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Electrochemiluminescence-Microscopy for microRNA Imaging in Single Cancer Cell Combined with Chemotherapy -Photothermal Therapy Huai-Rong Zhang, Wanxia Gao, Yong Liu, Yingnan Sun, Yanxialei Jiang, and Shusheng Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 6, 2019

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Analytical Chemistry

Electrochemiluminescence-Microscopy for microRNA Imaging in Single Cancer Cell Combined with Chemotherapy -Photothermal Therapy Huairong Zhanga, Wanxia Gaoab, Yong Liua, Yingnan Suna, Yanxialei Jianga, Shusheng Zhanga* a Collaborative

Innovation Center of Tumor Marker Detection Technology, Equipment and Diagnosis-Therapy Integration in Universities of Shandong, Shandong Province Key Laboratory of Detection Technology for Tumor Makers, School of Chemistry and Chemical Engineering, Linyi University, Linyi, 276005, China. b Shandong

Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, College of Chemistry and Chemical Engineering, Qingdao University, Qingdao, Shandong, 266071, China. ABSTRACT: In this work, a new technology using ECL as a microscopy to parallel image miRNA-21 in single cancer cell was built. Phorbol-12-myristate-13-acetate (PMA) loaded gold nanocages (Au NCs) was closed with DNA gate which could be recognized and opened by miRNA-21 in HeLa cell. PMA was then released and further induced HeLa cells to produce reactive oxygen species (ROS; including O2-•, •OH and H2O2 etc.). With H2O2 as coreactant and luminol as ECL active material, ECL imaging of intracellular miRNA-21 in single HeLa cell was obtained by EMCCD. Moreover, ROS therapy and photothermal therapy (PPT) of Au NCs@PMA probe were also motivated by in-situ miRNA-21 marker instead of the external source. The combined therapy leads to dramatically enhanced ability for cancer cell killing. Au NCs@PMA probe alone could not only achieve a high sensitivity and high resolution ECL-microscopy for imaging of intracellular miRNA-21 for the first time, but also realize the integrated diagnosis like ROS induced tumor damage and photothermal-induced intelligent therapy. This multifunctional platform is believed to be capable of playing an important role in future oncotherapy by the synergistic effects between chemotherapy and photothermal therapy.

INTRODUCTION Electrochemiluminescence (ECL) is a highly sensitive electrochemical method utilizing the radiative energy during the relaxation of electronically excited products to the ground state after electrochemical oxidation or reduction1-2. The smart combination of chemiluminescence and electrochemistry brings ECL many potential advantages3. ECL technology are increasingly being used for bioanalysis assay because of featuring the merits of low background, high sensitivity, good temporal and spatial resolution4-7. In traditional way, ECL signal was amplified by photomultiplier tube and transformed into digital form. As a result, the employment of traditional electrochemiluminescence (ECL) technology in single cell detection was limited due to the signal acquisition mode8-10. Previous studies about ECL methods usually used for collecting the information of cell groups11-13. Nevertheless, single cell14-16 or single molecule17-18 analysis provide a comprehensive characterization of the diseases search, recognition and verification process in living cells. Now, as the ECL signal can be collected by electron multiplying charge coupled device (EMCCD), ECL-microscopy has the potential to provide a new technology to study individual cancer cell19-20 and single nanoparticle21-22. For example, Xu’s group employed ECL microscopy to image the electrocatalysis of a novel bimetallic nanoparticle, the gold-platinum Janus nanoparticle. The research suggests that materials with Janus structure could weaken the surface poison and hold promise in electrocatalysis23-24. Besides, Francesco Paolucci and Neso Sojic’s group used ECL as a surface-confined microscopy to

