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Nature-Inspired Smart DNA Nanodoctor for Activatable in Vivo Cancer Imaging and in Situ Drug Release Based on Recognition-Triggered Assembly of Split Aptamer Yanli Lei, Jinlu Tang, Hui Shi, Xiaosheng Ye, Xiaoxiao He, Fengzhou Xu, Lv'an Yan, Zhenzhen Qiao, and Kemin Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03283 • Publication Date (Web): 03 Nov 2016 Downloaded from http://pubs.acs.org on November 5, 2016

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Analytical Chemistry

Nature-Inspired Smart DNA Nanodoctor for Activatable in Vivo Cancer Imaging and in Situ Drug Release Based on RecognitionTriggered Assembly of Split Aptamer Yanli Lei, Jinlu Tang, Hui Shi,* Xiaosheng Ye, Xiaoxiao He,* Fengzhou Xu, Lv’an Yan, Zhenzhen Qiao and Kemin Wang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, China ABSTRACT: DNA-based activatable theranostic nanoprobes are still unmet for in vivo applications. Here, by utilizing the “induced-fit effect”, a smart split aptamer-based activatable theranostic probe (SATP) was first designed as “nanodoctor” for cancer-activated in vivo imaging and in situ drug release. The SATP assembled with quenched fluorescence and stable drug loading in its free state. Once binding to target proteins on cell surface, the SATP disassembled due to recognition-triggered reassembly of split aptamers with activated signals and freed drugs. As proof of concept, split Sgc8c against CEM cancer was used for theranostic studies. Benefiting from the design without blocking aptamer sequence, the SATP maintained an excellent recognition ability similar to intact Sgc8c. An “incubate-and-detect” assay showed that the SATP could significantly lower background and improve signal-to-background ratio (~4.8 times of “always on” probes), thus affording high sensitivity for CEM cell analysis with 46 cells detected. Also, its high selectivity to target cells was demonstrated in analyzing mixed cell samples and serum samples. Then, using doxorubicin as a model, highly-specific drug delivery and cell killing was realized with minimized toxicity to nontarget cells. Moreover, in vivo and ex vivo investigations also revealed that the SATP was specifically activated by CEM tumors inside mice. Especially, contrast-enhanced imaging was achieved in as short as 5 min, thus laying a foundation for rapid diagnosis and timely therapy. As a biocompatible and target-activatable strategy, the SATP may be widely applied in cancer theranostics.

By integrating imaging and therapeutic functionalities in a single agent, cancer theranostics provide a novel solution to achieve early diagnosis followed by tailored therapy.1-4 Ideal theranostic agents should have two fundamental properties. One is biocompatibility and biodegradability, because selftoxicity is the deadly obstacle to clinical uses.5-7 The other is target-acitivatable nature, including signal activation and drug release triggering. Due to the “silence” of imaging and therapy will be kept until targets show up, unspecific signals and side effects can be minimized, thus contributing to highly-sensitive detection and precise therapy.8-10 Among various potential theranostic tools, DNA-based nanostructures exhibit unique merits. First, DNA molecules are inherently biocompatible and biodegradable.5-7 Next, benefiting from flexible three-dimensional shapes based on base pairing, hydrogen bonding and π-stacking, DNA is particularly advantageous in programmable and controllable example, nanotrains,14 structure assembly.11-13 For 15 5 nanoflowers, and DNA dendrimers have been developed as safe and effective carriers for anticancer drugs. More importantly, DNA aptamers, which can fold into well-defined shapes to recognize certain targets such as tumor cells, provide a powerful molecular tool for theranostics.16 And its property of induced-fit conformational changes can greatly facilitate the design of target-switchable structures.8, 17, 18 These features open up exciting avenues for DNA nanostructures in biomedicine. But currently, DNA theranostics are still in the

