localized drug delivery systems, which reduced drug dose, administration frequency and side effects, as opposed to repeatedly intravenous injections to human circulatory system. Therefore, a “sense-and-treat” localized drug delivery system based on DNA Nanostructure might have great potential in tumor diagnosis and treatment. The major cause of cancer-associated mortality is tumor metastasis.13 The tumor cells, which could detach from primary tumors, would travel a long journey before lodging at a new location


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and become a metastatic tumor. Consequently, these cells are called circulating tumor cells (CTCs).14-15 CTCs were extremely rare in the circulatory and were difficult to be destroyed.16 Thus, developing improved methods to combat CTCs spreading without disturbance to normal cell might be a potential approach to decrease cancer mortality.17-18 However, there is few research about destruction CTCs specifically.19-22 Herein, we developed a DNA Nanodevice-based “sense-and-treat” strategy which was suitable for destruction of CTCs by chemotherapy and photodynamic therapy synergistically. As shown in Scheme 1, the DNA Nanodevice was immobilized on a supporter and made up of the hairpin switch aptamer (HSA)

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accompany with a DNA tetrahedron. The DNA Nanodevice could

sense the existence of CTCs and treated CTCs with anticancer agents. Therefore, DNA Nanodevices immobilized supporter could be placed near tumors. DNA tetrahedrons have showed the capability to promote cellular internalization of anticancer agents, increase drug loading capacity and realize synergetic therapy, which enhanced the destructive ability of anticancer agents. The presence of target cell promoted the formation of hairpin structure of aptamer. Afterward, an aptamer accompanied with the DNA tetrahedron would release from the supporter. The chemotherapy drugs (doxorubicin, Dox) loaded in DNA tetrahedron would destroy the CTCs specifically. Moreover, the photosensitizer labelled on DNA tetrahedron would be activated by lights and generated toxic 1O2, once DNA Nanodevices bound CTCs flow through the superficial blood vessel. The synergetic therapy might destroy CTCs and inhibit metastasis more effective.23-25


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Scheme 1. Illustration of the “Sense-and-Treat” DNA Nanodevice for synergetic destruction of circulating tumor cells. The DNA Nanodevice would sense the existence of CTCs and bind on them. The chemical drug and photosensitizer in DNA Nanodevice might destroy CTCs synergistically. The chemotherapy drug Dox which loaded in the dsDNA of DNA Nanodevice would treat CTCs. Moreover, after irradiation by the household LED, the photosensitizer labelled at vertexes of DNA tetrahedron would be activated and generate toxic 1O2. In contrast, normal cell could flow across the surface without triggering the release of DNA Nanodevice.

RESULTS AND DISCUSSION In our “sense-and-treat” drug delivery strategy, the core was that CTCs would take anticancer agents away from the supporter precisely. Therefore, in the sense part, the DNA Nanodevices were fixed on magnetic beads for evaluation of the sensing principle. In the treat part, to


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demonstrate this strategy might provide a grafted device to destroy CTCs with synergetic therapeutic methods, the vessel was emulated by a micro-channel. The evaluation of DNA Nanodevices on magnetic beads was shown in Scheme 2. When target cell is nonexistence, the aptamer could hybridize with part of the DNA tetrahedron and cDNA. Conversely, when target cells interacted with DNA Nanodevices immobilized magnetic beads, the DNA hybridization part could be disturbed by the target cell, and the aptamer which accompany with a DNA tetrahedron would move away from magnetic beads (Scheme 2).