image single cells and their membrane proteins25-26. However, the challenge of ECL-microscopy still exists for intracellular tumor-biomarkers imaging without nano-electrode27. This work herein shows the ECL-microscopy was used as optical readout for intracellular miRNA-21 imaging in single cancer cell, a measurement offset the limitations of traditional ECL technology. Micro-ribonucleic acid (miRNA) are small noncoding singlestranded RNA molecules that regulate gene expression28-29. The expression of miRNA was associated with the regulation and progression of numerous cancers such as breast30, prostate31, lung32 and ovarian33. In contrast to the tightly regulated patterns of miRNA expression during development and in normal tissues, miRNAs are often misregulated under tumor condition. Therefore, miRNA biomarkers can be used to signify the early stage of tumor34-35. Previous methods provide a number of selectivity and sensitivity ways for detecting miRNAs in cancer cell36-38. However, rare studies used miRNA as biomarker in tumor diagnosis combined with therapeutics, which could have a great prospect to be an efficient method39-41. Due to such limitations, our group developed an original miRNA–activated chemotherapy-PPT system, which employed the in-situ miRNA-21 instead of the external light source to activate the combined therapy and efficiently kill cancer cells. Photothermal treatment (PPT) is a procedure based on localized heating due to light absorption for selective destruction of abnormal cells42-44. Generally, near-infrared (NIR, 700-1100 nm) light is preferred for such an application,

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as it can penetrate soft tissues deeply owing to the relatively low absorption/scattering by hemoglobin and water. The key component of this technique is a photothermal transducer that can absorb and convert NIR light into heat through a nonradiative mechanism with high efficiency. In recently studies, PPT has been actively explored as a minimally invasive approach to cancer therapy45-46. Au nanocages (Au NCs) with hollow interiors and porous walls have recently received increasing interests in tumor therapy due to their spectacular physical and chemical properties47-48. Au nanocages (Au NCs) are of particular interest for PPT because their LSPR peaks can be easily and precisely tuned into the NIR region. Besides, their hollow interiors can also be potentially loaded with drugs to provide a multifunctional carrier for biomedical applications4950. The availability of Au NCs loaded with therapeutic drugs offers a great benefit to therapeutic applications as Au NCs can be monitored with an optical imaging technique while the drug is released at the targeted site in a controllable fashion51-52. As expected, the released ROS can also greatly enhance the efficacy of PPT cancer treatment. Herein, based on the excellent properties of ECL-microscopy and Au NCs@PMA probe, a novel strategy combined the diagnosis and therapy of tumor was built. EXPERIMENTAL SECTION Chemicals and Regents Phorbol 12-myristate 13-acetate (PMA, 99%), luminol and tri (2-carboxyethyl) phosphine hydrochloride (TCEP) were obtained from Sigma-Aldrich Inc. (USA). Cell Counting Kit-8 (CCK-8) was obtained from Beyotime Biotechnology (Shanghai, China). Phosphate Buffer Saline (PBS, pH=7.4) containing 2 mM Tris-HCl, 10 mM NaCl, 5 mM MgCl2 was used to disperse Au NCs probe. Annexin V-EGFP/PI Apoptosis Detection Kit, Annexin V-FITC/PI Apoptosis Detection Kit were purchased from Shanghai yeasen Science&Technologies Co., Ltd. FTO coated glass (1.1 mm thickness) was purchased from Kaivo (Zhuhai, China). Au NCs (50 nm) was purchased from XFNANO, INNC. All aqueous solutions were prepared using RNAase-free water. All the DNA were purchased from Sangon Biotech (Shanghai) Co., Ltd. with the following sequences: AS1411

5’-SH GGGGGGTTGGTGGTTGTGGT GGTGG-3’

DNA Gate -1

5’-TCAACATCAGTCTGATAAGCTA-3’

DAN Gate -2

5’-SH- TAG CTTATCAGA-3’

Instrument ECL imaging was obtained by EMCCD (Andor, DU-897UCS0), spectrograph (Andor, SR-500i-A) and fluorescence inversion microscope system (Nikon, Ti-U). Transmission electron microscope (TEM) images were obtained from JEM2100 microscope (Hitachi, Japan). CCK-8 assay was performed on a microplate reader (EP0CH2, China). The cell images were performed on a TCS SP8 II laser confocal microscope (Nikon C2plus, Germany). Cell flow cytometry study was carried on Flow cytometry (Cytoflex, A00-1-1102). Photothermal curve were obtained from Haite photoelectric laser exciter (FLMM0808-861-005W). The Designed and Fabrication of the FTO Electrode