infancy stage and studies are focused on simple improvement of targeting and drug loading in therapy.5, 14 Specifically, imaging quality has been over ignored, which is a critical defect since timely and high-fidelity diagnosis is a precondition for therapeutic decision-making.2, 19 Another problem is widespread use of “always on” strategy, in which contrast imaging and selective treatment is achieved by aptamer-oriented accumulation of signal reporters and drug payloads in tumors.5, 8 The lack of target-activatable nature will inevitably result in high-background and low-contrast image, delayed diagnosis and toxicity to normal tissues. An encouraging attempt at activatable theranostics was recently made with a switchable aptamer triggering hybridization chain reaction (HCR).8 But due to the complex multistep activation mechanism as well as high demands for assembly time and DNA dosage of HCR, this strategy might be difficult for in vivo use. There is still a challenge in developing activatable theranostic DNA nanoprobes for in vivo applications. In the context of aptamer-based cancer detection, several activatable designs have been developed, such as competitive binding-based probes18, 20-22 and split probes23-25. The competitive binding-based design is commonly used and has been demonstrated to work inside mice in our group.18, 20, 21 A representative example is a hairpin-shaped activatable aptamer probe (HAAP), which was designed as an extended aptamer sequence with tailored short oligonucleotides to break binding conformation through hybridization with aptamer. Once

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encountered targets, the HAAP could be induced to reform binding conformation based on interaction between aptamer and target cells, thus providing an effective target-activatable strategy.18 Nevertheless, due to the use of original or extended aptamers, competitive designs have problems like long sequence, inhibited activity and false positive. As an alternative, cancer cell-specific split aptamers have recently been engineered, showing advantages of short length, low cost and simple structures.23, 24 Particularly, split fragments can assemble into a desired shape only in the presence of target cells, thus suggesting a good anti-interference performance.24 For example, two sets of split aptamers against CCRF-CEM leukemia and SMMC-7721 liver cancer respectively,23, 24 have been developed in our group and utilized for tumor cell detection with high sensitivity and specificity. However, owing to inactivity at physiological temperature, split aptamers have been stalled in applications in vitro. We thereupon envisage that if the thermosensitivity could be improved, split aptamers with target-switchable nature would afford a brand-new activatable theranostic platform with simplicity, biocompatibility and in vivo applicability. Inspired by nature, we herein design a novel activatable theranostic probe based on the target-induced shape change of split aptamers and the drug loading capacity of doublestranded DNA. As illustrated in Scheme 1, the split aptamerbased activatable theranostic probe (SATP) is assembled from a long DNA strand (Apt-L) and a short one (S). Apt-L consists Scheme 1. The Structure and Working Principle of SATP in Cancer Theranosticsα

α Split aptamer-based activatable theranostic probe (SATP) is assembled from a long DNA strand (Apt-L) and a short one (S) with a fluorophore (F) and a quencher (Q) attached respectively. In the free state, the SATP is double-stranded with signal inactivated and drug loaded. Once encountering the target cell, the SATP can disassemble based on aptamer binding-triggered conformational alteration, thus leading to fluorescence activation and doxorubicin (Dox) release.

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of two split fragments (Apt-a and Apt-b) deriving from an intact aptamer and a linker DNA labeled with a fluorophore (F). Because of hybridization of the linker with the S strand attached by a quencher (Q), the SATP is partly doublestranded, which keeps F close to Q and thus results in quenched fluorescence in the free state. Meanwhile, the double helix region is designed rich in CG bases, providing sites for doxorubicin (Dox) intercalation.5, 12 By varying the number of CG base pairs, drug loading can be precisely tuned. Once encountering target cancer cells, Apt-a and Apt-b act like snail tentacles for sensing, which reassemble to a target-induced binding shape and then force disassembly of the SATP. Consequently in response to the specific target-probe interaction, a fluorescence signal is activated, accompanied by drug release in situ like threatened octopus. As an activatable probe, the SATP is expected to enhance imaging quality and reduce unspecific toxicity. Moreover, by using the linker to convert interaction of split aptamers from intermolecular to intramolecular, the design might solve the problem of thermosensitivity. This would not only break through the limit of current split aptamer strategies for in vivo uses, but also enrich target-activatable designs.