Scheme 2. Sensing principle of DNA Nanodevices on magnetic beads. The formation of DNA Nanodevices was verified by agarose electrophoresis and dynamic light scattering (DLS). The self-assembly process was carried out eﬃciently by the presence of clean single bands and the DNA Nanodevices were formed as designed (Figure 1A). DLS measurement indicated that the size of DNA Nanodevices was ca. 9 nm (Figure 1B). Based on the strong and specific interaction between biotin and streptavidin, the DNA Nanodevices were immobilized on magnetic beads. All the DNA sequences in this paper were listed in Table S1 of Supporting information.4, 26


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Figure 1. The formation of DNA Nanodevices. (A) Agarose electrophoresis analysis of the formation of DNA Nanodevices. 1) Marker; 2) Tetra-1; 3) Tetra-2; 4) Tetra-3; 5) Tetra-4; 6) Aptamer; 7) Aptamer+ cDNA; 8) Control; 9) Control probe: Control + cDNA; 10) DNA tetrahedron: Tetra-1 + Tetra-2 + Tetra-3 + Tetra-4; 11) DNA Nanodevice: cDNA + Aptamer + Tetra-1 + Tetra-2 + Tetra-3 + Tetra-4; 12) Control DNA Nanodevice: cDNA + Control + Tetra-1 + Tetra-2 + Tetra-3 + Tetra-4; 13) Marker. (B) DLS measurement of HSA probes and DNA Nanodevices. The Size of HSA probe was about 0.6896 ± 0.1272 (nm). The Size of DNA Nanodevice was about 9.293 ± 2.527 (nm).

The binding of dye-modified DNA Nanodevices with different cells was monitored by the Flow cytometry assays. As shown in Figure 2, the DNA Nanodevices showed better recognition characteristics than the HSA probes. When DNA Nanodevices sense the existence of target SMMC-7721 cells, a higher fluorescence was observed compared with HSA probes. The signal ratio of DNA Nanodevice/control DNA Nanodevice was 8.4; while the signal ratio of HSA probe/control probe was 2.6. Conversely, the fluorescence intensity on Bel-7404 and PC-3M1E8 (control cells) were not changed significantly. Flow cytometry samples were also used for confocal imaging (Figure S1, Figure S2 and Figure S3 of Supporting information). In accordance with Figure 2, for target SMMC-7721 cells, DNA Nanodevices showed higher fluorescence compared with HSA probes, and none fluorescence of Bel-7404 and PC-3M-1E8


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control cells was observed. Therefore, the DNA Nanodevice showed better capability in distinguishing tumor cells than the HSA probe. Meanwhile, DNA Nanodevices could enter into target SMMC-7721 cells with time extending (Figure S4 and Figure S5 of Supporting information), this might enhance the destructive ability of DNA Nanodevices. The binding ability of different concentrations of DNA Nanodevices and probes for immobilization onto magnetic beads was also investigated (Figure S6).

Figure 2. The Flow cytometry results of the binding of dye-modified DNA Nanodevices with different cells. They showed the selective binding of DNA Nanodevices (BHQ1-labelled-cDNA + FAM-labelled-Aptamer + FAM labelled DNA tetrahedron) and HSA probes (BHQ1-labelledcDNA + FAM-labelled-Aptamer) to SMMC-7721 target cells, but not Bel-7404 or PC-3M-1E8 cells. Control DNA Nanodevice: BHQ1-labelled-cDNA + FAM-labelled-Control + FAM labelled DNA tetrahedron. Control probe: BHQ1-labelled-cDNA + FAM-labelled-Control. Temperature: 37oC. Concentration of probes: 10 nM. Ex: 488nm, Em: 530nm. SMMC-7721 and Bel-7404 were all human hepatocellular carcinoma cell lines, PC-3M-1E8 was human prostatic carcinoma cell line.


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All above results made sure that the sensing principle of the DNA Nanodevice was in consonance with we expected, i.e. DNA Nanodevices could sense the existence of target cells and recognize them specifically. Subsequently, to investigate whether the DNA Nanodevice may destroy CTCs with chemotherapy and photodynamic therapy synergistically, a microfluidic chip was used to emulate the vessel which could provide a micro-channel in the treat part, on account of the behavior of CTCs. As shown in Scheme 3, the surface of micro-channel was coated with avidin, and then DNA Nanodevices were immobilized on the surface through biotin-avidin interaction (Section S6 of Supporting

information).

