Structure-Switching Aptamer Triggering Hybridization Chain Reaction

Jun 5, 2015 - The ability to probe low-abundance biomolecules or transport a high-load drug in target cells is essential for biology and theranostics...
4 downloads 7 Views 584KB Size
Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Letter

Structure-switching Aptamer Triggers Hybridization Chain Reaction on Cell Surface for Activatable Theranostics Yu-Min Wang, Zhan Wu, Si-Jia Liu, and Xia Chu Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 05 Jun 2015 Downloaded from http://pubs.acs.org on June 8, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Structure-switching Aptamer Triggers Hybridization Chain Reaction on Cell Surface for Activatable Theranostics Yu-Min Wang, Zhan Wu, Si-Jia Liu, Xia Chu* State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (P. R. China) ABSTRACT: The ability to probe low-abundance biomolecules or transport high-load drug in targeting cells is essential for biology and theranostics. We develop a novel activatable theranostic approach by using a structure-switching aptamer triggered hybridization chain reaction (HCR) on cell surface, which for the first time creates an aptamer platform enabling real-time activation and amplification for fluorescence imaging and targeting therapy. The aptamer probe is designed not to initiate HCR in its free state but trigger HCR on binding to target cell via structure switching. The HCR not only amplifies fluorescence signals from a fluorescence-quenched probe for activatable tumor imaging, but accumulates high-load prodrugs from a drug-labeled probe and induced its uptake and conversion into cisplatin in cells for selective tumor therapy. In vitro assay shows that this approach affords efficient signal amplification for fluorescence detection of target protein PTK7 with a detection limit of 1 pM. Live cell studies reveal that it provides high-contrast fluorescence imaging and highly sensitive detection of tumor cells, while renders highefficiency drug delivery into tumor cells via an endocytosis pathway. The results imply the potential of the developed approach as a promising platform for early-stage diagnosis and precise therapy of tumors.

The ability to monitor expressions of biomolecules and deliver reagents of bioactivity in living cells is essential for understanding biology and developing efficient theranostic tools.1 In therapeutic development for a particular disease such as cancer, there is significant interest in theranostic approaches that permit concurrent monitoring and treatment. With increased insights into delivery kinetics and biological response, the theranostic approaches may help move the filed of medicine toward an era of more effective personalized and precise treatment.2 Unfortunately, probing low-abundance biomolecules or transporting high-load drug in specific tumor cells, and, in particular, their combination, still remain a major challenge. Current cancer theranostic approaches typically rely on targeting tumor-associated antigens with antibodies or other ligands, which allows an accumulation of signal reporters or drug payloads in tumors and thus renders contrast imaging and selective treatment.3 However, the lack of activatable designs used to give less differential signal intensity and drug concentration for tumors relative to normal tissues, leading to limited imaging sensitivity and increased side effects.4 The pursuit of activatable designs, therefore, represents a major effort in cancer theranostics. Aptamers are single-stranded RNA or DNA with unique intramolecular conformations for selective binding to various targets.5 Because of their advantages over antibodies in terms of size, synthetic accessibility and chemical modification, aptamers are under extensive development as potential diagnostic or therapeutic agents.6 Besides their direct applications to conjugation with therapeutic or imaging agents for theranostics,7 aptamers are able to be incorporated in DNA assembly or nanodevices for signal amplification, which may enhance their capacity for low-abundance molecule imaging and high-load drug delivery.8 On the other hand, aptamers are useful for design of activatable imaging agents because they

undergo structure switching in response to ligand binding, which may efficiently convert the targeting of tumors into high-contrast signals.9 Though these activatable probes afford a high contrast with improved detection speed,10 they do not involve a signal amplification step, which limits their sensitivity and efficacy for molecular imaging and therapy. Aptamer based approaches combining target-specific activatable designs and signal amplification have currently not realized for cancer theranostics. Scheme 1. theranostics.