The fabrication process of FTO chip is similar to the previous report53. The shape of FTO was designed according to the demand and ablated by laser. Firstly, a piece of quartz FTO was incised into pieces (3 cm *3 cm) which were washed by boiling 2-propanol which contained 2 M KOH for 20 min, followed by rinsing thoroughly with deionized water and dried at 60 °C. After that, the degassed PDMS liquid was loaded on glass mold which coincided with the shape of quartz FTO piece at 80 °C for 40 min to curdle. After cooled, PDMS was stripped from the mold, and two connected holes were punched as reservoirs at the central of PDMS by puncher. Then, designed channel structure was obtained. Afterwards, the PDMS layer was attached to the quartz glass piece surface and pressed firmly. After cleaning with ultrapure water and ethanol, the detection chip was obtained and stored for further use. Preparation of Au NCs@PMA Probe 1 mL Au nanocages (Au NCs) and PMA solution (0.01 mL, 5 µg mL-1) were cultured at room temperature for 6 h to get Au NCs@PMA materials. AS1411 aptamer (0.005 mL, 10 µM) and DNA gate -2 (0.1 mL, 10 µM) solution were then cultured with obtained Au NCs@PMA overnight. NaCl solution (0.005 mL, 0.1 M) were added every two hours during culture for three times. After that, DNA gate -1 (0.1mL, 10 µM) were added in the obtained solution and cultured for 30 min at 37 °C. The obtained mixture was centrifuged, washed thrice with ultrapure water and resuspended in PBS (0.1 M). At last, the Au NCs@PMA probe closed by DNA gate was stored at 4 °C without light for further experiment. Cell Culture HeLa cells and LO-2 cells were cultured in DMEM medium containing 1% streptomycin and penicillin, 10% fetal bovine serum (FBS) and kept at 37 °C in a moist atmosphere constituted by 5% CO2 and 95% air. The cells in the exponential growth phase were digested for 1 min by trypsin (0.1%, m/v), and then separated from the cell culture medium. The obtained solution was centrifuged at 1000 rpm for 3 min and washing thrice with a sterile phosphate buffer solution (PBS, 10 mM, pH=7.4). After that, the cell precipitate was then resuspended in DMEM for further use. Electrochemiluminescence Imaging for miRNA-21 The PDMS layer was attached to the special shape of the FTO conductive electrode surface and pressed firmly. HeLa cells were incubated in FTO chip at 37 °C for 6 h. After HeLa cell were attached on the FTO electrode, Au NCs@PMA probe was added and cultured at 37 °C for 2 h. After that, 40 µL luminol solution (0.001 M) was added into detection cells. Electrochemiluminescence imaging were available by EMCCD after add 2.5 V voltage. Cytotoxicity Experiments HeLa cells and LO-2 cells were both incubated with Au NCs probe (1mg mL-1), Au NCs@PMA probe respectively for 2 hours, 4 hours, 6 hours, 8 hours, 12 hours and 24 hours in the cell incubator. CCK-8 (5 mg mL-1) was added in each well and kept at 37 °C for 1 h. Microplate reader was used to measure the absorbance of each well at 450 nm. Cell viability were obtained according to the equation: Cell viability (%) = (Atest/Acontrol)*100%. Flow Cytometry Study LO-2 cells (0.2 mL, 1×106 cells mL-1) were incubated with Au NCs and Au NCs@PMA probe for 24 h respectively. And

