Biostable Aptamer Rings Conjugated for Targeting Two Biomarkers on

Article Cite This: Chem. Mater. 2017, 29, 10312−10325

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Biostable Aptamer Rings Conjugated for Targeting Two Biomarkers on Circulating Tumor Cells in Vivo with Great Precision Haiyan Dong,†,‡,§ Longyu Han,† Zai-Sheng Wu,† Ting Zhang,† Jingjing Xie,∥ Ji Ma,† Jie Wang,† Tao Li,† Yu Gao,† Jingwei Shao,† Patrick J. Sinko,⊥ and Lee Jia*,† †

Cancer Metastasis Alert and Prevention Centre and Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou, Fujian 350002, China ‡ Fujian Key Laboratory for Translational Research in Cancer and Neurodegenerative Diseases Institute for Translational Medicine, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian 350108, China § Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, Fuzhou, Fujian 350108, China ∥ School of Pharmaceutical Sciences and Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiang’an South Road, Xiamen, Fujian 361102, China ⊥ Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey 08854-8020, United States S Supporting Information *

ABSTRACT: Cancer metastatic spread is life-threatening and caused by circulating tumor cells (CTCs) that are very difficult to precisely capture in vivo. Here we show that two aptamer rings targeting different CTC biomarker epitopes conjugated on dendrimers capture CTCs with enhanced precision even in the presence of 108 interfering cells, or blood cells, and in mice or patient samples when compared with their single aptamer counterparts. The aptamer-conjugate inhibited in vivo metastasis and demonstrated enhanced biostability by resisting biodegradation caused by the endogenous nucleases. The capture arms of the aptamer conjugates could simultaneously and specifically seize two biomarkers (EpCAM and Her2). The double seizure resulted in significant cell-cycle arrest, apoptosis, and growth inhibition of the captured CTCs. The aptamer-conjugate highly penetrated and accumulated in mouse tumors. This study provides the first conceptual evidence that two aptamer rings, inexpensive but bioequivalent to their antibodies, can be biocompatibly conjugated to specifically capture and down-regulate CTCs in vivo with the enhanced biostability. chemoprevention.3−5 Characterization of the patient-derived CTCs indicated that CTCs usually possess different types of surface biomarkers.6−9 Therefore, we hypothesized that simultaneously targeting more than one surface protein on a CTC may greatly enhance the precision and potency of seizing and restraining the CTCs in vivo, resulting in their apoptosis and eventual prevention of metastasis. Current CTCs capture applications focus on in vitro detection, characterization for

1. INTRODUCTION Metastatic spread is a complex life-threatening process caused by the rare circulating tumor cells (CTCs).1 Current pharmaceuticals still cannot specifically eradicate them. The CTCs-based cancer metastatic organ tropism inspired us to create a quadruple-combined drug for comprehensively controlling the CTCs-based metastasis with a success in animal models without adverse effects.2 Our analysis of the molecular and cellular similarities and differences between embryonic implantation to uterine endometrium and CTCs adhesion to vascular endothelium motivated us to test abortifacients and abortion botanical medicine extracts for safe premetastatic © 2017 American Chemical Society

Received: July 20, 2017 Revised: November 23, 2017 Published: December 7, 2017 10312

DOI: 10.1021/acs.chemmater.7b03044 Chem. Mater. 2017, 29, 10312−10325

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Chemistry of Materials

Figure 1. De novo design and synthesis of ds aptamer rings (cCAP1 and cCAP2) and their functionality and biostability. (a) Complementary DNA (L1) hybridized its template L1-T, while biotinated AP1 (AP1-bio) hybridized template 1. The hybridization products were modified by T4 DNA polymerase to configure single circular DNAs, namely, C1 and CAP1. The latter two hybridized each other to form the ds circular CAP1 (cCAP1), which is composed of the unique capture arm (Region 1 containing SYL3C for targeting EpCAM) and the circular body (Region 2 resistant to T4 DNA polymerase). (b−e) The linear AP1 and AP2 for binding to CTC surface biomarkers EpCAM and Her2, respectively. (b) FAM-containing AP1 binds to BT474 and MDA-MB-231 (both EpCAM+) but not A549 (EpCAM‑); (c) CY5-containing AP2 binds to BT474 and A549 (both Her2+) but not MDA-MB-231 (Her2‑). The sham probes (P1−P6) did not bind to EpCAM+ or Her2+ cell lines. (d and e), Quantitative fluorescence ratios of BT474 and MDA-MB-231 or BT474 and A549 to nontarget HELF cells for binding to AP1 (d) or AP2 (e) and sham probes. The results demonstrated that only BT474 binds to both AP1 and AP2. (f) Structure of cCAP2 that is composed of the capture arm (Region 1 10313


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Chemistry of Materials Figure 1. continued

