Artificial Antibody with Site-Enhanced Multivalent Aptamers for

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Artificial antibody with site-enhanced multivalent aptamers for specific capture of circulating tumor cells Lukuan Liu, Kaiguang Yang, Hang Gao, Xiao Li, Yuanbo Chen, Lihua Zhang, Xiaojun Peng, and YuKui Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05259 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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Analytical Chemistry

Artificial antibody with site-enhanced multivalent aptamers for specific capture of circulating tumor cells Lukuan Liu,†,‡,||,§ Kaiguang Yang,†,§ Hang Gao, †,‡ Xiao Li, † Yuanbo Chen, †,‡ Lihua Zhang,*,† Xiaojun Peng, || Yukui Zhang† CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China || State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China †

ABSTRACT: Isolation of circulating tumor cells (CTCs) from blood holds the great potential to diagnose cancers and discover therapeutic targets. Herein, we reported a novel kind of artificial antibody, the cell-imprinted hydrogel with site-directed modification of aptamer (APT-CIH) to achieve the specific capture of CTCs. Cell-imprinted sites could be used not only to recognize target cells, but also as efficient scaffolds for assembling aptamers to enhance the capture efficiency and selectivity. Due to the synergistic effect of conformation recognition and multivalent interaction between the aptamers and target cells, APT-CIH showed high capture efficiency and selectivity to SMMC-7721 cells. In the coexistence of the 1000 times leukaemia Jurkat cells, the enrichment factor of APT-CIH could reach as high as 21.6 ± 3.1 towards target cells, while that relied only on cell imprinting or aptamer affinity was 8.1 ± 5.0 or 10.1 ± 1.3 respectively. Furthermore, the capture efficiency could reach to 58.2% ± 10.9% with 1000 SMMC-7721 cells spiked in 1 mL blood. Moreover, 92% of the captured cells could be released, beneficial to carry out further biological and clinical study of CTCs. These results demonstrated that APT-CIH might have great potential in CTCs analysis.

Circulating tumor cells (CTCs) are cancer cells shed from the primary or metastatic tumor sites thereby circulating in the peripheral blood.1 Therefore, the isolation and characterization of CTCs hold the great potential to diagnose cancer and discover therapeutic targets.2-4 So far, the gold standard for isolating CTCs is using antibody-coated magnetic beads, based on structure and chemical affinity matching between the epitope on the antibody and the antigen on the cell surface,5, 6 by which CTCs could be captured from blood with high selectivity, but are mostly dependent on the epithelial cell adhesion molecule (EpCAM). Therefore, the false negative results might be caused by the down regulation of EpCAM expression during the epithelial-mesenchymal transition. As an artificial antibody, aptamers are single-stranded oligonucleotides with high affinity to target cells. Compared with natural antibodies, aptamers are screened by using whole cells as targets, without considering the surface characteristics of the cells, thus avoiding the false negative result of natural antibodies to some extent. Therefore, aptamers have been widely used to capture CTCs.7 Initially, the binding between aptamers and cells was monovalent, resulting in the low capture efficiency.8 Recently, some researches showed that the receptors on the cell surface were clustered, and the binding between the receptors and the aptamers was also multi-site.9 Therefore, multivalent effect, by integrating multiple aptamers into one entity, could be used for enhancing the capture efficiency. Some entities, such as dendrimers and micro/nanoparticles, have been used as scaffolds to assembling multivalent aptamers.10, 11 To date, with the target cells as the templates, the cell imprinting could generate recognition sites

that match the conformation and properties of the cells thereby specifically identifying target cells.12, 13 Therefore, we hypothesized that these cell-imprinted sites could be employed as novel entities to assemble multivalent aptamers and the cell capture efficiency and selectivity could be greatly improved due to the synergistic effect of the cell imprinting and multivalent aptamers. To modify the multivalent aptamers within the cell-imprinted sites, reactive groups that could bind to the aptamers needed to be introduced into these sites in advance. Generally, imprinted sites only possess structural and affinity groups to target cells, but do not possess reactive groups to aptamers. A convenient solution is to mix reactive monomers into the imprinted materials during the polymerization, to provide immobilization groups for aptamers. However, in this case, the aptamers were indiscriminately modified on the surface of the imprinted materials, and could hardly be clustered to form the multivalent effect in the imprinted sites, eventually resulting in weak capturing selectivity. Herein, we present an artificial antibody, the cell-imprinted hydrogel with the site-directed modification of aptamer (APTCIH), to provide the synergistic effect of conformation recognition and multivalent binding for the specific capture of CTCs. To achieve the conformation recognition, we designed the recognition sites toward the target cell by cell imprinting technique. To achieve multivalent binding, the aptamer was site-directed modified within the imprinted site via a trifunctional cleavable crosslinker (TCC).14