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Analytical Chemistry HeLa cells (0.2 mL, 1 × 106 cells mL-1) were also incubated with Au NCs probe and Au NCs@PMA probe for 24 h. Besides, HeLa cells treated with Au NCs@PMA probe were also treated with photothermal effect for 15 min. All the LO-2 cells and HeLa cells apoptosis rates were determined by Annexin V FITC/PI Apoptosis Detection Kit and quantified by flow cytometry. Briefly, cells were harvested after incubation and stained with 5 µL Annexin V-FITC for 20 min and 5 µL PI for 30min with in the dark before analyzed by flow cytometry. The live cells were negative for both Annexin V-FITC and PI (Annexin V-FITC−/PI−). Cells at the early stage of apoptosis are negative for PI but positive for Annexin V-FITC (Annexin V-FITC+/PI−), whereas dead cells and late-stage apoptotic cells could be stained for both PI and Annexin V-FITC (Annexin VFITC+/PI+). Confocal Microscopy Images After LO-2 cells (0.2 mL, 1×105 cells mL-1) and HeLa cells (0.2 mL, 1 × 105 cells mL-1) incubated with Au NCs@PMA probe. Cell apoptosis images were determined by Annexin VEGFP/PI Apoptosis Detection Kit and obtained by laser confocal microscopy. Briefly, cells were harvested after incubation and stained for 15 min with 0.5 µL Annexin VEGFP and 20 min with 2 µL PI in the dark before analyzed by confocal microscopy. The live cells were negative for both Annexin V-EGFP and PI (Annexin V-EGFP−/PI−). Cells at the early stage of apoptosis are negative for PI but positive for Annexin V-EGFP (Annexin V-EGFP+/PI−), whereas dead cells and late-stage apoptotic cells could be stained for both PI and Annexin V-EGFP (Annexin V-EGFP+/PI+). RESULTS AND DISSCUSSION Characterization of Au NCs The important goals of this work were to develop an ECLmicroscopy method, which combined diagnosis and therapy. According to scheme 1, Au NCs filled with phorbol 12myristate 13-acetate (PMA) were closed by DNA-1 and DNA2 biogate. After Au NCs were endocytosis by HeLa cells, DNA1 was designed to hybridize with miRNA-21 and disengaged from Au NCs. PMA was then released and stimulated HeLa cancer cells to produce reactive oxygen species (ROS; including O2-• , •OH and H2O2 etc.). A visible ECL imaging of HeLa cell was obtained for miRNA-21 detection in luminol solution with H2O2 as coreactant27. After imaging, photothermal effect of Au NCs and ROS induce the apoptosis of HeLa cells with high-efficiency. Finally, the challenge of a simple, specificity, sensitive technique about ECL imaging of miRNA21 in single HeLa cell, and tumor therapy by photothermal and ROS were built for the first time. For this, it is essential to choose a multi-functional probe. Gold nanocages (Au NCs) are finally employed in this platform as efficient carriers and used for photothermal therapy. The reason should be owe to their biocompatibility, hollow plasmonic nanostructures and tunable light absorption/scattering properties50-52. The Au NCs used in this work were characterized by transmission electron microscopy (TEM) technology and shown in Fig. 1A, which reflect the good distribution and uniform size with the average diameter around 50 nm. The holes on Au NCs were bright which means the mesoporous structure could be successfully used for drug delivery. The thermal response of the Au NCs solutions during 808 nm laser irradiation at ∼ 5 W/cm2 was measured directly in the cell culture media. As shown in Fig. 1B, the Au NCs suspensions exhibited rapid temperature