containing HB5 for targeting Her2) and the circular body resistant to T4 DNA polymerase. (g) Urea-PAGE gel analysis showed stains of AP1, CAP1, L1, C1, and cCAP1 that was obtained from hybridization of CAP1 with C1 (left panel); the right panel showed the stains of cCAP2 components. (h) Urea-PAGE gel analysis for biostability comparison between AP1 and cCAP1 after their incubation with T4 DNA polymerase (5 μM) for different times. Note, AP1 was degraded fast. (i, j) Flow cytometric analysis showed that cCAP1 bound to BT474 and MDA-MB-231 cells (i) and cCAP2 bound to BT474 and A549 cells (j) more than their counterparts did after overnight incubation. (k, l) Comparison in binding specificity and potency between cCAP1 and EpCAM-antibody (k) or between cCAP2 and Her2-antibody (l); cCAP1 and cCAP2 showed their binding trend similar to their antibody counterparts. Briefly, aptamers were incubated with cells in the cell culture at 37 °C overnight in the dark and determined by a FACScan cytometer (BD Biosciences). 2.5. Synthesis of Single Stand (ss) or Double Strand (ds) Aptamer Rings. The methods were similar to what we described before.23 First, the linear aptamers were phosphorylated with ATP and PNK according to the manufacturer’s recommendations. Second, the ligation of phosphorylated linear aptamers was performed by hybridization with the corresponding templates. They were incubated with T4 DNA ligase followed by ethanol precipitation. Third, the templates and unreacted linear aptamers were digested by T4 DNA polymerase. After purification by PAGE gel, the ss aptamer rings (CAP1 or CAP2) were dissolved in water and quantified by mass spectroscopy (Figure S2, Supporting Information). The ds aptamer rings were the hybridization products of the ss aptamer rings and their corresponding circular DNAs (C1 or C2). The synthetic procedures for constructing the cCAP1 and cCAP2 were illustrated in Figure 1a and Figure S3a (Supporting Information). Briefly, the AP1-biotin or AP2-biotin was hybridized with its template T1 or to T2-1 and T2 to form the CAP1 or CAP2; the latter was partially hybridized with C1 or C2 to become cCAP1 or cCAP2. 2.6. Verification of ss or ds Aptamer Rings. To determine if the linear aptamers became the circular ss or ds ones, PAGE gel electrophoresis was performed using the protocol we described previously.23,30 2.7. Biostability of Aptamer Rings. The methods were similar to what we described previously,18,23,30 and in vivo tumor and organ images were conducted following the previous study.31 First, the circular or linear aptamers were treated with T4 DNA polymerase for 0, 10, 20, 40, 60, 90, or 120 min. After exonuclease digestion, PAGE gel analysis, mass spectroscopy, flow cytometry experiments, and in vitro cell imaging, as well as in vivo tumor and organ imaging, were performed to evaluate biostability of ss circular, ds circular, or linear aptamers. 2.8. Synthesis of Single or Dual DS Aptamer Ring Conjugates. To improve synthesis productivity, the classical streptavidin−biotin interaction was used for linking G4.5 with aptamers. Streptavidin-modified G4.5 (G4.5-Stre) was synthesized by the covalent conjugation of the amino group of streptavidin (Stre) onto G4.5-COOH using cross-linking reagents EDC and NHS. Biotinmodified aptamers (bio-APs) and biotin-modified inserting fragment (bio-In) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). The ds circular bio-cCAPs were synthesized according to the above-mentioned procedures (see Figure 1a and Figure S3a, Supporting Information). Then, the ds bio-cCAPs were mixed with G4.5-Stre followed by stirring for 4 h. Finally, the products were purified by dialysis. The synthesized ds aptamer ring conjugates were named as ds aptamer ring1 (anti-EpCAM) conjugated with G4.5 (G4.5-cCAP1), ds aptamer ring2 (anti-Her2) conjugated with G4.5 (G4.5-cCAP2), and dual ds aptamer rings-coated G4.5 (cCAP1-G4.5cCAP2). 2.9. Characterization of Aptamer Ring Conjugates. The PAGE gel analysis was applied to confirm the conjugation of ds aptamer rings with G4.5 surface. FTIR spectra were used for changes in active groups (Nicolet 360 Fourier Transform IR spectrometer, Nicolet Instruments, Inc.). The mean particle size and zeta potential of aptamer ring conjugates were determined by using Zetasizer Nano ZS Particle Size and Zeta Potential Analyzer (Malvern Instruments Ltd., U.K.). The surface morphology and particle size were determined by

diagnostic purposes,10−12 and/or culturing.13−16 Nonetheless, we developed the dual-antibody conjugates to capture the rare CTCs in patients and nude mice with significantly enhanced specificity.17−20 Unfortunately, the antibody-based biotechnology suffers bioinstability, immunogenicity, high cost, and poor tissue penetration. Linear aptamers, which are considered to be “chemical antibodies”, have surfaced as an important tool for targeting cell surface biomarkers since the invention of SELEX (systematic evolution of ligands by exponential enrichment).21 In contrast to antibodies, linear aptamers possess advantages such as nanosize, structural flexibility, sequence programmability, thermal stability, and nonimmunogenicity.22 Their characteristics attracted us to develop single-stranded (ss) DNA catenanes via the formation of a linking duplex.23 These two interlocked DNA rings are capable of operating as an independent unit. In view of the bioinstability of most DNA, RNA, and peptide aptamers degraded by nucleases and proteases,22,24 the established technique motivated us to further develop circularized double-strand (ds) DNA aptamers that show greater biostability than the linear ones. We then engineered such two ds aptamer rings with different biomarker-targeting units, one containing SYL3C (selected by SELEX targeting EpCAM) and another HB5 (targeting Her2), and conjugated such functionalized aptamer rings onto the same PAMAM dendrimers as a single molecular entity to identify more fingerprints of the CTC phenotype. Here, we report the design, synthesis, and characterization of the novel dual ds aptamer ring conjugate, which can simultaneously target EpCAM and Her2 epitopes on CTCs in the presence of millions of interfering normal cells that do not express EpCAM or Her2, blood cells, and patients and mice with greatly enhanced biostability and capture precision.

2. EXPERIMENTAL SECTION More detailed information can be found in the Supporting Information. 2.1. Sequence Design of Linear DNA Aptamers, Templates, and Probes. All sequences were designed with the mFold program (http://mfold.rna.albany.edu/) to predict and optimize their secondary structures. All DNA sequences used in this work are listed in Table S1, and structures of Aptamer 1 (AP1) and Aptamer 2 (AP2) are shown in Figure S1 (Supporting Information). The control ss probes 1, 2, and 3 were designed for targeting PTK7 protein,25 LOVO cells,26 and MUC1 protein,27 respectively. They were all labeled with fluorophore FAM. The control ss probes 4, 5, and 6 were designed as the same as the probes 1, 2, and 3, except that they were labeled with fluorophore CY5 (Table S1, Supporting Information). 2.2. Cell Culture. The cell lines and the culture methods were similar to what we described before.2,28 2.3. Cell Surface Biomarkers Analysis. Flow cytometry assay was conducted to determine expression of EpCAM, and Her2 on cell surface following the methods we described previously.18 2.4. Analysis of Recognition and Binding between Cells and Aptamers. Flow cytometry assay was performed as reported.29 10314