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Hepatocellular carcinoma is one of the most malignant cancers in the world. SMMC-7721 cells are the typical human hepatocellular carcinoma cell line and the aptamers against SMMC-7721 cells were successfully developed.15 The dissociation constant of the aptamers was 2.18 nM, and the aptamers could recognize not only SMMC-7721 cells, but also the in vivo tumor and tumor tissue section.16 Therefore, SMMC7721 cells were employed as the cell templates in this study. As shown in Scheme 1, SMMC-7721 cells were derivatized by the esterification between the amine groups of cells and the succinimide ester of TCC, and the methacryloyl groups of TCC remained only on the derivatized cells after the removal of unreacted and weakly adsorbed TCC. Then acrylamide and N,N'-methylene diacrylamide were covalently polymerized with methacryloyl groups on the derivatized cells to form an recognition layer. Afterwards, the cell templates were removed by reducing the disulfide bond of TCC, thereby exposing the cell-imprinted sites with thiol groups. Finally, the aptamers were introduced into the sites by click chemistry between the acryloyl groups of aptamers and the thiol groups in the imprinting site, to form site-enhanced heterogeneous hydrogel with multivalent effects. Scheme 1. Preparation of cell-imprinted hydrogel with the site-directed modification of aptamer (APT-CIH) for capture and release of SMMC-7721 cells.

EXPERIMENTAL SECTION Chemicals and Reagents. The sequences of the affinity aptamers that could capture SMMC-7721 cells and react with the thiol groups in the imprinted sites (Acrylite-ZY sls-FITC aptamers) were 5’- Acrylite -TTT TTT ACG CGC GCG CGC ATA GCG CGC TGA GCT GAA GAT CGT ACCGTG AGC GCG TTT TTT T-FITC-3’, synthesized by Takara Biotechnology (Dalian) (Dalian, China). The crosslinkers with trifunctional groups (TCC) were synthesized according to the method reported by Horikawa et al, with the detail synthesis steps shown in the Supporting Information.14 Preparation of cell-imprinted hydrogel with the sitedirected modification of aptamer (APT-CIH). Firstly, 40 mg TCC in phosphate buffer (10mM or 50 mM, PB, pH 7.4) reacted with the amine groups on the surface of SMMC-7721 cells. Secondly, acrylamide, methylene diacrylamide, α,α′Azodiisobutyramidine dihydrochloride in deionized water were polymerized with TCC on the cell surface at 60 °C. After gelation, the hydrogels were then peeled off and incubated with 0.25% trypsin solution at 37°C to remove SMMC-7721 cell. Thirdly, tris(2-carboxyethyl)phosphine (TCEP, 20mM) was reacted with the disulfide linkages of the TCC on the surface of

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the hydrogels at 56°C for 1 h. Then potassium carbonate, TCEP and Acrylite-ZY sls-FITC aptamers (5 μL, 25 μM) in deionized water were added and reacted with the thiol groups in the imprinted sites of the hydrogels at 55°C for 4 h. Finally, APTCIH was obtained. As control groups, no-imprinted hydrogels without aptamer (NIH), aptamer-functionalized NIH (APTNIH), cell-imprinted hydrogels without aptamer (CIH) were also prepared ((The detail preparation methods of the hydrogels and the capture and release experiments were shown in the Supporting Information).