In

this

“sense-and-treat”

localized

drug

delivery

system,

pyropheophorbide-a (Pyro) and Dox were chosen as the model anticancer agents of photodynamic therapy and chemotherapy, respectively. When the CTCs flowed across the micro-channel, they would take the anticancer agents loaded DNA Nanodevices away, the chemotherapy drug Dox would destroy target cells. Meanwhile, upon light irradiation, Pyro would brighten targeted cells and produce cytotoxic 1O2, meanwhile normal cells were still undetectable and unharmed. In order to achieve a better performance, the flow rate of introduce tumor cells in micro-channel was optimized. On the basis of data in Figure S7 and Table S2 of Supporting information, a flow rate of 150 µL/min was chosen.


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Scheme 3. Sensing principle of DNA Nanodevices on surface of micro-channel. Pyro was covalently linked at vertexes of DNA Nanodevices. A commercially 1O2 sensor(1, 3diphenylisobenzofuran, DPBF) was used to evaluate the efficiency of 1O2 generation of Pyro labelled probe. After irradiating for 4 min, the generation of 1O2 increased to the plateau (Figure 3). Flow cytometry assays (Figure 4) and confocal imaging (Figure 5) indicated an extremely specific binding of the DNA Nanodevice with Pyro to SMMC-7721 cells, rather than Bel-7404 cells (Figure S8 of Supporting information) and PC-3M-1E8 (Figure S9 of Supporting information). Moreover, Dox was chosen as the chemotherapy agent which could be loaded in the dsDNA of DNA Nanodevice. Based on fluorescence assay, the ratio of Dox and DNA Nanodevice was about 34:1.


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Figure 3. The efficiency of 1O2 generated by pyropheophorbide-a. The concentration of DPBF and Pyro/Pyro labeled probe were 100 mM and 1 µM respectively. The photobleaching during labeling process may reduce the 1O2 generated by the Pyro labelled on the probe.

Figure 4. The selective binding of Pyro labelled DNA Nanodevices (cDNA + Pyro- labelledAptamer + Pyro labelled DNA tetrahedron) and HSA probes (cDNA + Pyro- labelled-Aptamer) to target SMMC-7721cells, rather than negative Bel-7404 or PC-3M-1E8 control cells. Control DNA Nanodevice: cDNA + Pyro-labelled-Control + Pyro labelled DNA tetrahedron. Control probe: cDNA + Pyro-labelled-Control. Temperature: 37 oC, Concentration of probes: 10 µM, Flow rate: 150 µL/min, Ex: 633 nm, Em: 661nm.


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Figure 5. Confocal imaging of SMMC-7721 cells which were collected from outlet. DNA Nanodevices: cDNA + Pyro-labelled-Aptamer + Pyro labelled DNA tetrahedron. HSA probes: cDNA + Pyro- labelled-Aptamer. Ex: 633 nm, Em: > 660 nm. Probes concentration: 10 µM, Temperature: 37 oC, Scale bar: 20 µm. The MTS assay was use to perform the cytotoxicity study of Pyro labelled and Dox loaded DNA Nanodevices. A household LED light was used to irradiate cells by photodynamic therapy, when cells flowed in the capillary. After irradiating for 15 min, cell samples were cultured in dark for one day before investigating the cytotoxicity. Once DNA Nanodevices with Pyro (group c) were immobilized in the micro-channel, the cell viability of SMMC-7721 cancer cells was decrease to about 40% after white LED light irradiation (Figure 6). Since the LED light could excite Pyro on DNA Nanodevices, which lead to cancer cells dying of photodynamic therapy. In contrast to Pyro labelled HSA probes (group b), which left about 70% SMMC-7721 target cancer cells alive after LED light irradiation. Therefore, the DNA Nanodevices have enhanced the destructive ability of Pyro labelled HSA probes. Moreover, after loading Dox into Pyro labelled DNA Nanodevice (group e), there were only ca. 25% cancer cells still alive after irradiating with LED, the synergetic therapy showed better therapeutic activity than either chemotherapy or photodynamic therapy alone. Otherwise, the cell viability was not influenced by the LED


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irradiation, when cells flowed through a micro-channel without DNA Nanodevices. Meanwhile, the target cells showed high cell viability without irradiation. In addition, this drug delivery system showed outstanding selectivity, the control Bel-7404 cells and PC-3M-1E8 cells kept high cell viability (Figure S10 of Supporting information).