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Figure 2. Synthesis and physicochemical characterization of the ds aptamer ring conjugates. (a) FTIR spectra. (b) Synthesis of cCAP1-G4.5-cCAP2: synthesis procedures of cCAP1-bio (panel a1), cCAP2-bio (panel a2), and G4.5-streptavidin include ①, base-paring hybridization of AP1-bio/T1, L1/L1-T, AP2-bio/T2/T2-1, or L2/L2-T; ②, the 5′-to-3′ end ligation of CAP1-bio and CAP2-bio by T4 DNA ligase; and hybridization between cCAP1-bio and C1, or cCAP2-biotin and C2 to form cCAP1-bio and cCAP2-bio, respectively. The latter two react with G4.5-streptavidin to form single ds aptamer ring-conjugated G4.5 (G4.5-cCAP1 and G4.5-cCAP2) or their dual counterpart cCAP1-G4.5-cCAP2. (c) Size and zeta potential of the aptamer ring conjugates. (d) Urea-PAGE gel electrophoresis verified the conjugation of each cCAP to G4.5; M, DL200 DNA marker; 1, cCAP1; 2, cCAP2; 3, cCAP1-biotin-streptavidin; 4, cCAP2-biotin-streptavidin; 5, cCAP1-biotin-streptavidin-cCAP2; 6, G4.5-cCAP1; 7, G4.5-cCAP2; 8, 10315


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Chemistry of Materials Figure 2. continued

cCAP1-G4.5-cCAP2. (e) AFM image; (f) SEM image; and (g) fluorescent image (confocal microscope) of cCAP1-G4.5-cCAP2. (h) UV and (i) fluorescent spectra of the conjugates (note: cCAP1-G4.5-cCAP2 showed the peaks at both 518 nm (for cCAP1) and 670 nm (for cCAP2)). (j) The linear standard curves of AP1 and AP2 for quantitation. 2.14. Analyses of Cell Viability, Cell Cycle, and Apoptosis after Capture. The analyses were conducted as we described previously.2 After the cells were exposed to the conjugates at various concentrations for 24 or 48 h, the cell viability, cell cycle, or apoptosis was evaluated by MTT assay, flow cytometry, DAPI staining, or AO/ EB staining. The details were described in the Supporting Information. 2.15. In Vivo Capturing CTCs. The method was similar to what we described previously.20 Briefly, BT474, MDA-MB-231, or A549 cells were stained with Hoechst 33258 for 30 min, and then the stained cancer cells 2 × 104 were intravenously injected into the nude mice followed by administration of the conjugates 5 min later. Thirty minutes later, the mouse blood was withdrawn and the mixture of cells and WBCs was collected via centrifugation after separation of BT474 from RBCs lysis.6 The cells were examined directly by flow cytometry or observed under the confocal microscope to demonstrate the presence of cell nucleus. All the animal procedures complied with the guidelines of the Institutional Animal Care. 2.16. In Vivo Tumor Metastasis Assay. The method was similar to what we described previously.32 2.17. In Vivo Monitoring CTCs. To dynamically monitor distribution of the injected conjugates over mouse body, we replaced the FAM with CY5 because of FAM’s weak tissue penetration. We used the SI Imaging Ami X equipment to observe the real-time dynamic distribution of the conjugates (Figure S4, Supporting Information). We injected cancer cells (5 × 104 in 200 μL PBS) into the nude mice. After 5 h, the conjugates were injected to the mice separately, and the fluorescence intensities of mouse body and organs were measured by the imaging equipment. 2.18. In Vivo Fluorescence Imaging. BALB/c nude mice bearing BT474 breast tumor xenografts were obtained by subcutaneous (s.c.) injection of 1 × 107 BT474 cells into the site near the breast of the mice. After the tumor reached 1−2 cm, the conjugates were injected intravenously into the mice via the tail vein. At specified times, the fluorescence images of mouse body or organs were taken by a SI Imaging Ami X. Control mice were injected with the PBS buffer. 2.19. Capturing CTCs in Patient Blood. The method was similar to what we described previously.20 The study was reviewed and approved by the hospital institutional review board (IRB). Briefly, blood samples were drawn from 10 patients and collected into tubes with the anticoagulant EDTA following separation as we described previously.6 Then, the conjugates were incubated with the blood samples pretreated with 1% BSA overnight. The mixture of captured CTCs and WBCs were obtained after separation of RBCs using the above-mentioned method. Finally, the cells were examined directly by flow cytometry or observed under confocal microscope after staining with DAPI. 2.20. Statistical Analysis. Data were presented as means ± standard deviations of three determinations (n = 3). Statistical analysis was done by Student’s t-test and one-way analysis of variance by using SPSS statistical software (version 19.0). The symbols *, **, and *** represented the comparison between samples within the same group but treated at different levels; NS, #, ##, and ### represented the comparison between different groups; and NS, *, or #, ** or ##, and *** or ### indicate no significant difference, P < 0.05, P < 0.01, and P < 0.001, respectively.