EXPERIMENTAL SECTION Chemicals and Materials. All DNA probes used in this study were custom-designed and then synthesized by Takara Bio Inc. Sequences of the oligos are listed in Table S1. Dulbecco’s phosphate buffered saline (D-PBS), bovine serum albumin (BSA) and yeast tRNA were purchased from SigmaAldrich. Cell Titer 96 Cell Proliferation Assay was purchased from Promega. Hoechst 33342 was purchased from Invitrogen Life Technologies Corporation. Mouse serum and doxorubicin hydrochloride were obtained from Dingguo reagent company (Beijing, China). All other reagents were of the highest grade available. Deionized water was obtained by the Milli-Q ultrapure water system. Binding buffer was prepared by adding 1 mg/mL BSA and 0.1 mg∕mL yeast tRNA into DPBS containing 4.5 mg/mL glucose and 5 mM MgCl2. Cells. CCRF-CEM (T lymphoblast, human acute lymphoblastic leukemia) cells, SMMC-7721 (human hepatocellular cancer) cells, and L02 (human hepatocyte cell line) cells used in this study were obtained from Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Ramos (B lymphocyte, human Burkitt’s lymphoma) cells were purchased from the Cancer Institute & Hospital of Chinese Academy of Medical Sciences (Beijing, China). Cells were cultured in RPMI 1640 medium supplemented with 13% fetal bovine serum (FBS, heat inactivated) and 100 IU/mL penicillin−streptomycin. All cells were maintained at 37℃ in a 5% CO2 atmosphere. The cell density was determined using a hemocytometer, and this was performed prior to any experiment. Animals. Male athymic BALB/c (Balb/C-nu) mice were obtained from Hunan SJA Laboratory Animal Co., Ltd. They were 4–6 weeks old at the start of each experiment and weighed 20–25 g. All animal operations were in accord with institutional animal use and care regulations, according to protocol No. SCXK (Xiang) 2013-0004, approved by the Laboratory Animal Center of Hunan Province. Preparation and Characterization of SATP. The oligos S and Apt-L were first mixed at a molar ratio of 2: 1 (unless


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specifically mentioned) in binding buffer (without BSA and yeast tRNA). Then, the mixture was heated at 95℃ for 4 min, cooled immediately on ice for 5 min and finally left hybridizing at room temperature for 30 min to form SATP with partly double strand region. Control probes were prepared using the same procedure. The formation of SATP was determined on an F-7000 fluorescence spectrophotometer (Hitachi, Japan) by measuring the fluorescence signal of Cy5labeled Apt-L. To investigate the fluorescent stability of SATP in complex biofluids, probes were incubated at 37℃ in mouse serum for about 240 min and the fluorescence intensity of Cy5 was monitored with the time extended (Ex=610 nm; Em=670 nm). Doxorubicin Loading. The SATP-3* was incubated in an aqueous solution of doxorubicin (Dox: 2 µM) for 30 min at various molar ratios of SATP-3* to Dox. The fluorescence spectra of Dox were then examined by an F-7000 fluorescence spectrophotometer (Hitachi, Japan; Ex = 480 nm, Em = 520– 660 nm). Flow Cytometry Assays. Generally, DNA probes, with or without drug loading, were incubated with 1.5×105 cells in 200 µL of binding buffer at 20℃ for 60 min in the dark and then immediately determined with a FACScan cytometer (BD Biosciences, USA) by counting 10,000 events. To investigate the sensitivity of this strategy, samples with varying cell numbers ranging from 0 to 4.6×105 in 200 µL of binding buffer were determined. Each sample was detected with flow cytometer at high rate for 165 s. Every group was repeated for 3 times. Laser Scanning Confocal Microscopy Imaging. Fluorescent images were acquired on a FV500 confocal microscope (Olympus, Japan) with a 100× oil immersion objective. Excitation wavelength and emission filters were described as follows. Hoechst 33342: Ex=405 nm, Em=430– 460 nm bandpass; Calcein-AM: Ex=488 nm, Em=505–525 nm bandpass; Dox: Ex=488 nm, Em=505 nm long-pass; Cy5: Ex=633nm, Em=660 nm long-pass. Specifically in the selective detection of mixed cell samples, Ramos cells were pre-stained by calcein-AM and then mixed with an equal amount of CEM cells. Next, the mixed cell samples were incubated with SATP-3* in binding buffer at 20℃ for 60 min in the dark and then imaged after settlement on the bottom. To investigate the specific cellular uptake of SATP-3*-Dox by CEM cells, 1×105/mL of CEM or Ramos cells were incubated with 2 µM of SATP-3*-Dox (SATP-3*/Dox=0.6) or free Dox in medium (without FBS; 37℃, 5% CO2) for 2 h. Then, cells were centrifuged at 2,000 rpm for 4 min and resuspended in medium (without FBS; 37℃, 5% CO2) for further 3 h. Finally, after staining with Hoechst 33342 for 15 min, cells were centrifuged at 2,000 rpm for 4 min and resuspended in binding buffer for imaging. In Vitro Cytotoxicity Assays. Cells (5×104 cells per well) were incubated with 1.2 µM of SATP-3*, free Dox or SATP3*-Dox (Dox: 2 µM) in medium (without FBS; 37℃, 5% CO2) for 2 h. Then, 80% of the supernatant was removed and replenish fresh medium (13% FBS) for further cell growth (48 h). Cell viability was determined using Cell Titer 96 Cell Proliferation Assay. The absorbance (490 nm) was recorded using a Bio-RAD (Benchmark, USA). Cell viability was calculated as described by the manufacturer. In Vivo and ex Vivo Fluorescence Imaging. Four-week-old