CONCLUSION In summary, by using the DNA Nanodevice we developed a “sense-and-treat” localized drug delivery system, which could destroy CTCs with chemotherapy and photodynamic therapy synergistically. Unlike the aptamer only labelled with photosensitizer, the DNA Nanodevice showed several advantages: 1) promoting cellular internalization of anticancer agents demonstrated by confocal imaging27-28; 2) increasing drug loading capacity demonstrated by flow cytometry assays and confocal imaging; 3) realizing synergetic therapy demonstrated by MTS assay. With the increasing numbers of DNA Nanostructures1-2, more drug delivery systems with higher CTCs destructive ability could be established based on “sense-and-treat” strategy.


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Figure 6. Cell viability by MTS assay. After 15 min household LED irradiation, the cytotoxicity in vitro was measured after incubation for 24 h in cell medium. a) the micro-channel just immobilized with cDNA. b) the micro-channel immobilized with Pyro labelled HSA probes (cDNA + Pyro-labelled-Aptamer). c) the micro-channel immobilized with Pyro labelled DNA Nanodevices (cDNA + Pyro-labelled-Aptamer + Pyro labelled DNA tetrahedron). d) the microchannel immobilized with Dox loaded DNA Nanodevices (cDNA + Aptamer + DNA tetrahedron + Dox). e) the micro-channel immobilized with Pyro labelled and Dox loaded DNA Nanodevices (cDNA + Pyro-labelled-Aptamer + Pyro labelled DNA tetrahedron + Dox). Cell: SMMC-7721, Luminous flux: 6.0×104 lux, LED +: LED light irradiation, LED -: without irradiation.

MATERIALS AND METHODS Materials and reagents. Avidin, Bovine serum albumin (BSA), yeast tRNA and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Sigma (Shanghai, China). PDMS was purchased from Dow Corning Corporation Midland, MI (Bejing, China). 1, 3-diphenylisobenzofuran (DPBF) was purchased from J&K (Guangzhou, China). Doxorubicin hydrochloride (DOX) was purchased from Hualan Chemistry Technology Co., Ltd (Shanghai, China). Pyropheophorbide-a (Pyro) was purchased from Xianhui Pharmaceutical Co. Ltd. (Shanghai, China). Fetal bovine serum (FBS) was purchased from Tianhang Biological Technology Co., Ltd. (Zhejiang, China). RPMI 1640 cell medium was purchased from Sangon Biotechnology Company, Ltd. (Shanghai, China). Magnetic beads were purchased from Invitrogen (Dynabeads® M-280 Streptavidin, Catalog number: 11205D, Shanghai, China). Perfluoroalkoxy resin (PFA) was purchased from Iwase


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(EXLON-tubing, Japan, No. 5F1M18, 0.3mm×0.5mm). Household LED was purchased from Langtai Company (Jiangsu, China, LED bead: 0.5W, Color temperature: 6000-6500K. The household LED light consist of 96 LED beads and the power is 48W in total). All the DNA sequences in this research were purchased from Sangon Biotechnology Inc. Thy were purified by HPLC and characterized by ESI-MS. We used chemicals as received. We used Milli-Q-purified water for all experiments. Preparation of DNA Nanodevices. The ratio of DNA sequences which make up the DNA Nanodevice, cDNA: ZY1-linker/ZY1linker-Control: Tetra-1: Tetra-2: Tetra-3: Tetra-4 was 1.5:1:1:1:1:1. These DNA sequences for formation of the DNA Nanodevice were mixed in buffer (50 mM MgCl2 in DPBS) at 95 °C for 5 minutes, and directly cooled to 4 °C overnight. Electrophoresis was used to analyze each sample at 80 V through a 3% agarose gel for 60 min in 1×TBE buffer. Digital camera was used to photograph DNA bands which were visualized via UV illumination. ZataSizer DLS detector (Malvern Instruments Ltd. U.K.) was used to measure the DNA Nanodevice size and distribution at 25 °C. Cell culture. SMMC-7721 (human hepatocarcinoma cell), Bel-7404 (human hepatocarcinoma cell) were purchased from Cell Bank of the Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). PC-3M-1E8 (human prostate cancer) was gained from Cell Bank of Biological College in Hunan University (Changsha, China). All cells were cultured in10% fetal