SEM (Nova Nano SEM 230) and AFM (Agilent AFM 500, tapping probe 40 N m−1, in AC mode) images. The fluorescence images were obtained by using a laser confocal microscope to make sure the FAM or CY5 labeled ds aptamer rings conjugated to the G4.5 surface (Olympus Fluo View 1000). The SEM instrument was operated at an accelerating voltage of 0.5 kV. The conjugates were diluted with dd H2O and dropped to the copper grid followed by drying for 20 min. The copper grid was then fixed to the conductive resin on the SEM sample column followed by morphological analysis using the resin as the background. UV absorption value (λ = 260 nm) and fluorescence intensity were measured using UV spectrophotometer (Shimadzu, Japan) and a Cary Eclipse fluorescence spectrometer (Varian Ltd., U.S.A.). AP1 and AP2 (10 μM) were employed to determine the amount of cCAP conjugation by using the calibration curve of AP1 or AP2 standard solutions through fluorescent measurement of FAM (λex = 492 nm and λem = 518 nm) and CY5 (λex = 645 nm and λem = 670 nm) with the fluorescence spectrometer. We explained the IR spectra as follows: the 3500−3000 cm−1 represented the stretching vibration of the N−H bond of the amide group in G4.5-cCAP1, G4.5-cCAP2, and cCAP1-G4.5-cCAP2 when the O−H bond in the carboxyl group of G4.5 was changed to the N− H after G4.5 amidation (Figure 2a, peak 1). The peaks within the 1800−1700 cm−1 characterized the stretching vibration of the CO bond of the carboxyl groups (Figure 2a, peak 2). The intensity of the characteristic peaks in the 1700−1400 cm−1 range derived from the stretching vibration of glycosidic bond and ring vibrations of cytosine of DNA was significantly increased after G4.5 was conjugated with cCAP1 or cCAP2, separately, or with both (Figure 2a, peaks 3, 4). Moreover, the fingerprint region (1500−500 cm−1) in the spectra of G4.5-cCAP1, G4.5-cCAP2, and cCAP1-G4.5-cCAP2 were different from the spectra of G4.5, suggesting the differences in chemical entity between G4.5 and its aptamer ring conjugates. 2.10. Stability of Aptamer Ring Conjugates. The stability of aptamer ring conjugates in water at different pHs was measured by the above-mentioned UV spectral analysis. The stability of aptamer ring conjugates in blood was evaluated by the above-mentioned fluorescence spectral analysis and PAGE analysis. Human serum was prepared from blood samples of healthy volunteers. G4.5-cCAP1, G4.5-cCAP2, and cCAP1-G4.5-cCAP2 were incubated with human serum at 37 °C for various times followed by the fluorescence measurement using the Cary Eclipse fluorescence spectrometer, and % aptamer ring conjugates remaining was calculated by using the equation (see Supporting Information). 2.11. Confocal Microscope Analysis of Specific Recognition between Target Cells and Aptamer Ring Conjugates. The method was similar to what we described in our previous study of antibody.18 2.12. Flow Cytometric Analysis of Specific Recognition between Target Cells and Aptamer Ring Conjugates. The method was similar to what we described in our previous study with antibody.20 Briefly, aptamer ring conjugates were incubated with the cells in the binding buffer at 37 °C overnight. The binding between the cells and the conjugates was determined by using the flow cytometry. The capture efficiency (% positive cells) of aptamer ring conjugates was defined as the number of captured FAM+CY5+ BT474 cells, FAM +CY5- MDA-MB-231 cells, or FAM-CY5+ A549 cells in Q2, Q3, or Q1 plot quadrant divided by the number of the cells shown in the P1 gate. 2.13. Capture Specificity in the Presence of Interfering Cells, RBCs, or Healthy Blood. The method was similar to what we reported previously.20 BT474, MDA-MB-231, or A549 cells were spiked into HELF cells or RBCs at mixture ratios of 1:103, 1:105, 1:107, and 1:108, or 2 and 4 mL healthy blood, and incubated overnight.

3. RESULTS AND DISCUSSION 3.1. Sequence Design of Linear Aptamers and Their Specific Binding to Cell Surface Biomarkers. We chose the BT474 cell line because it expresses high levels of EpCAM and Her2 as well as drug resistance protein.33,34 We first used the fluorophore PE-linked antibody EpCAM and Her2 to 10316


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secondary or tertiary structures to recognize surface biomarkers. Figure 1g compared the differences in PAGE gel electrophoresis between cCAP1 (or cCAP2) and its components, confirming the successful assembly of cCAP1/cCAP2. It is noteworthy that CAP1 and cCAP1 or CAP2 and cCAP2 showed the same retentions on the same gel, indicating that all aptamer rings run at the same rate, but slower than the linear one because of their spherical morphology, size and charges.23 To examine the biostability and functionality of cCAP1, cCAP1 was incubated with 5 μM of T4 DNA polymerase in PBS at 37 °C for as long as 120 min, and then the incubation medium was loaded to (1) the PAGE gel for degradation analysis; (2) the test cell lines were examined for binding functionality analysis. Figure 1h showed that the circular cCAP1 was much more stable than the linear AP1. Similarly, the results showed that cCAP2 was more biostable than AP2 (Figure S7a, Supporting Information). Flow cytometry analysis showed that the binding functionality of cCAP1 (Figure 1i) or cCAP2 (Figure 1j) remained unchanged after their overnight incubation with BT474 and MDA-MB-231 or BT474 and A549. Moreover, cCAP1 and cCAP2 seemed to be more potent than CAP1 and CAP2 in binding to their corresponding cells probably because the formers possess the capture arm (Figure 1i,j). In short, the aptamer rings appeared to be much more biostable than their linear counterparts (AP1 and AP2) that degraded fast and lost their functionality as shown in the gel assay (Figure 1g,h; Figure S7a, Supporting Information), mass spectroscopy analysis (Figure S7b, Supporting Information), flow cytometry functionality assay (Figure S7c,d, Supporting Information), confocal microscopic analysis (Figure S8a, Supporting Information), in vivo stability and binding assay (Figure S8b, Supporting Information), and tissue distribution assay (Figure S8c, Supporting Information). Moreover, cCAP1 and cCAP2 possess more target functionality than their counterparts CAP1 and CAP2 as shown in the in vitro binding assays (Figure 1i,j) because the formers have the capture arm. The binding affinity between the aptamers and antibodies for the same biomarkers, i.e., EpCAM and Her2, were also compared. The results showed that the antibodies and their corresponding aptamers (EpCAM-Ab vs cCAP1; Her2-Ab vs cCAP2; Figure 1k,l) demonstrated the similar trends in affinity toward their biomarkers although the binding potency of the antibodies differs from that of the aptamers. Additionally, the market price of 10 μg of EpCAM-Ab is about 8-fold higher than 10 μg of SYL3C aptamer ring ($123 versus $15), which is in line with the literature.22 3.3. Characterization, Biostability and Specificity of ds Aptamer Ring Conjugates. As described above and in the Experimental Section and shown in Figure 1a and Figure S3a (Supporting Information), we hybridized AP1-bio with template T1 and L1 with LT1 based on base paring rules to produce cCAP1-bio (Figure 2b-a1). We used the same protocol to produce cCAP2-bio (Figure 2b-a2). We then chose carboxyl PAMAM dendrimer G4.5 as the carrier for both cCAP1-bio and cCAP2-bio aptamers. The dendrimers showed suitable biocompatibility, charge, and large number of surface functional groups, which make them easier for conjugation with the two cCAPs.4,20 For high conjugation reaction, we functionalized G4.5 with streptavidin in the presence of the cross-linking reagents EDC (1-ethyl-3-(3-dimethly aminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide) (Figure 2b-a3). The G4.5-streptavidin reacted with cCAP1-bio and cCAP2-bio aptamers to form