Illustration

ACS Paragon Plus Environment

of

SATHCR

for

activatable

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Herein we develop a novel activatable theranostic approach based on structure-switching aptamer triggered hybridization chain reaction (SATHCR) on cell surface, as illustrated in Scheme 1. As a proof of concept, an aptamer sgc8, which has been identified to bind to a membrane protein tyrosine kinase-7 (PTK7) closely associated with cancers,11 is chosen as the model system to demonstrate this approach. In the SATHCR approach, the aptamer probe acts as not only a molecular recognition probe, but also an activatable initiator for hybridization chain reaction (HCR) with two hairpin probes.12 To this end, the aptamer probe AP is designed to have a hairpin structure with the HCR initiator sequence blocked by a part of the DNA aptamer sequence. The two hairpin probes are designed as a signal probe H1 with a Cy5 fluorophor tag quenched by a BHQ3 label and a drug probe H2 conjugated with a synthesized prodrug, c,c,t-[Pt (NH3)2 Cl2 (OH) (O2CCH2CH2 COOH)].13 In the absence of target protein, the aptamer probe is not able to trigger HCR with these two hairpin probes, without activated fluorescence signal for diagnostic imaging and internalized drug probes for tumor therapy. When binding to target cell-surface protein, the aptamer probe undergoes structure switching and activates a single-stranded initiator sequence, which then triggers a real-time HCR with probes H1 and H2. An amplified fluorescence signal is activated in the chain-like HCR product due to the separation of fluorophores from quenchers, enabling real-time fluorescence imaging of tumor cells. The HCR product also accumulates a high load of prodrug, which is internalized into cells and induces selective cytotoxicity to tumor cells via reduction of the conjugated prodrug into cisplatin by cytosolic thiols.13 To our knowledge, this is the first time that an aptamer-based theranostics approach has been developed for real-time fluorescence activation and amplification for tumor imaging as well as in situ accumulation of prodrug payloads on cell surface for targeting therapy. This unique real-time imaging strategy offers a prominent advantage of high contrast for diagnostic imaging. Moreover, because the prodrug shows low toxicity in blood with low thiol concentration and is selectively activated after uptake in targeting cells, this approach also affords substantial improvement of selectivity for tumor therapy. On the other hand, because the use of small DNA probes confers less immunogenicity and higher penetration rate than preassembled large DNA assembly8, this in situ activation strategy may enable highly efficient tumor theranostics. The SATHCR approach may thus provide a useful platform for early-stage diagnosis and more precise and efficient therapy of tumors. To demonstrate the feasibility of the activatable theranostic approach, we firstly investigated the response of SATHCR to target protein PTK7, as shown in Figure 1a. Reaction of two hairpin probes H1 and H2 merely gave a very weak fluorescence peak, indicating that these probes had a low fluorescence background and there was no HCR between two probes in the absence of initiator sequence. Incubation of the aptamer probe AP with two hairpin probes also did not generate enhanced fluorescence, testifying that the AP probe was unable to initiate the HCR with H1 and H2. An intense fluorescence peak at 660 nm, a typical emission for the activated signal probe, appeared after incubation of 50 nM target protein with the AP probe plus H1 and H2 probes. This observation implied the activation of the AP probe in response to target protein as an initiator for the HCR with H1 and H2. A control

Page 2 of 5

experiment for incubation of 50 nM target protein with probes H1 and H2 did not show a remarkable change in the fluorescence signal, which manifested that target induced structure switching of the AP probe was essential for activation of the fluorescence signal. These results gave clear evidence for the proposed design of SATHCR. On the other hand, no appreciable fluorescence activation was observed for incubation the probes AP plus H1 and H2 with a complex matrix such as the cell growth medium containing 10% bovine serum. This finding suggested the specificity of the SATHCR approach to target protein. Additionally, the fluorescence response to 50 nM target protein in 10% bovine serum merely showed slightly deviation from that in response to 50 nM target protein in the assay buffer, indicating the complex matrix had little interference to the detection sensitivity for SATHCR. To validate signal amplification of this approach, a control experiment in which 50 nM target protein was incubated with the AP probe plus probe H1. Note that in the absence of probe H2, the AP probe activated by target protein only hybridized with probe H1 with no chain reaction between H1 and H2, which did not yield signal amplification. It was observed that the fluorescence peak intensity was only ~1.7×105 in the amplification-free case, much smaller than the peak intensity (~5.8×105) obtained using SATHCR. This result implied that substantial fluorescence signal amplification was achieved using our approach.