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bovine serum added RPMI 1640 culture medium with 100 U/mL streptomycin-penicillin. All cells were cultured in a humidified incubator at 37 °C containing 5 wt %/vol CO2. The washing buffer was prepared by adding certain quality of glucose and MgCl2 in DPBS to make the final concentration of glucose and MgCl2 were 4.5 g/L and 5 mM, respectively. And the binding buffer was prepared by adding certain quality of yeast tRNA and BSA in washing buffer to make the final concentration of yeast tRNA and BSA were 0.1 mg/mL and 1 mg/mL, respectively. Before any experiment, we determined the cell desity by a hemocytometer. Flow cytometric analyses. SMMC-7721, Bel-7404 and PC-3M-1E8 cells were washed with D-Hank’s twice, detached with 0.5% trypsin and 0.02% EDTA mixture, then dispersed in binding buffer. The density of cells was about 106 cells/mL. A FACScan flow cytometer was used to analyze cell by counting 10 000 events. The DNA Nanodevice immobilized on the magnetic beads. Magnetic beads were firstly washed with DPBS for 3 times, then incubated with the DNA Nanodevice/control DNA Nanodevice for 1 h and washed three times before incubating with cells. 200 µL cells (2× 105) were incubated with 2.5 µL magnetic beads with the probes immobilized on the surface in binding buffer without any light for 30 min. Then the magnetic beads were separated by an external magnetic field, and the supernatant was used for flow cytometric analyses. The DNA Nanodevice immobilized in micro-channel. The surface of micro-channel was modified with avidin first, and then the DNA Nanodevice/DNA control Nanodevice was immobilized by the interaction between avidin and biotin. 2× 105 cells in 200 µL binding buffer


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were dropped at the inlet of the channel, and aspirated at the outlet of the channel by a syringe pump (KD Scientific, Inc.). Cells collected from the outlet of the channel were used for flow cytometric analyses. Confocal imaging of cells bound with DNA Nanodevices. The cell samples used for flow cytometric analyses were also used for confocal imaging. The Olympus FV1000-TY1318 with 40× objective was used to collect the cellular fluorescent images. A thin glass slide which could drop cells dispersion was used for confocal imaging. The 488 nm laser and 633 nm laser were the excitation source for FAM labelled probes and Pyro probes throughout the experiments, respectively. Design and fabrication of experimental device. As shown in Scheme 3, this experimental device was consist of three parts: 1) a microfluidic chip which could provide a micro-channel used to immobilize the DNA Nanodevice; 2) a 100 cm perfluoroalkoxy resin (PFA) capillary could be irradiated by a household LED to emulate the superficial blood vessel; 3) a syringe pump was used to drive the cells flowing in the microchannel and capillary. The micro-channel was kept in a box without light and maintained a stable temperature by a heating board. The capillary was irradiated by household LED light when cells flowed in the capillary. The syringe pump was also placed in dark place. The microfluidic chip was composed of two layers: a piece of slide glass and a chaotic-mixer1 PDMS slice. The glass support was cleaned with piranha solution. The PDMS replica with a 50 cm long herringbone mixing channel (wh = 1 × 0.1 mm) was produced via soft-lithography using