quantitatively examine the abundances of EpCAM, Her2, and other biomarkers on our test cell lines. We found that a single biomarker (e.g., EpCAM) was expressed on different cell lines with different abundances (Figure S5, Supporting Information). Furthermore, a single cell could express various surface biomarkers with different abundances (Figure S5, Supporting Information). For example, under the same culture conditions, BT474 expressed high levels of both EpCAM and Her2 (EpCAM+/Her2+). Whereas, MDA-MB-231 expressed only lower EpCAM (EpCAM+/Her2‑), A549 expressed only lower Her2 (EpCAM-/Her2+), and normal HELF expressed neither EpCAM nor Her2 (EpCAM-/Her2‑) compared to BT474. We designed two linear aptamers AP1 and AP2 as ss oligonucleotides of 79 bases and 89 bases, respectively (Table S1 and Figure S1, Supporting Information). The AP1 is composed of SYL3C, a sequence for constructing a ring (Region 2) that could significantly enhance biostability of the entire aptamer, and the inserted green fluorophore FAM. The AP2 is composed of HB5, a sequence for constructing Region 2, and the inserted red fluorophore CY5. The FAM and CY5 provided a controlled setting for visualizing the binding of the two aptamers to EpCAM and Her2, respectively. To assess the specific binding between AP1 and EpCAM or between AP2 and Her2, we incubated BT474, MDA-MB-231, A549, and HELF cell lines with AP1, AP2, and their control ss probes P1−P6 (see Experimental Section), separately, and measured the corresponding fluorescence intensities resulted from the specific binding between cell surface biomarkers and these probes after washing out the free unbound probes. Figure 1b,c showed that the fluorescence intensities resulted from binding between BT474 and AP1 or BT474 and AP2 were significantly higher than those from the control probes P1−P6. MDA-MB-231 bound only to AP1, and A549 bound only to AP2; HELF did not bind to either AP1 or AP2 (Figure S6, Supporting Information). For a quantitative comparison between the target and nontarget cell lines in their binding to various probes, the fluorescence intensity ratios of BT474, MDA-MB-231, and A549 to HELF cells were calculated after their binding to different probes. Figure 1d,e showed that the ratios of FBT474/FHELF and FMDA‑MB‑231/FHELF for binding to AP1 were about 6- and 4-fold higher than to the control probes, respectively, and that the ratios of FBT474/FHELF and FA549/ FHELF for binding to AP2 were respectively about 350- and 150fold higher than to the control probes, indicating the high binding specificity between AP1 and EpCAM or between AP2 and Her2. 3.2. Preparation of Biostable ds Aptamer Rings (cCAP1 and cCAP2) with High Binding Specificity. As shown in Figure 1a, the complementary DNA (L1) was hybridized with its template L1-T. Meanwhile, biotin was linked to AP1 (AP1-bio), and AP1-bio was hybridized with template 1 (T1). After phosphorylation at the 5′-end, ligase was added to catalyze the closure reaction between the 3′- and the 5′-ends of the ss to form C1 and CAP1.35 The latter two hybridized each other to form the ds circular CAP1 (cCAP1), which possesses a unique capture arm (Region 1 that contains AP1 for targeting EpCAM; Figure S3b, Supporting Information) and a circular body (Region 2 as the steric hindrances to degradation; Figure S3b, Supporting Information). Figure 1f showed the structure of cCAP2, which is similar to that of cCAP1 except for the capture arm (Region 1; Figure S3c, Supporting Information) containing HB5 that targets biomarker Her2. The capture arm can change into unique 10317


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Figure 3. Enhanced targeting specificity and super biostability of cCAP1-G4.5-cCAP2. (a) Confocal imaging of the enhanced binding of cCAP1G4.5-cCAP2 to target BT474; only cCAP1-G4.5-cCAP2 showed the full red-green merged image for its binding to BT474 that possesses both EpCAM and Her2, while G4.5-cCAP1 and G4.5-cCAP2 showed either green or red image for binding to BT474 and MDA-MB-231 or BT474 and A549, respectively. cCAP1-G4.5-cCAP2 did not bind HELF. (b) Quantitative analysis by flow cytometry of the enhanced capture specificity of cCAP1-G4.5-cCAP2 to BT474 in comparison with MDA-MB-231 and A549 bound to G4.5-cCAP1 (lower panel) or G4.5-cCAP2 (middle panel). The % shown in Q2, Q1, and Q3 plots represents the percent cells captured by cCAP1-G4.5-cCAP2, G4.5-cCAP2, and G4.5-cCAP1, excluding those free unbound conjugates. (c, d) Super biostability examined by UV and fluorescence of cCAP1-G4.5-cCAP2 at different pHs (c) and in human serum (d) at 37 °C for 6 days. 10318


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Figure 4. Enhanced specificity of cCAP1-G4.5-cCAP2 in capturing cancer cells and affecting cell viability. (a, b) Significantly enhanced specificity of cCAP1-G4.5-cCAP2 in capturing cancer cells in comparison with its counterparts analyzed by flow cytometry. The cancer cells were spiked into HELF at the ratios of 1:107 and 1:108 (a) or blood (b). (c, d) Concentration-dependent inhibition (c) and apoptotic effect (d) of cCAP1-G4.5cCAP2 on various cell lines in comparison with its counterparts using the G4.5 as a negative control. (e) Concentration-dependent induction by cCAP1-G4.5-cCAP2 of target BT474 cells into S and G2/M cycle phases; (f) AO/EB staining of BT474 after incubation with cCAP1-G4.5-cCAP2 and its counterparts for 24 h. cCAP1-G4.5-cCAP2 caused BT474 to have more apoptosis (yellow dots; for comparison with other cells and aptamer conjugates). (g) Anexin V-FITC/PI apoptotic analysis showed significant early and later apoptosis of BT474 cells caused by cCAP1-G4.5-cCAP2 in comparison with its counterparts.