Figure 1. (a) Fluorescence spectral responses obtained from reactions of H1 and H2 (black), AP with H1 and H2 (blue), AP with H1 plus 50 nM PTK7 (pink), H1 and H2 with 50 nM PTK7 (red), as well as AP, H1 and H2, 50 nM PTK7 (brown), 50 nM PTK7 in 10% fetal bovine serum (cyan) or 10% fetal bovine serum supplemented cell culture (green). (b) Fluorescence responses of SATHCR to PTK7 of varying concentrations.

A further investigation was performed using gel electrophoresis analysis (Figure S1). We observed that binding of target protein to the AP probe resulted in a remarkable shift of the band for the probe, evidencing the high-affinity binding of the AP probe to target protein. No new bands appeared for reactions of probes H1 and H2 with the AP probe or target protein. In contrast, reaction of target protein with the AP probe plus probes H1 and H2 gave a broad bright band with a maximum size over 1500 base-pairs, implying the formation of a large molecule-weight product. This result confirmed the successful HCR triggered by target protein, validating the proposed design for the activatable theranostic approach. The SATHCR approach was found to give activated fluorescence signals dynamically correlated to the concentrations

ACS Paragon Plus Environment

Page 3 of 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

of target protein through a over five-decade range from 5 pM to 1 µM (Figure 1b). A quasi-linear correlation was obtained for the peak intensities at 660 nm to the logarithmic protein concentrations in the range from 5 pM to 50 nM with a detection limit estimated to be 1 pM (Figure S2). This detection limit was much better than existing aptamer-based methods for this target,14 indicating the advantage of sensitivity enhancement for the SAT-HCR approach. Next, we investigated the feasibility for the SATHCR approach on cell surface (Figure 2). After incubation for 4 h of the AP probe plus probes H1 and H2 with CEM cells, which had overexpressed protein PTK7 on the surface,15 we observed that very bright fluorescence was not only obtained around the cell surface but also inside the cells. This observation testifies the realization of the HCR on the cell membrane with a concomittant internalization of the HCR product in the cells, which supported the potential of the SATHCR approach for theranostic applications. In contrast, very low fluorescence contrast was obtained for Romas cells with low expression of PTK7 on the surface.15 This result gave direct evidence for high selectivity of SATHCR in fluorescence activation imaging of targeting cells. An amplification-free control experiment by incubating the cells with the AP probe plus probe H1 showed that the fluorescence brightness on the cell surface or inside CEMs cells was much lower than that obtained using SATHCR. This finding manifested efficient signal amplification with SATHCR for activatable fluorescence imaging of tumor cells. In addition, because the fluorescence signals inside cells gave indicators of intracellular delivery efficiency for the conjugated prodrugs, the higher fluorescence contrast inside CEMs cells obtained using SATHCR also suggested that it afforded improved efficiency for transporting the prodrug into targeting cells, implying its capacity in targeting delivery of high-load drugs for tumor therapy.

Figure 2. Confocal microscopy images. (a) CEM incubated with AP, H1 and H2; (b) CEM incubated with AP and H1; (c) Romas incubated with AP, H1 and H2.