male BAL /c nude mice received a subcutaneous injection of 5×106 in vitro propagated cancer cells into the backside. Tumors were then allowed to grow for 3–4 weeks to 1–2 cm in diameter. Before imaging, BALB/c nude mice, with or without tumors, were anesthetized with anesthetic. Once the mice were anesthetized to be motionless, a 300 µL volume of binding buffer containing 0.35 nmol of labeled probes and 4.5 nmol of unlabeled random oligonucleotide was injected intravenously via tail vein. Subsequently, time-lapse fluorescence imaging was taken by an IVIS Lumina II in vivo imaging system (Caliper Life Sicence, USA). For ex vivo organ imaging, the mice injected with different probes were killed by cervical dislocation under general anesthesia at 90 min post-injection. Excised tumor tissues or normal tissues at the similar location were imaged by the IVIS Lumina II in vivo imaging system. A 640 nm bandpass filter and a Cy5.5 longpass filter were selected to be used as the excitation filter and the emission filter, respectively.

RESULTS AND DISCUSSION As proof of concept, a pair of split fragments (Sgc8c-3a and Sgc8c-3b) deriving from an intact Sgc8c aptamer24 were used to construct a SATP. The Sgc8c was evolved by cell-SELEX against human leukemia CCRF-CEM cells,26 targeting protein tyrosine kinase-7 on cell surface.27 The split Sgc8c was recently engineered in our group, and a previous study based on fluorescence resonance energy transfer (FRET) demonstrated that free Sgc8c-3a and Sgc8c-3b could be induced by target proteins on cell surface to assemble into recognition configuration like intact Sgc8c.24 In view of the vital role of the double helix region in regulating stability and activation of the SATP, five probes (SATP-1, 2, 3, 4, and 5) with different S/Linker sequences were designed by employing Cy5-BHQ2 as the F-Q pair (Table S1). Specifically, with the stability of double helix region gradually elevated from SATP-1 to 5, the background signal was expected to decrease stepwise while the probe activation might be increasingly difficult. Fluorescence spectra tests revealed a successful assembly and disassembly of the SATP in buffer (Figure S1). By increasing the dose ratio of S to Apt-L, the background signal was greatly lowered. This suggests an advantage of the SATP over the traditional hairpin-shaped activatable aptamer probe (HAAP)18 in giving a much higher sensitivity for cancer diagnosis. Then, to find the optimized SATP sequence, flow

Figure 1. Optimization of SATP sequences. (A) Wash-free and direct analysis of CEM or Ramos cells by flow cytometry after incubation with SATP-1, 2, 3, 4, and 5 respectively. (B) The corresponding histogram of the fluorescence ratios of CEM to Ramos cells for probes in (A). (Probe concentration: 25 nM.)