single ds aptamer ring-conjugated G4.5, i.e., G4.5-cCAP1 (targeting EpCAM), G4.5-cCAP2 (targeting Her2), or dual ds aptamer rings conjugated with G4.5 (targeting both EpCAM and Her2). The diameters of G4.5-cCAP1, G4.5-cCAP2, and cCAP1G4.5-cCAP2 were approximately 100, 120, and 110 nm measured by dynamic light scattering (Figure 2c-1), indicating that the conjugation of cCAP1 or cCAP2 with G4.5 did not

significantly change the diameter and size of G4.5, probably because the nanosize aptamers could imbed into the space between the branched dendrimers. However, the conjugation completely changed the zeta potential of G4.5 from the positive to negative charges owing to the contribution from the negative charges of AP1 and AP2 (Figure 2c-2). Fourier transform infrared (FTIR) spectra showed characteristic peaks of each nanomaterial (Figure 2a), indicating the successful synthesis of 10319


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bind to HELF (Figure 3a, the far right panel, and Figure S11a,b,Supporting Information). Figure 3c,d exhibited good biostability of cCAP1-G4.5cCAP2 at different pH buffers (Figure 3c; Figure S12a,b,Supporting Information), and in serum (Figure 3d; Figure S12c,d,Supporting Information) at 37 °C monitored by using UV or fluorescence for up to 6 days. The conjugates seemed to be very stable at pH 7 and in serum that contains nucleases and lacks stabilizing salts such as magnesium. The PAGE gel electrophoresis provided additional evidence to demonstrate the stability of conjugates (Figure S12e,f,Supporting Information): no degraded cCAP bands showed on the gel from the samples incubated at 37 °C in blood with 9 h of shaking. 3.5. Enhanced Specificity of cCAP1-G4.5-cCAP2 in Seizing Cancer Cells. We evaluated the specificity of these conjugates in capturing the target cancer cells spiked into healthy blood (2 or 4 mL), HELF cells at mixture ratios of 1:103, 1:105, 1:107, and 1:108, or RBCs at mixture ratios of 1:103 and 1:105 by using flow cytometry.20 The same batch BT474 captured by cCAP1-G4.5-cCAP2 in the Q2 region was statistically significantly more than that by G4.5-cCAP1 and G4.5-cCAP2. Moreover, the same cCAP1G4.5-cCAP2 captured BT474 two times more than MDA-MB231 and A549 cells (Figures S13−15,Supporting Information) in the presence of the interfering HELF, blood cells, or RBCs (Figure 4a,b). The results indicated that cCAP1-G4.5-cCAP2, by simultaneously seizing two types of cell surface biomarkers, captured 2 times more targeted cells in comparison with its single ring counterparts. Figure 4c further demonstrated that the tightly seized cells BT474 lost their viability significantly more than other cell lines, and the effect was dependent on cCAP1-G4.5-cCAP2 concentrations. Other conjugates suppressed cell viability lesser than cCAP1-G4.5-cCAP2. PAMAM G4.5 affected viability of the four cell lines nonspecifically and only at 25 μg mL−1. DAPI staining that reveals nuclear condensation related to apoptosis showed that cCAP1-G4.5cCAP2 drove BT474 into DAPI positive status significantly more than other cell lines probably because of the dual capture effect. G4.5-cCAP1 and G4.5-cCAP2 specifically made their corresponding target cells MDA-MB-231 and A549 DAPIpositive more than other cell lines (Figure 4d; also Figure S16b, Supporting Information). Flow cytometry analysis following the propidium iodide (PI) staining revealed that BT474 cells captured by cCAP1-G4.5-cCAP2 were significantly arrested at cell cycle S and G2/M phases. G4.5-cCAP1 and G4.5-cCAP2 did not significantly affect the BT474 cell cycle (Figure 4e). MDA-MB-231 cells were arrested by cCAP1-G4.5-cCAP2 and G4.5-cCAP1 at S and G2/M phases, while A549 cells were arrested by cCAP1-G4.5-cCAP2 and G4.5-cCAP2 at S and G2/ M phases (Figure S17, Supporting Information). These conjugates did not affect the cell cycle of the HELF cells. Figure 4f shows that both the untreated and the G4.5-treated BT474 cells stayed uniformly green with normal and homogeneous nuclei after AO/EB staining, indicating good viability of the cells. By contrast, the BT474 cells treated with G4.5-cCAP1 or G4.5-cCAP2 exhibited some yellow-green, and the BT474 cells treated with cCAP1-G4.5-cCAP2 showed more yellow-green, suggesting that the cells restrained by the conjugates tend to go to apoptosis.2 The A549 and MDAMB-231 cells treated with G4.5-cCAP2 and G4.5-cCAP1, as well as cCAP1-G4.5-cCAP2, showed their apoptotic nuclei condensation and fragmentation (Figure S18a, Supporting Information). The HELF cells, however, remained apoptosis-