Further flow cytometry assay revealed selective fluorescence activation on PTK7-overexpressed cells and substantial sensitivity enhancement for the SATHCR approach (Figure S3). To demonstrate the sensitivity of this approach for cell detection, we used the number of events appearing in the upright region obtained using flow cytometry assay for quantifying the number of CEM cells. It was found that the number of positive events detected by SATHCR showed linear correlation to the number of CEM cells with a detection limit of 16 cells (Figure S4). This low detection limit was much better

than that obtained using an activatable aptamer probe,10 further disclosing high sensitivity of SATHCR for tumor cell detection. Because of its ability for activatable fluorescence imaging in real-time and wash-free manner, this approach could be used to monitor the fluorescence activation events to shed light on the HCR kinetics on a single cell (Figure S5). It was found that, after 10 min, a few bright spots appeared around cell surface, indicating the initiation of HCR on the cells. After 70 min, bright spots appeared inside the cells, suggesting the uptake of the HCR product in the cells. The higher rates of HCR reaction on cell surface than those for cellular uptake of the HCR product implied the possibility of substantial accumulation of fluorescence reporters and prodrug payloads on cell surface before their internalization into cells. This gave direct evidience for efficient amplification using the SATHCR approach for tumor theranostics. Further investigation by pretreating the cells with NaN3, an inhibitor for ATPase involved in all energy-dependent endocytic pathways,16 revealed that fluorescence activation was only obtained around the cell surface with no uptake of the HCR product into the cells (Figure S6). This finding manifested that cellular internalization of the HCR product followed a normal endocytosis mechanism. Closer interrogation of subcellular localization of the fluorescence signals evidenced that the HCR product was colocalized with lysosomes (Figure S7), which was consistent with the endocytic pathways for cellular uptake of the HCR product. It was noteworthy that entrapment of the HCR product in lysosomes provided an efficient avenue for activating the prodrug payloads conjugated in probe H2, because a higher concentration of biothiols in lysosomes17 could facilitate reduction of the prodrug into active cisplatin drug.

Figure 3. Cytotoxicity assay for CEM (a) and Ramos (b) cells using different approaches.

Having demonstrated the ability of SATHCR for diagnostic imaging of tumor cells, we then investigated its efficacy for targeting tumor therapy. To this end, we synthesized a prodrug, c,c,t-[Pt (NH3)2 Cl2 (OH) (O2CCH2CH2 COOH)] using a previously reported method,13 and conjugated it to probe H2 to achieve targeting delivery and therapy (Figure S8-S10). The use of such a prodrug furnished additional advantage of minimized nonspecific cytotoxicity, because the prodrug merely had low toxicity in blood with low thiol concentration but restored high toxicity to tumor cells after it selectively internalized in targeting cells and reduced to cisplatin. As shown in Figure 3, after incubating the cells with the pro-

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

drug or cisplatin, it was observed that cisplatin had almost the same dose-dependent cytotoxicity to both CEM and Ramos cells with little selectivity. We also found that the prodrug exhibited little cytotoxicity, which was consistent with previous findings and could be ascribed to lower uptake efficiency of the negatively charged prodrug.13,18 In contrast, after cells were incubated with the AP probe plus probes H1 and H2, we observed that the dose-dependent toxicity to CEM cells was much higher than cisplatin, while much lower cytotoxicity was obtained for Ramos cells. This result demonstrated that the SATHCR approach afforded high efficacy and selectivity for targeting tumor therapy. Control experiments by incubating the cells with probes H1 and H2 showed that these probes displayed little toxicity, implying that the selective toxicity of SATHCR to CEM cells was specifically induced by the aptamer probe. In addition, we found that incubation of the cells with a drug-modified aptamer probe AP2 also displayed selective dose-dependent toxicity to CEM cells but was much lower than SATHCR. This finding testified that the SATHCR approach provided improved efficacy for targeting tumor therapy. Further ICP-MS analysis of intracellular drug concentrations revealed that the drug load was much higher in CEM cells treated using SATHCR than those incubated using the drug-modified probe AP2, and the drug concentration of nontargeting cells was very low (Figure S11). This data clearly demonstrated that the developed approach provided an useful platform for highly selective and efficient delivery of drugs in targeting tumors as compared to aptamer-directed drugs or non-targeting anti-tumor drugs. In conclusion, we have developed a novel activatable theranostic approach by using a structure-switching aptamer triggered HCR on cell surface, which realizes real-time activation and signal amplification for sensitive fluorescence imaging and efficient targeting therapy of tumors. In vitro assay shows that this approach affords efficient signal amplification for fluorescence detection of target protein PTK7 with a detection limit down to 1 pM. Live cell studies suggested that it provides high-contrast fluorescence imaging and highly sensitive detection of tumor cells using flow cytometry with a detection limit of 16 cells. Fluorescence imaging studies also revealed that the HCR product forms much faster on cell surface than cellular uptake, indicating its high efficiency for in situ signal amplification. Cytotoxicity assay and ICP-MS analysis suggested that this approach renders high-efficiency delivery of drug in targeting tumor cells. Collectively, the results imply that the developed approach may hold great potential for early-stage diagnosis and efficient targeting therapy of tumors.