RESULTS AND DISCUSSION Compared with cells such as bacteria and yeast, mammalian cells are more fragile, and thus are difficultly to be used as the template to fabricate effective imprinted sites.17 Herein, firstly, SMMC-7721 cells adhered and stretched adequately in the culture dish in the shape of spindles (Figure 1a). To maintain cell integrity and conformation during the imprinting process, the esterification was performed in PB with the optimized concentration. As shown in Figure 1b, the conformation of esterified cells was well maintained after the reaction with TCC in 50 mM PB. Otherwise, the cell integrity and morphology changed obviously when the esterification processed in 10 mM PB, resulting in the failure to generate conformation matched imprinted sites (Figure S-1a). By the copolymerization of methacryloyl groups between the derivatized cells and the functional monomers, the cellimprinted hydrogel was prepared. As shown in Figure 1c, the imprinted sites with thiol groups were left on the surface of the cell-imprinted hydrogel after the cell templates were removed. By further site-directed functionalization, as shown in Figure 1d, the aptamers modified with acryloyl groups and FITC were clicked inside the imprinted sites. The graft density of the aptamers was measured at 260 nm by the UV-Vis spectrophotometer and could reach 49.9 ± 0.5 pmol/cm2. For a reasonable comparison, by controlling the amount of TCC during polymerization, APT-NIH was also prepared, and the graft density was 46.7 ± 0.5 pmol/cm2, comparable with that of APT-CIH. By contrast, as shown in Figure S-1b, only smooth surface was obtained on the NIH, and the CIH was also prepared (Figure S-2).

Figure 1. Micrographs of SMMC-7721 cells on culture dish before (a) and after reaction with TCC in 50 mM PB (b); surface morphology of cell-imprinted hydrogel after removing cell templates (c). Dark field micrograph of APT-CIH with FITCmodified aptamers (d). Scale bars represent 100 μm.

According to our initial hypothesis, the synergistic effect of conformation recognition and multivalent aptamers should obviously improve the potential interaction between enhanced sites and target cells. To verify the site-enhanced multivalent binding property, we investigated the potential of APT-CIH to capture CTCs. As shown in Figure 2a and Figure 3a, many cells were captured on APT-CIH, and the capture efficiency reached up to 94.7% ± 0.9% (n=3) when 1×105 SMMC-7721 cells were incubated on the hydrogels, comparable to that of the antibodyassisted substrates.18-20 By contrast, as shown in Figure 2b and

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Analytical Chemistry Figure 3a, few SMMC-7721 cells adhered on NIH, with the capture efficiency of only 16.4% ± 0.4% (n=3), demonstrating that the limited ability of NIH to capture target cells. On APTNIH, more SMMC-7721 cells were captured, with capture efficiency up to 66.7% ± 1.7% (n = 3) (Figure 2c and Figure 3a), attributed to the specific affinity interaction provided by aptamers, but still lower than APT-CIH, due to the monovalent interaction between aptamers and target cells and lack of microstructures on the hydrogel to promote the adhesion of the target cells. On CIH, the capture efficiency reached 84.1% ± 8.3% (n = 3), contributed by the conformation recognition of imprinted sites (Figure 2d and Figure 3a), but lower than APTCIH due to the lack of specific affinity to SMMC-7721 cells provided by aptamers. These results demonstrated that both the microstructures and the specific affinity were indispensable in identifying and capturing SMMC-7721 cells. When the cell counts dropped to the range from 10000 to 100, as shown in Figure 3a, APT-CIH still showed the highest capture efficiency, 78.0% ± 1.6% (n = 3), which further proved the superiority.

Figure 2. Micrographs and of SMMC-7721 cells on APT-CIH (a), NIH (b), APT-NIH (c) and CIH (d). Scale bars represent 100 μm.