each nanomaterial (for major FTIR peak assignments, see Experimental Section). The PAGE gel electrophoresis staining provided additional evidence for the successful conjugation of cCAP1 and cCAP2 to G4.5 (Figure 2d). The final conjugation products G4.5cCAP1 (lane 6), G4.5-cCAP2 (lane 7), and cCAP1-G4.5cCAP2 (lane 8) stayed at the same retention slot as they were loaded at the first place without degraded species as shown in lanes 6−8, indicating the entity completeness and stability of the conjugates. The PAMAM-free entities that moved faster on the gel were cCAP1 (lane 1), cCAP2 (lane 2), cCAP1biostreptavidin (lane 3), cCAP2-biostreptavidin (lane 4), and cCAP1-biostreptavidin-cCAP2 (lane 5). The morphology of cCAP1-G4.5-cCAP2 was shown by atomic force microscopy (AFM; Figure 2e) and scanning electron microscopy (SEM; Figure 2f) images, respectively. Briefly, both methods showed the diameters of these aptamer conjugates were 100 ± 20 nm (Figure 2e,f; Figure S9a− d,Supporting Information). The cCAP1-G4.5-cCAP2 did not show significant increase in particle size in comparison with the single cCAP-coated G4.5. Laser microscopic imaging showed cCAP1-G4.5-cCAP2 in green fluorescence in the FAM channel and in red in the CY5 channel, respectively, and in light brown when the two channels were merged (Figure 2g). By contrast, the fluorescence images showed G4.5-cCAP1 in green only and G4.5-cCAP2 in red only (Figure S9e,f,Supporting Information). The observation indicated the successful conjugation. Ultraviolet absorption values at λ260 nm increased significantly as the result of the nucleic acids conjugated onto G4.5 (Figure 2h). Fluorescence spectrum peaks at λ518 nm and λ670 nm represented FAM-labeled cCAP1 and CY5-labeled cCAP2. As shown in Figure 2i, only the ds cCAP1-G4.5-cCAP2 exhibited two peaks at λ518 nm and λ670 nm, indicating the successful conjugations of cCAP1 and cCAP2 to G4.5 (Figure 2i). HPSEC analysis (Figure S10c, Supporting Information) showed the distinct peaks of each conjugate. To quantitatively analyze the amount of cCAP1 and cCAP2 conjugated onto G4.5, we used AP1 and AP2 as the internal standards to establish the fluorescent standard calibration curves (Figure S10a, b, Supporting Information) with linearity R2 = 0.99 within the concentration range from 0.05 to 1.25 μM (Figure 2j). We used the curves to determine that per milligram of G4.5 carried 1.19 nmoles of cCAPs. The payload capacity of G4.5 seemed to be higher than what we achieved previously using mesoporous silica nanoparticles as the carrier,36 indicating the payload advantage of dendrimers. 3.4. Enhanced Binding Specificity and Biostability of cCAP1-G4.5-cCAP2. To test functionality of the conjugates, we incubated them with the test cell lines for 24 h and quantitatively measured the binding between the conjugates and the cells after removing the unbound free conjugates. The confocal imaging demonstrated that cCAP1-G4.5-cCAP2 could recognize BT474 cells as shown by the merged color, by red CY5 and green FAM, as well as by blue DAPI (staining the cell nuclei; Figure 3a). Flow cytometric analysis showed that the binding potency between cCAP1-G4.5-cCAP2 and BT474 was about 90%, almost twice as much as the binding potency between cCAP1-G4.5-cCAP2 and MDA-MB231 or A549 (Figure 3b, upper panel). G4.5-cCAP1 and G4.5-cCAP2 bound to their corresponding biomarkers on MDA-MB-231 and A549 cells with the binding potency almost equivalent to their binding to BT474 (Figure 3b). These conjugates did not 10320


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Figure 5. Capturing CTCs in nude mice by cCAP1-G4.5-cCAP2 and its tissue distribution analysis. (a) Experimental procedures: the Hoechstlabeled BT474 cells were injected into the nude mice for 5 min followed by injection of the conjugates (50 μg per mouse) before blood withdraw. The blood samples were separated and analyzed by confocal microscopy and flow cytometry following purification. (b) Fluorescence images of the BT474 cells captured in vivo by cCAP1-G4.5-cCAP2 and its single counterparts. The merge 1 combines images of Hoechst, FAM, and CY5 together, while the merge 2 combines images of merge 1 with a microscopic photo (for binding between other cell lines and the conjugates). (c) Flow cytometric analysis showed numbers of FAM+CY5+ BT474 captured in vivo by cCAP1-G4.5-cCAP2 and its single counterparts. (d) BT474-induced metastatic nodes in lungs of mice treated differently. (e) In vivo fluorescence images of tissue distribution of cCAP1-G4.5-cCAP2 and its single counterparts at 1 h after intravenous injection of BT474 to nude mice. (f) Mean fluorescence intensity per gram of tumor weight. (g) Relative fluorescence intensity of different organs of BT474-bearing mice at 9 h post-tail-vein injection of cCAP1-G4.5-cCAP2 and its single counterparts (see Figure S18b). (h) In vivo fluorescence images of tissue distribution of cCAP1-G4.5-cCAP2 and its single counterparts at 9 h after intravenous injection to BT474-bearing nude mice.

as we conducted previously,5,37 further displayed the apoptotic degree of BT474 quantified by % DNA after the conjugate

free following the conjugate treatments (Figure S18a, Supporting Information). The Annexin V-FITC/PI staining, 10321


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Figure 6. Ex vivo analysis of CTCs captured by cCAP1-G4.5-cCAP2 from blood of breast cancer patients. (a) Experimental procedures: cCAP1G4.5-cCAP2 and its single counterparts were separately mixed with patient bloods that were analyzed by laser confocal microscopy and flow cytometry following purification. (b) Fluorescence images of the real CTCs captured by cCAP1-G4.5-cCAP2. (c) Flow cytometric analysis of captured CTCs from the blood of patient 1 by cCAP1-G4.5-cCAP2 and its single counterparts. (d) Number of CTCs captured by cCAP1-G4.5cCAP2 and its single counterparts from blood of 10 breast cancer patients for quantitative analysis.

3.6. Capturing and Inhibiting CTCs in Vivo and Conjugate Tissue Distribution. To evaluate the specificity and precision of the aptamer ring conjugates in capturing CTCs in vivo, we stained cancer cell lines with Hoechst 33258 for 30 min and injected these cells (2 × 104 per mouse) into the nude mice followed by administration of these conjugates (50 μg), blood drawn, and separation. The results were analyzed by flow cytometry and confocal microscopy as we described previously (Figure 5a).18

treatments (Figure 4g). By comparison, cCAP1-G4.5-cCAP2 produced the concentration-dependent apoptosis more statistically significant than G4.5-cCAP2 and G4.5-cCAP1 in BT474 cells. The potency of cCAP1-G4.5-cCAP2 in capturing and restraining A549 and MDA-MB-231 appeared to be equivalent to that of G4.5-cCAP2 and G4.5-cCAP1 (Figure S19, Supporting Information). The above data demonstrated the specificity and potency of the conjugated dual ds aptamer rings in seizing the target cells were enhanced by 2 times at least in comparison with their single counterparts. 10322