REFERENCES (1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13) (14)

(15)

ASSOCIATED CONTENT Supporting Information

(16)

Additional experimental details and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

(17)

AUTHOR INFORMATION (18)

Corresponding Author [email protected]

Page 4 of 5

(a) Weissleder, R. Science 2006, 312, 1168-1171. (b) Srinivasarao, M.; Galliford, C.V.; Low, P. S. Nat. Rev. Drug Discov. 2015, 14, 203-219. (a) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nat. Nanotechnol. 2007, 2, 751-760. (b) Conway, J. R.; Carragher, N. O.; Timpson, P. Nat. Rev. Cancer 2014, 14, 314-328. (a) Allen, T. M. Nat. Rev. Cancer 2002, 2, 750-763. (b) Andersen, E. S.; Dong, M.; Nielsen, M. M.; Jahn, K.; Subramani, R.; Mamdouh, W.; Golas, M. M.; Sander, B.; Stark, H.; Oliveira, C. L. P.; Pedersen, J. S.; Birkedal, V.; Besenbacher, F. K.; Gothelf, V.; Kjems, J. Nature 2009, 459, 73-76. (a) Kobayashi, H.; Choyke, P. L. Acc. Chem. Res. 2011, 44, 83-90. (b) Bhuniya, S.; Maiti, S.; Kim, E. J.; Lee, H.; Sessler, J. L.; Hong, K. S.; Kim, J. S. Angew. Chem. Int. Ed. 2014, 53, 4469-4474. (a) Huang, Y. C.; Ge, B.; Sen, D.; Yu, H. Z. J. Am. Chem. Soc. 2008, 130, 8023-8029. (b) Liu, J.; Cao, Z.; Lu, Y. Chem. Rev. 2009, 109, 1948-1998. (c) Goulko, A. A.; Li, F.; Chris, Le. X. Trends Anal. Chem. 2009, 28, 878-892. (d) Cella, L. N.; Sanchez, P.; Zhong, W.; Myung, N. V.; Chen, W.; Mulchandani, A. Anal. Chem. 2010, 82, 2042-2047. (e) Lu, C. H.; Li, J.; Lin, M. H.; Wang, Y. W.; Yang, H. H.; Chen, X.; Chen, G. N. Angew. Chem. Int. Ed. 2010, 49, 84548457. (f) Helwa, Y.; Dave, N.; Froidevaux, R., Samadi, A.; Liu, J. ACS. Appl. Mater. Interfaces 2012, 4, 2228-2233. (a) Keefe, A. D.; Pai, S.; Ellington, A. Nat. Rev. Drug Discov. 2010, 9, 537-550. (b) Tan, W.; Donovan, M. J.; Jiang, J. H. Chem. Rev. 2013, 113, 2842-2862. (a) Wang, S.; Kong, H.; Gong, X.; Zhang, S.; Zhang, X. Anal. Chem. 2014, 86, 8261−8266. (b) Kruspe, S.; Hahn, U. Angew. Chem. Int Ed. 2014, 53, 10541-10544. (a) Zhu, G.; Zheng, J.; Song, E.; Donovan, M.; Zhang, K.; Liu, C.; Tan, W. Proc. Natl. Acad. Sci. USA 2013, 110, 7998-8003. (b) Zhu, G.; Zhang, S.; Song, E.; Zheng, J.; Hu, R.; Fang, X.; Tan, W. Angew. Chem. Int. Ed. 2013, 52, 5490-5496. (a) Nutiu, R.; Li, Y. J. Am. Chem. Soc. 2003, 125, 