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Figure 2. Signal-to-background ratio comparison of the optimized SATP-3* with other three “always on” probes. (A) Wash-free and direct analysis of CEM or Ramos cells by flow cytometry after incubation with different probes. (B) The corresponding histogram of the fluorescence ratios of CEM to Ramos cells for probes in (A). (Probe concentration: 25 nM.)

cytometry assays of target CEM cells were performed after incubation with different probes by using human lymphoma Ramos cells as control. As shown in Figure 1A, the five probes were all activated by CEM cells presenting a higher fluorescence response than Ramos cells. It was further confirmed that the signal elevation was indeed caused by cell recognition-induced disassembly of the SATP (Figure S2). Moreover, with the probe stability increased, a gradual decrease of fluorescence intensities was detected for both CEM and Ramos cells, in line with our expectations. As a result, the highest signal-to-background ratio was realized by SATP-3 (Figure 1B). Next, to improve sensitivity, BHQ3, a more efficient quencher for Cy5, was used to replace BHQ2 in SATP-3 and consequently, an optimized SATP-3* was constructed for in vitro CEM cell analysis. As displayed in Figure 2A, a comparison of the SATP-3* with other three “always on” probes, including Cy5-Sgc8c, Cy5-Apt-L-3 and Cy5-SATP-3, was carried out. The “always on” probes showed roughly equal contrasts in differentiating target and nontarget cells. This suggested that the sequence alteration from Sgc8c to SATP-3 did not harm cell recognition ability. Significantly, by using the activatable mechanism of SATP-3*, a signal-tobackground ratio as high as ~20.7 was achieved (Figure 2B). This contrast was ~4.8 times of the “always on” probes, promising a highly-selective and highly-sensitive analysis of CEM cells.

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Then, a SATP strategy-based quantitative analysis of CEM cells was performed to investigate the sensitivity of SATP-3*. The samples with various CEM cell numbers from 0 to 460,000 cells were prepared by gradient dilution. As seen from Figure 3A, along with the reduction of cell number, the SATP-3* labeled CEM cells locating in the upper right region (UR) were detected to decrease gradually. The calibration curve implied a good linear relationship in the range of 46 to 460,000 cells with a regression equation of logY =0.9221logX + 0.1047 (Figure 3B). The smallest cell number detected in our real experiments was 46 cells in 200 µL solution, which was lower than most of that obtained using the reported activatable aptamer probes.18, 22 We next investigated the specificity of the SATP strategy. Control Probe-3*, which was designed with the loop region of split Sgc8c subjected to an arbitrary change such that it had little affinity to target cells, was used as a negative control for SATP-3*. Noticeably, in free states, Control Probe-3* had a little higher fluorescence intensity than SATP-3*, perhaps due to the random sequence in Control Probe-3* influenced the hybridization of Apt-L and S (Figure S3). After optimization of the ratio of S to Apt-L and time for incubation (Figure S4), different cells were analyzed with SATP-3* and Control Probe-3*. As illustrated in Figure 4A, due to recognitioninduced shape change, SATP-3* showed much higher labeling of CEM cells than Control Probe-3*. In contrast, fluorescence responses of SATP-3* to control cells, including Ramos cells, liver cancer 7721 cells and normal liver L02 cells, were a little lower than Control Probe-3* due to the latter’s higher background. Obviously, SATP-3* was not activated by nontarget cells. Confocal microscopy images further demonstrated that SATP-3* was activated on the surface of target cells (Figure S5). The result accorded with the reports that the target of Sgc8c was membrane protein.14, 18, 27 Then, a successful application of SATP-3* to image target cells in mixed cell samples was achieved, in which CEM cells surrounded by red fluorescence were clearly distinguished from green Ramos cells (Figure 4B). Also, the high efficiency for target activation was confirmed by selectively detecting CEM cells in mouse serum with SATP-3*. Although mouse serum provided a much more complex and unfavorable surrounding for detection than binding buffer, obvious fluorescence elevation was still observed for SATP-3* treated