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WBCs (Figure 6b). The CTCs were further verified by using our established method, i.e., immunocytochemistry in conjunction with flow cytometry,6,20 which characterizes patient CTCs by three-color immunocytochemistry. Only cells that were DAPI+CD45−cytokeratin+ with the appropriate size and morphology were identified as patient CTCs. The conjugate bound to the patient CTCs, resulting in the FAM-green or CY5-red under the confocal microscope. The merged image displayed the multifluorescent stains, making the CTCs more distinguishable from WBCs. Whereas, the WBCs showed only DAPI blue because of no binding between WBCs and the conjugates. The healthy blood samples show no immunocytochemistry colors under the confocal microscope, and negative FAM-green and CY5-red cell counts by flow cytometry because no CTCs exist in the healthy blood, which would otherwise have bound to the conjugates and showed images. By the way, CD45+ WBCs were confirmed by CD45 staining assay, but the data was not shown here. The aliquots of the same blood samples were re-examined by flow cytometry to quantify the number of CTCs captured by these conjugates. The representative dot plot prepared from the patient 1 showed that cCAP1-G4.5-cCAP2 captured 23 CTCs and G4.5-cCAP2 and G4.5-cCAP1 captured 11 and 8 CTCs, respectively (Figure 6c). Statistical analysis further demonstrated that cCAP1-G4.5-cCAP2 captured 16 ± 5 CTCs (n = 10) and G4.5-cCAP2 and G4.5-cCAP1 captured only 8 ± 3 and 5 ± 2 CTCs, respectively (Figure 6d; see Figures S21 and 22, Supporting Information), confirming that the dual cCAP conjugates were superior to their single counterparts in capturing the CTCs.

The confocal photos showed the big BT474 mixed with many WBCs (the far left or right panels of Figure 5b; for other cells, see Figure S20a, b, Supporting Information). cCAP1G4.5-cCAP2 bound to the circulating BT474 as the result of both EpCAM and Her2 recognition. G4.5-cCAP1 and G4.5cCAP2 bound to the circulating BT474 cells as the result of either EpCAM or Her2 recognition, but not both. The merged 1 and 2 images clearly showed the advantage of cCAP1-G4.5cCAP2 in binding to the targets over its counterparts, and the advantage was further confirmed by the flow cytometry analysis (Figure 5c), which showed that cCAP1-G4.5-cCAP2 captured 114 FAM+CY5+ cells in the Q2 region, G4.5-cCAP2 captured 51 FAM-CY5+ cells in Q1, and G4.5-cCAP1 captured 30 FAM +CY5- cells in the Q3 region. But, these conjugates did not recognize the control HELF cell lines (Figure S20c, Supporting Information). The BT474-inoculated mice were also examined for their lung metastasis 3 weeks after receiving G4.5 and the conjugates. Figure 5d showed that the cCAP1-G4.5-cCAP2 inhibited the BT474-induced metastatic nodes in mouse lungs more significantly than G4.5-cCAP1 and G4.5-cCAP2. To dynamically monitor distribution of the conjugates over the mouse body, we injected the conjugates (80 μg per mouse) into the nude mice 4 h postinjection of BT474 cells (5 × 104 per mouse) to the mice. Figure 5e showed strong fluorescent signals peaked at 1 h postinjection of cCAP1-G4.5-cCAP2, which concentrated at some tissues of the mice. Other conjugates exhibited relatively weak signals. After exploring the dynamic distribution profile of the conjugates, we implanted BT474 into the nude mice and investigated the in vivo binding specificity of the conjugates for BT474 when the tumors formed. cCAP1-G4.5-cCAP2 could distinguish the tumor region from the surrounding normal tissue 1 h postinjection (Figure S4a, Supporting Information). Its fluorescence signals increased gradually, reached its maximum at 9 h, and maintained the intensity for another 9 h. To compare the binding potency between the conjugates, we collected each tumor from the mice and quantified the fluorescent intensity per gram of tumor weight 9 h postinjection of the conjugates following our established protocol for tissue distribution.4,38 As shown in Figure 5f, cCAP1-G4.5-cCAP2 exhibited the fluorescence intensity per gram of tumor weight significantly higher than its counterparts. Further analysis on tissue distribution of these conjugates revealed that cCAP1-G4.5cCAP2 deposited in BT474 tumor significantly higher than its deposition in other tissues (Figure 5g and Figure S4b, Supporting Information), indicating the specific deposition of cCAP1-G4.5-cCAP2 toward the EpCAM+Her2+ tumor. The functionality of the conjugates as a tumor probe was further examined in the BT474-bearing nude mice by real time observation of the conjugate tropism using the imaging equipment. As shown in Figure 5h and Figure S4b (Supporting Information), by 9 h postinjection, the conjugates were highly concentrated at the implanted BT474 tumor. cCAP1-G4.5cCAP2 exhibited the strongest signal as the result of its high binding potency toward EpCAM+Her2+ BT474. 3.7. Capturing CTCs in Breast Cancer Patient Blood. Ten patient blood samples were obtained following protocols approved by the institutional review board. The conjugates were separately (50 μg) added into 1 mL aliquots of each blood sample after purification. Figure 6a illustrates the clinical analysis procedures as we described previously.20 cCAP1-G4.5cCAP2 precisely distinguished the patient CTCs from their

4. CONCLUSIONS The present studies constructed the novel dual aptamer ring conjugates to simultaneously recognize and seize two surface biomarkers on one type of CTC. Such unique molecular architecture can significantly withstand degradation by nucleases and precisely capture the target CTCs in the presence of millions of interfering normal cells and in patient and animal bloods. The conjugate with its enhanced functionality and biostability provides a more easily scalable and low-cost clinical approach to restraining CTCs and preventing CTCs-based cancer metastasis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b03044. All the methods, the designed sequences, the structures of AP1 and AP2, and the supplementary data in this work (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(J.L.) E-mail: [email protected]; cmapcjia1234@163. com. ORCID

Yu Gao: 0000-0002-1137-7669 Lee Jia: 0000-0001-6839-5545 10323


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Chemistry of Materials Author Contributions

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L.J. and H.D. designed the study, and Z.-S.W. designed all DNA sequences. H.D. developed methods, collected, analyzed, and interpreted data, and organized figures. L.J. and H.D. wrote the manuscript. L.H. and J.W. performed the experiments. J.X. and J.S. reviewed the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of China (Grant No. 2015CB931804); National Natural Science Foundation of China (NSFC) (Grant Nos. U1505225, 81773063, 81273548, 81571802, and 81702988); Natural Science Foundations of Fujian Province of China (Grant Nos. 2016J06020 and 2017J05137); Fujian Development and Reform Commission project (Grant No. 829054 (2014; 168)), and the Fundamental Research Funds for the Central Universities (Grant No. 20720160061).

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