4771-4778. (b) Wang, Y.; Li, Z.; Hu, D.; Lin, C. T.; Li, J.; Lin, Y. J. Am. Chem. Soc. 2010, 132, 9274-9276. (c) Chen, T. T.; Tian, X.; Liu, C. L.; Ge, J.; Chu, X.; Li, Y. J. Am. Chem. Soc. 2015, 137, 982-989. Shi, H.; He, X.; Wang, K.; Wu, X.; Ye, X.; Guo, Q.; Tan, W.; Qing, Z.; Yang, X.; Zhou, B. Proc. Natl. Acad. Sci. USA 2011, 108, 39003905. (a) Boudeau, J.; Miranda-Saavedra, D.; Barton, G. J.; Alessi, D. R. Trends Cell Biol. 2006, 16, 443-452. (b) Shin, W. S.; Kwon, J.; Lee, H. W.; Kang, M. C.; Na, H. W.; Lee, S. T.; Park, J. H. Cancer Sci. 2013, 104, 1120-1126. (a) Dirks, R. M.; Pierce, N. A. Proc. Natl. Acad. Sci. USA 2004, 101, 15275-15278. (b) Huang, J.; Gao, X.; Jia, J.; Kim, J. K.; Li, Z. Anal. Chem. 2014, 86, 3209-3215. 32. (c) Xuan, F.; Hsing, I. M. J. Am. Chem. Soc. 2014, 136, 9810-9813. Dhar, S.; Daniel, W. L.; Giljohann, D. A.; Mirkin, C. A.; Lippard, S. J. J. Am. Chem. Soc. 2009, 131, 14652-14653. (a) Chen, Y.; Munteanu, A. C.; Huang, Y. F.; Phillips, J.; Zhu, Z.; Mavros, M.; Tan, W. Chemistry 2009, 15, 5327-5336. (b) Yin, J.; He, X.; Wang, K.; Qing, Z.; Wu, X.; Shi, H.; Yang, X. Nanoscale 2012, 4, 110-112. (c) Yin, J.; He, X.; Wang, K.; Xu, F.; Shangguan, J.; He, D.; Shi, H. Anal. Chem. 2013, 85, 12011-12019. (d) Li, L.; Wang, Q.; Feng, J.; Tong, L.; Tang, B. Anal. Chem. 2014, 86, 5101-5107. Shangguan, D.; Li, Y.; Tang, Z.; Cao, Z. C.; Chen, H. W.; Mallikaratchy, P.; Sefah, K.; Yang, C. J.; Tan, W.; Qiu, L.; Chen, T.; Ocsoy, I.; Yasun, E. Proc. Natl. Acad. Sci. USA 2006, 103, 11838-11843. Chen, J.; Chen, S.; Zhao, X.; Kuznetsova, L. V.; Wong, S. S.; Ojima, I. J. Am. Chem. Soc. 2008, 130, 16778-16785. Xu, X.; Xie, K.; Zhang, X. Q.; Pridgen, E. M.; Park, G. Y.; Cui, D. S.; Shi, J.; Wu, J.; Kantoff, P. W.; Lippard, S. J.; Langer, R.; Walker, G. C.; Farokhzad, O. C. Proc. Natl. Acad. Sci. USA 2013, 110, 18638-18643. Kumar, A.; Huo, S.; Zhang, X.; Liu, J.; Tan, A.; Li, S.; Jin, S.; Xue, X.; Zhao, Y.; Ji, T.; Han, L.; Liu, H.; Zhang, X.; Zhang, J.; Zou, G.; Wang, T.; Tang, S.; Liang, X. J. ACS Nano 2014, 8, 4205-4220.

ACKNOWLEDGMENT This work was supported by NSFC (21275045, 21190041), NCET-11-0121 and NSF of Hunan (12JJ1004).

ACS Paragon Plus Environment

Page 5 of 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Table of Contents (TOC)

ACS Paragon Plus Environment