cells released from APT-CIH could be further cultured. As shown in Figure 3b, the counts of proliferated SMMC-7721 cells released from APT-CIH showed the exponential increase with time, similar to that of the untreated cells. These results showed that the released cells had unaffected viability and proliferation capacity and could be used for the downstream profiling. The capture selectivity of the hydrogels for SMMC-7721 cells was further studied by mixing SMMC-7721 cells with leukaemia Jurkat cells in different ratios. The enrichment factor, calculated as the ratio of the capture efficiency of the template, SMMC-7721 cells, to the capture efficiency of interferece, Jurkat cells, was used to as the evaluation parameter.22 As shown in Figure 3c, APT-CIH showed the highest enrichment factor, up to 30.6 ± 2.6 when the ratio between Jurkat cells and SMMC-7721 cells was 1:1. Even in the presence of 1000 times Jurkat cells, SMMC-7721 cells could be captured with the enrichment factor of up to 21.6 ± 3.1 (n = 3), with the capture efficiency towards SMMC-7721 cells as 75.0% ± 3.8% (n=3), demonstrating the high selectivity of APT-CIH even in a complex sample. By contrast, the enrichment factors of NIH, APT-NIH and CIH were 0.03 ± 0.04, 10.1 ± 1.3 and 8.1 ± 5.0 respectively, much lower than that of APT-CIH, due to the absence or weak affinity interaction between the hydrogels and SMMC-7721 cells. These results showed that APT-CIH exhibited excellent capture and anti-interference ability to target cells. Finally, APT-CIH was applied to capture tumor cells from blood. SMMC-7721 cells in amounts ranging from 1000 to 10000 were spiked into 1 mL of whole blood. The linear correlation between the counts of spiked and captured cells (R2 = 0.997, n = 3) was observed (Figure 3d), with the capture efficiency as 58.2% ± 10.9% (n = 3) at 1000 cells/mL, better than antibody-based strategy (38%-44%),23 and could be further improved by introducing tumor specific functional monomers in the imprinting sites and designing the hyperbranched crosslinker to increase the grafting density of the aptamers. Furthermore, for CTCs with different phenotypes, epithelial and mesenchymal tumor cells could be used as the template respectively or together, to further ensure the capture specificity.

CONCLUSIONS

Figure 3. Capture efficiency of APT-CIH, CIH, APT-NIH and NIH for 105, 104, 103, and 102 SMMC-7721 cells (a). Proliferation of SMMC-7721 cells released from APT-CIH and untreated control SMMC-7721 cells (b). Enrichment factors of APT-CIH, CIH, APT-NIH and NIH for SMMC-7721 cells mixed with Jurkat cells in different ratios (c). Regression analysis of the counts of SMMC-7721 cells captured by APT-CIH versus the counts of cells spiked in blood (d). Bright and dark field micrographs of one SMMC-7721 cell captured on APT-CIH from the blood (insert). Scale bars represent 50 μm. Error bars represent standard deviations, n = 3.

Moreover, the captured cells could be released by the complementary sequences of the aptamers with the efficiency up to 94.0% ± 1.9% (n=3), higher than that of the antibodybased capture methods, such as 85% for A549 human lung adenocarcinoma cell line from NanoVelcro Chips and 92% for PC3 prostate cancer cell line from anti-EPCAM antibodymodified microfluidic chips (Figure S-3).21 Furthermore, the

In summary, we prepared an artificial antibody with siteenhanced multivalent aptamers hydrogel based on cell imprinting and site-directed aptamer modification. Due to the synergistic effect of conformation recognition and multivalent interaction between the aptamers and target cells, APT-CIH showed the highest capture efficiency and selectivity to SMMC-7721 cells. Moreover, in the presence of non-target cells as interferences, APT-CIH also exhibited better capture and anti-interference performances than the materials that relied solely on cell imprinting or aptamer affinity. Furthermore, the captured cells could be released and their viability was well remained, which were important to the further characterization of CTCs. All these results demonstrate that APT-CIH has great potential to become a powerful artificial antibody to pave a new way to achieve the selective capture of CTCs from blood.

ASSOCIATED CONTENT Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website. Detail synthesis steps of the trifunctional crosslinkers; Detail preparation methods of the hydrogels and the capture and release experiments; Micrographs of NIH, APT-NIH and CIH; Micrographs of the cells before and after release process of the APT-CIH (PDF)

AUTHOR INFORMATION Corresponding Author *Address: 457 Zhongshan Road, Dalian 116023, China. Tel/Fax: +86-411-84379720. E-mail: [email protected].

Author Contributions §These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully acknowledge funding from National Key Research and Development Program of China (2017YFA0505003 and 2016YFA0501401), National Natural Science Foundation (21575143, 91543201 and 21725506) and CAS Key Project in Frontier Science (QYZDY-SSW-SLH017). Kaiguang Yang is the member of Youth Innovation Promotion Association, CAS (2017222).

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Analytical Chemistry

Table of Contents (TOC)

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