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a facsimile on the SU-8 wafer. Patterned SU-8 master was fabricated by Dalian Institute of Chemical Physics, Chinese Academy of Science. The 10:1 PDMS pre-polymer mixture was used for making the PDMS facsimile. After pouring the mixture onto the SU-8 wafer, the PDMS facsimile was solidified at 75 °C for 2 hours. After solidification, the PDMS facsimile was shed from the master. At the beginning and the end of the micro-channel, we punched inlet and outlet wells, respectively. The PDMS facsimile was then reversibly attached to the support to form a whole microfluidic chip. All solutions were introduced into the micro-channel with a syringe pump at the outlet of the micro-channel. Avidin (1 mg/mL) absorbed in the micro-channel at 200 µL/s and then washed the microchannel by DPBS at 300 µL/s for three times. Similarly, then the DNA Nanodevice was immobilized at 200 µL/s by the interaction between avidin and biotin, and the channel was washed with DPBS for three times at a flow rate of 300 µL/s. Calculate the doxorubicin loading content. Briefly, the doxorubicin (Dox) loading content was determined by comparing molecule concentration before and after loading, after calculating the surface coverage of DNA Nanodevices according to our previous method.22 The fluorescence of Dox was measured using F-7000 fluorescence spectrophotometer, and a standard curve was made by using known concentrations of Dox. The standard curve of the Dox was F = 1.0959 C + 7.5984, R=0.9997. F was the fluoresence signal of Dox. C was the concentration of Dox. The Dox loading content was estimated using the following equation: Dox loading content = (Cbefore – Cafter) / CDNA Nanodevices


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Cbefore and Cafter were measured as the molar concentration of Dox before and after incubating with DNA Nanodevices immobilized magnetic beads. Cytotoxicity investigation. The MTS assay was use to perform the cytotoxicity study with the CellTiter (Promega, Madison, WI). SMCC-7721, Bel-7404 and PC-3M-1E8 cell lines were cultured in a 96-well cell culture plate with the density of 20 000/well, 250 µL. Cell samples were divided into five groups as follows: Group a, cells collected from the micro-channel without Pyro labelled DNA probes and treated without/with LED irradiation when they flowed in the capillary; Group b, cells collected from the micro-channel with Pyro labelled HSA probes and treated without/with LED irradiation when they flowed in the capillary; Group c, cells collected from the micro-channel with Pyro labelled DNA Nanodevices and treated with/without LED irradiation when they flowed in the capillary; Group d, cells collected from the micro-channel with Dox loaded DNA Nanodevices and treated without/with LED irradiation when they flowed in the capillary; Group e, cells collected from the micro-channel with Dox loaded DNA Nanodevices which were also labeled Pyro and treated without/with LED irradiation when they flowed in the capillary. Pyro labelled probes must be kept in dark before irradiating by household LED. The household LED light irradiated the cells when they flowed in the capillary for 15 min. The illuminance was 6.0×104 lux. The flow rate was 150 µL/min. Afterwards, cells were incubated in incubator for 24 h at 37 o

C. Finally, CellTiter reagent solution (30 µL/well) was added and incubated for 3 h at 37 oC.

The multi-function plate reader Infinite® M1000 (Tecan, Switzerland) was used to determine the absorbance at 570 nm.


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ASSOCIATED CONTENT Supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. DNA Sequences, flow cytometry analysis in microfluidic chip, confocal imaging by FAM labelled HSA probes and DNA Nanodevices, the binding ability of probes and DNA Nanodevices with time extending, the binding ability of different concentrations of DNA Nanodevices and probes, optimize the flow rate of cell dispersion in micro-channel, confocal imaging of cells by Pyro labelled DNA Nanodevices and probes, cell viability by MTS assay. AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected]; Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the General Program of National Natural Science Foundation of China (21190040, 21675047, 21175035), the National Basic Research Program of China (2011CB911002). REFERENCES 1.

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Table of Contents


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âSense-and-Treatâ DNA Nanodevice for Synergetic Destruction of

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