Figure 3. Quantitative detection of CEM cells by SATP-3*. (A) Flow cytometry assays of CEM cells with decreasing cell amounts in 200 µL binding buffer after incubation with SATP-3*. (B) Calibration curve illustrating the relationship between the amount of CEM cells counted by hemocytometer and the number of CEM cells detected with SATP-3*. (Probe concentration: 25 nM.)


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Figure 4. Highly-specific detection of CEM cells by SATP-3*. (A) Wash-free and direct analysis of different cells by flow cytometry after incubation with SATP-3* or Control Probe-3*. (B) Fluorescent confocal images of the mixture of CEM and Ramos cells after incubation with SATP-3*. Ramos cells were pre-stained by calcein-AM (green).

*

Figure 5. Drug loading capacity of SATP-3 . (A) Fluorescence spectra of Dox solutions (500 nM) added with SATP-3* at various ratios of SATP-3* to Dox. (B) Flow cytometry assays of CEM or Ramos cells after incubation with SATP-3* or SATP-3*-Dox respectively.

CEM cells, suggesting potential detection ability both in vitro and in vivo (Figure S6). The above study proved the high selectivity and sensitivity of the SATP strategy in cancer cell diagnosis. We, thereupon, investigated its function of targeted therapy by employing doxorubicin (Dox) as a model drug. As shown in Figure 5A, the fluorescence signal of Dox gradually decreased with the added SATP-3*, implying successful drug loading of SATP3*-Dox.5, 14 And a saturated Dox intercalation was observed at a molar ratio of ~0.6 (SATP-3*/Dox), which was routinely used in the following therapy study. To investigate whether intercalated Dox would influence the cell recognition of SATP-3*, the affinity of SATP-3*-Dox to CEM cells was assessed using flow cytometry. As shown in Figure 5B, equal fluorescence intensity was detected for CEM cells treated with SATP-3*-Dox and SATP-3*. It was indicated that drug loading did not affect the binding of SATP-3* to its target. We next evaluated the selective drug delivery of SATP-3*Dox into target cells through confocal microscopy imaging. As indicated in Figure 6A, strong fluorescence emitted from Dox was observed only in positive CEM cells treated with SATP-3*-Dox, but not in negative Ramos cells. In addition, by analyzing the fluorescence co-localization of Dox and Hoechst 33342, it was deduced that Dox could be effectively transported to the nucleus. In contrast, free Dox suffered nonselective permeation to both target CEM cells and

nontarget Ramos cells (Figure S7). These data disclosed a highly-selective drug delivery of SATP-3*-Dox to CEM cells. It was speculated that only target cells could initiate the structural reassembly of SATP-3*-Dox and break the double helix region, thus releasing Dox. A quantitative cell toxicity test further demonstrated that the SATP-3*-Dox only killed CEM cells, which greatly decreased the nonspecific cytotoxicity of Dox (Figure 6B). In particular, SATP-3* without Dox loading displayed negligible cytoxicity to both cells, suggesting good biocompatibility of DNA vehicles. The target-activatable drug release function and excellent biocompatibility property paved a way for clinical translation with precise therapy of cancer. Finally, an evaluation of the SATP strategy for activatable in vivo cancer imaging was conducted. In view of the impact of complex biofluids and physiological temperature in real living systems, SATP-5*, a more stable probe consisting of Cy5-Apt-L-5 and BHQ3-S-5, was used in case of unexpected disassembly. A series of in vitro experiments revealed that SATP-5* had similar properties to SATP-3*, including effective drug loading, specific cell binding and selective toxicity to target cells (Figure S8, S9). Has to be mentioned here is an advantage of SATP over the classical HAAP. The stability of SATP could be adjusted for various applications, just by altering the double helix region and with no need to consider aptamer sequences. And a comparison study also indicated that SATP-5* held a better fluorescent stability in serum than HAAP, promising a better performance in vivo (Figure S10). Then, the SATP-5* was intravenously injected into CEM tumor-bearing mice for time-lapse fluorescent imaging. To investigate the specificity, Control Probe-5* and Ramos tumor-bearing mice were used as negative controls. As displayed in Figure 7A, at 5 min post-injection, the CEM tumor site was clearly lighted up by the SATP-5*, as a result of rapid blood circulation and cell recognition-triggered in situ structural and signal activation. With the time extended, the fluorescent signal of CEM tumor first increased and then decreased. The tumor was observed to be brightest at 15 min, which further supported the work principle of SATP. In contrast, no obvious signal activation in tumor sites or similar positions of normal mice was detected for all other control