increases, with the finally temperature exceeding 65 °C. Within 1 min, the Au NCs solution reached ∼ 45 °C, upon increasing the exposure time to 5 min, the thermal response of Au NCs was found to reach a maximum of 65 ° C. The temperature-time curve displayed a strong thermal response of Au NCs which most due to the degree of overlap between the Au NCs LSPR bands and the NIR laser. At last, those properties make Au NCs could successfully be used to carry PMA and be used for PPT. Electrochemiluminescence-microscopy for Intracellular miRNA-21 Imaging For intracellular miRNA imaging by ECL-microscopy, HeLa cells were grown on designed FTO chip. After that, Au NCs@PMA probe was added into HeLa cells and cultured until Au NCs@PMA probe was endocytosed by HeLa cells. As the miRNA-21 in HeLa cell can open the DNA gate on Au NCs@PMA probe, PMA was released and caused HeLa cells to produce ROS. H2O2, as one of the component of ROS, can penetrate to the extracellular and react with luminol solution. In order to obtain the best ECL image of HeLa cells, the concentration of luminol, incubation time between HeLa cell and Au NCs@PMA probe were optimized (Fig. S1A, B). As a result, ECL imaging of HeLa cells was obtained by 2h incubation time and 0.001M luminol solution. Then, strong ECL signal after added a voltage of 2.5 V. ECL signal was finally collected by EMCCD and the ECL imaging of miRNA21 in single HeLa cell were obtained (Fig. 2A, B and Fig. S2A, B). Notice that, by contrast with HeLa cells, there is no ECL signal of LO-2 cell after carried the same experimental conditions as HeLa cells (Fig. 2C, D). Such a result, intrinsically associated unique design of DNA gate which DNA gate-1 designed to partly hybridize with DNA gate-2 but fully hybridized with miRNA-21. DNA Gate-2, synthesized with sulfhydryl at the end of 5’, can modified on Au NCs by Au-S bond. DNA Gate-1 hybridized with DNA gate -2 and formed DNA gate to close the holes on Au NCs and encapsulate PMA inside Au NCs. After HeLa cell endocytose Au NCs@PMA probe, miRNA-21 in HeLa cell could hybridized with Gate-1 DNA which fell off from Au NCs@PMA probe. PMA was then released from Au NCs@PMA probe and induce HeLa cell to produce ROS which finally react with luminol to produce ECL imaging. However, DNA gate cannot be opened in normal cell such as LO-2 cell due to the absence of miRNA-21, which means the ECL signal was negative to normal cells. ECL signal of HeLa cell after added reactive oxygen species scavenger was also studied and the result was shown in Fig. S3. The ECL intensity of HeLa cell after treated with reactive oxygen species scavenger was obviously lower than HeLa cell without treated with scavenger. This phenomenon proved the ECL sign was motivated by ROS which could be due to the opening of DNA gate. The result illustrates a simple, specificity, sensitive ECLmicroscopy was built for imaging intracellular biomarker at single cell level. Cytotoxicity of Au NCs@PMA Probe and Apoptosis of HeLa Cells Previous studies about ECL can hardly serve for fabrication a technology of diagnosis and therapy at the same time23, 25-27. Therefore, this work gives a proposal for ECL imaging and combined tumor therapy for the first time. To push the further studies about therapy of tumor, the cytotoxicity of Au NCs probe in HeLa cells line and non-cancerous LO-2 cells was studied by CCK-8 assay. According to the assay data in Fig. 3A, it could be concluded that Au NCs has no cytotoxicity to both

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LO-2 cell and HeLa cancer cell. Besides, Au NCs@PMA also has no cytotoxicity to LO-2 cell (Fig. S4).However, after treated with Au NCs@PMA probe and photothermal effect for 12 h, HeLa cells viabilities was less than 40% and the death of HeLa cell was observable (Fig. 3B). This phenomenon is probably due to the miRNA-21 in HeLa cell could open the designed DNA gate and PMA was released. The released PMA result in an immediate increase of intracellular ROS level. According to previous studies, cancer cells are more vulnerable to the increased intracellular ROS level which causes the apoptosis of HeLa cancer cell. To begin to understand how combination therapy can efficiently kill cancer cells, flow cytometry or confocal imaging were used to quantitatively express apoptosis of HeLa tumor cells. Cells were incubated with Au NCs, Au NCs@PMA probe for 24 h and irradiated with 808 laser for 15 min, followed by staining with Annexin V-FITC/PI kit. As shown in Fig. 4A and Fig. S5A, the apoptosis rate of the HeLa cells and LO-2 cell treated with only Au NCs was 2.64% and 0.77% respectively. This phenomenon illustrates the Au NCs was harmless to both tumor cell and normal cells which was coincidence with the result of CCK experiment. The specificity of Au NCs@PMA probe to LO-2 cells was also studies in this work and shown in Fig. S5B and Fig. S6. The result shows there is no apoptosis of LO-2 normal cells after treated with Au NCs@PMA probe which fully illustrate Au NCs@PMA probe are harmless to normal cells. Although ROS alone was proven effective for obtaining significant anticancer therapeutic effects (Fig. S7). We are currently attempting the introduction of Au NCs for PTT therapy, which further improve tumor killing efficiency. The apoptosis rate of HeLa cells treated with combination therapy was 31.35 %, (Fig. 4C), which was significantly higher than 20.66 % of single ROS therapy (Fig. 4B). The confocal laser experiment of Annexin V-EGFP/PI staining further confirmed that Au NCs@PMA probe induced high-efficiency apoptosis in HeLa cells. As shown in Fig. 5, early apoptotic HeLa cells were stained with Annexin-EGFP and the green fluorescence imaging was collected at 500-570 nm under excitation at 488 nm to obtain the early apoptotic cells information (Fig. 5B). Moreover, late apoptotic HeLa cells were stained with Annexin-EGFP and PI double staining, and the red fluorescence imaging was collected at 650-720 nm under excitation at 535 nm to obtain the late apoptotic cells information. (Fig. 5D). As expected, the displayed system dramatically enhanced ability for cancer cell killing, benefiting from the synergistic chemotherapy and photothermal therapy of Au NCs@PMA probe. CONCLUSION In this work, a new ECL-microscopy method is established to combine the diagnosis and treatment of tumor. Au NCs@PMA drug system not only possesses the improved ECL imaging of miRNA-21 in single cancer cell, but also able to combine chemotherapy and photothermal therapies to enhance the therapeutic effect. By combining ROS and photothermal therapy of Au NCs, highly effective chemotherapy-PTT dual therapy becomes possible. Compared with previous studies, this method provides a novel approach, starting with the study of the high sensitivity and low cost of ECL-microscopy for intracellular miRNA-21 imaging. At the same time, the porous and photothermal effects of the Au NCs are utilized to make the

platform realize combined chemotherapy and photothermal therapy.

Scheme 1. Schematic diagram of ECL-imaging and combined therapy

Figure 1. (A) Transmission electron microscopy (TEM) image of Au NCs. (B) The temperature increase induced by PPT heating of Au NCs solution upon exposure on 808 nm laser excitation.

Figure 2. (A) Bright image of HeLa cells. (B) ECL images of HeLa cells on FTO chip. (C) Bright image of LO-2 cells. (D) ECL image of LO-2 cells on FTO chip. ECL images of cells were recorded by EMCCD by applying 2.5 V voltage, the cells were incubated with Au NCs@PMA probes.

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Analytical Chemistry

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Figures for optimization conditions of experiment, ECL images of HeLa cells, ECL intensities vs. reactive oxygen species scavenger, cell viability of non-cancerous LO-2 cells, flow cytometry of LO2 cells, confocal laser scanning microscopy images of LO-2 cells and HeLa cells (PDF). Figure 3. (A) Cell viability of HeLa cells and non-cancerous LO-2 cells incubated with 5 µL Au NCs probe. (B) Cell viability of HeLa cells incubated with 5 µL Au NCs probe and Au NCs@PMAphotothermal effect respectively.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest. Figure 4. (A) Flow cytometry study of HeLa cells after incubation with Au NCs (1 mg mL-1) for 24 h. (B) Flow cytometry study of HeLa cells after incubation with Au NCs@PMA probe (1 mg mL1) for 24 h. (C) Flow cytometry study of HeLa cells after incubation with Au NCs@PMA probe (1 mg mL-1) for 24 h and photothermal effect for 15 min. All the HeLa cells were stained with Annexin VFITC/PI Apoptosis Detection Kit.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21535002, 21775063, 21605069, 2160573), the Postdoctoral Foundation of China (2016M600518), and the Natural Science Foundation of Shandong province (ZR2017ZC0226).

REFERENCES

Figure 5. Confocal laser scanning microscopy (CLSM) images of HeLa cells treated with Au NCs@PMA probe for 24 h and photothermal effect for 15 min. (A) Bright field: provides cell information. (B) The green channel was collected at 500-570 nm under excitation at 488 nm to obtain the early apoptotic cells information. (fluorescent dye, Annexin V-EGFP). (C) The red channel was collected at 650-720 nm under excitation at 535 nm to obtain the late apoptotic cells information. (fluorescent dye, PI). (D) Merged image: green channel, red channel, and bright field.

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polyethylene glycol for prolonging the circulation lifetime of gold nanocages for photothermal therapy. ACS Nano 2014, 8, 10414. (52) Cho, E. C.; Zhang, Y.; Cai, X.; Moran, C. M.; Wang, L. H. V.; Xia, Y. N. Quantitative analysis of the fate of gold nanocages in vitro and in vivo after uptake by U87-MG tumor cells. Angew. Chem. Int. Ed. 2013, 52, 1152. (53) Zhang, H. R.; Li, B. X.; Sun, Z. M.; Zhou, H.; Zhang, S. S. Integration of intracellular telomerase monitorin by electrochemiluminescence technology and targeted cancer therapy by reactive oxygen species. Chem. Sci. 2017, 8, 8025.

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TOC: ECL-imaging combined chemotherapy and photothermal therapy.

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