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Figure 6. Selective cytotoxicity of Dox delivered by the SATP strategy. (A) Fluorescent confocal images displaying a selective drug transportation of SATP-3*-Dox (Dox: 2 µM). (B) MTS assay results of the viability of CEM or Ramos cells after different treatments.

Figure 7. In vivo specific fluorescence imaging of CEM tumor with SATP-5*. (A) Time-lapse in vivo fluorescence imaging of nude mice bearing different tumors after intravenous injection with different probes. (Pink circles locate the tumor sites.) (B) Images of the isolated tumor tissues at 90 min post-injection. From top to bottom were CEM tumor with SATP-5*, normal tissue with SATP-5*, Ramos tumor with SATP-5* and CEM tumor with Control Probe-5*, respectively.

groups, irrespective of the disturbance of probe metabolism organs. Fluorescent images of isolated tumor tissues acquired at 90 min post-injection further verified the specific binding of SATP-5* to CEM cells in vivo (Figure 7B). Although the fluorescence from tumor sites was generally higher than normal muscles due to enhanced permeability and retention effect, the signal of CEM tumor injected with SATP-5* was much higher than other two control tumor groups. Unlike the activatable strategy, “always on” probes had to bear extremely high fluorescence background through the whole body at early stage, and presented poor image contrast until unbound probes were eliminated from nontarget tissues.18, 21 These results surely demonstrated that the SATP could successfully achieve fast, high-contrast and selective in vivo cancer imaging.

release. The design also solved the thermal inactivating problem of split aptamers. And its tunable stability facilitated in vivo application, in which rapid diagnosis with contrastenhanced image was achieved in 5 min. In addition, the strategy is expected to be available for a variety of drugs. For example, the CG-rich double helix region can be used to load other anthracycline drugs like epirubicin and daunorubicin.5, 14 Besides, the double helix region may be changed to poly A pairs for coralyne loading, or siRNA-antisense siRNA hybrids to silence gene expression.28, 29 As a promising and versatile platform, the SATP can thus be widely explored for rapid and precise cancer theranostics.

CONCLUSION

Supporting Information

In summary, a biocompatible activatable theranostic probe was first developed based on recognition-induced reassembly of split aptamers. Inspired by nature, the SATP design subtly combined “sensitive sense” and “stimuli resist”, providing a simple and effective DNA platform acting as “nanodoctor” for activatable in vivo cancer imaging and in situ drug release. By using split Sgc8c against CEM cells as the model, the SATP showed efficacious target-activated fluorescence activation. And high selectivity and sensitivity for CEM cell analysis was indicated with a detection limit of 46 cells. Using Dox as the model drug, the SATP realized highly-specific drug delivery and CEM cell killing as a result of target-triggered in situ

ASSOCIATED CONTENT Additional table and figures. The Supporting Information is available free of charge on the ACS Publications website.

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

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS


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This work was supported by the National Natural Science Foundation of China (Grants 21190044, 21322509, 21305038, 21575037, 21521063 and 21675046), and the Hunan Provincial Natural Science Foundation (Grant 2015JJ3044).

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