Extracellular Milieu and Membrane Receptor Dual-Driven DNA Nanorobot for Accurate in Vivo Tumor Imaging

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 May 2022Extracellular Milieu and Membrane Receptor Dual-Driven DNA Nanorobot for Accurate in Vivo Tumor Imaging Kun Yuan, Hong-Min Meng, Yanan Wu, Juan Chen, Hui Xu, Lingbo Qu, Lele Li and Zhaohui Li Kun Yuan College of Chemistry, Green Catalysis Center, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostic, Zhengzhou University, Zhengzhou 450001 , Hong-Min Meng *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] College of Chemistry, Green Catalysis Center, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostic, Zhengzhou University, Zhengzhou 450001 , Yanan Wu College of Chemistry, Green Catalysis Center, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostic, Zhengzhou University, Zhengzhou 450001 , Juan Chen College of Chemistry, Green Catalysis Center, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostic, Zhengzhou University, Zhengzhou 450001 , Hui Xu College of Chemistry, Green Catalysis Center, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostic, Zhengzhou University, Zhengzhou 450001 , Lingbo Qu College of Chemistry, Green Catalysis Center, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostic, Zhengzhou University, Zhengzhou 450001 , Lele Li CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190 and Zhaohui Li *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] College of Chemistry, Green Catalysis Center, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostic, Zhengzhou University, Zhengzhou 450001 https://doi.org/10.31635/ccschem.021.202100836 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Precise diagnostic approaches have great potential in cancer intervention and prognosis. Although diverse DNA nanoprobes have been reported for tumor diagnosis, precise tumor imaging in vivo still encounters a great challenge due to the scarcity of exquisite design methodology. Herein, by assembling three programmable modules on a DNA triangular prism, we engineered a DNA nanorobot for simultaneous recognition of extracellular pH and cancer cell membrane receptor in an intelligent manner. Since the design uses two heterogeneous types of biomarkers as inputs, pH-RE not only could discriminate target tumor cells from similar cell mixtures with a recognition accuracy as high as 98.8%, but also could perform precise tumor imaging in living mice by intravenous injection. We expect that this extracellular pH and membrane receptor dual-driven DNA nanorobot will facilitate the establishment of a novel design paradigm for precise cancer diagnosis and therapy. Download figure Download PowerPoint Introduction The development of accurate diagnosis strategies is highly significant for personalized medicine.1–3 With the discovery of diverse tumor biomarkers, detection of such targets has provided an opportunity for cancer diagnosis.4–6 However, most of these biomarkers are not exclusively expressed on the target cancer cells, but are also expressed on normal cells at relatively low levels or different subtypes of cells at similar levels.7–9 Therefore, approaches to profile the abnormal levels of multiple biomarkers are actively being pursued to precisely identify cancer cells as distinguished from normal cells. In the wake of the advance of molecular computing, increasing attention is being focused on designing nucleic acid-based nanorobots for reliable tumor recognition10 and therapy.11,12 These nanorobots combine both recognition units and analysis functions and can give an intelligent data readout in one step.13,14 For instance, some nanodevices use intracellular biomarkers [mRNA,15,16 miRNA,17,18 or adenosine 5′-triphosphate (ATP),19 etc.] as inputs for conducting logic operations, but most of these systems operate at the cellular level and suffer from vulnerable stability and impeded delivery. Additionally, DNA nanodevices have implemented their smart logic functions by evaluating protein receptors on the cell surface.20–25 However, such protein receptor-based logic nanodevices have the following limitations. First, the unknown spatial distribution of different membrane receptors severely hampers the spatially controlled logic operation at the surface of specific cell types, thus affecting the accuracy of tumor identification. Second, solely using surface receptor-targeting ligands may also increase the risk of on-target off-tumor effects. Recently, we explored the engineering of light-controlled DNA nanodevices for improved spatial-temporal control over molecular imaging26–29 and tumor targeting.30 However, one fundamental roadblock that limited the in vivo application is the poor tissue penetration of light. To the best of our knowledge, DNA-based logic devices serving as imaging tools for tumor diagnosis in vivo have not yet been reported. Thus motivated, here we designed a functional nucleic acid (FNA)-based DNA nanorobot (denoted as pH-RE) with improved accuracy for in vivo tumor imaging through bispecific recognition of extracellular pH (pHe) and cell surface receptor (Scheme 1). Decreased pHe in the extracellular milieu of solid tumors is emerging as a new target for cancer diagnosis31–33 due to its unparalleled spatial resolution34–36 in cancer intervention. In our design, two FNAs, named as i-motif and sgc8c aptamer, were chosen to specifically bind H+ and the protein tyrosine kinase 7 (PTK7) receptor, respectively.37,38 As presented in Scheme 1a, pH-RE is composed of a pHe-recognition module (i-motif/cF, pink block), a receptor-recognition module (sgc8c/cS, green block), and a report module (R/S/F) on the surface of a DNA triangular prism (TP). When pH-RE reaches the tumor microenvironment, the pH-recognition module (i-motif/cF) is protonated due to the exclusively decreased pHe, accompanied by release of the cF strand. Then, cF acts as the first input to partially digest the report module by a strand displacement reaction. Meanwhile, the PTK7 receptor on tumor cells induces the structure switch of the sgc8c/cS module to liberate cS, which acts as the second input to interact with the S strand of the report module. Therefore, upon the cascade removal of both the F and S gate strands in the report module, pH-RE is activated with restored fluorescence on specific tumor cell membranes (Schemes 1a–1c). The developed nanorobot possesses the following advantages: (1) Based on the distinctive microenvironment of solid tumor, where membrane receptors are surrounded by low pHe, the logic operation of pH-RE will not be affected by the spatial hindrance between two targets, resulting in dramatically improving the accuracy of tumor identification in an intelligent manner. (2) Two recognition modules are coassembled on one DNA skeleton, which can make the inputs to a circuit come from a single cell, lessening the false-positive signal greatly. (3) The logic design can reduce the on-target off-tumor effects before reaching the tumor site and upgrade the precision of the cell discrimination. Scheme 1 | (a) Schematic of the molecular structure and operational mechanism of pH-RE. (b) Schematic of using pH-RE for in vivo precise tumor imaging. (c) The truth table of pH-RE-based DNA computation. Download figure Download PowerPoint Experimental Methods Reagents All DNA strands were provided by Sangon Biotech Co., Ltd. (Shanghai, China), and the detailed information is given in Supporting Information Table S1. Yeast tRNA, bovine serum albumin, Hoechst-33342, and Synergy Brands, Inc. (SYBR) Gold were obtained from Sigma-Aldrich Co., Ltd. (St. Louis, MO). The cell culture medium (RPMI-1640) was supplied by Thermo Fisher Scientific (Waltham, MA). All other reagents were of analytical grade and used directly. Binding buffer was prepared by adding 5 mM of MgCl2, 4.5 mg/mL of glucose, 0.1 mg/mL of yeast tRNA, and 1 mg/mL of bovine serum albumin into Dulbecco’s phosphate-buffered saline. The pH of buffers was calibrated with a pH meter (Orion 3-Star, Thermo Fisher Scientific). Cell culture CCRF-CEM (human acute lymphoblastic leukemia cell line) and Ramos (human B-cell Burkitt’s lymphoma cell line) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, both of which were maintained at 37 °C under a 5% carbon dioxide atmosphere. Animals Female athymic BALB/c (BALB/c-nu) mice were obtained from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). At the start of all experiments, the mice were 6–8 weeks old and weighed 15–20 g. The animal operations were conducted according to protocol no. SYXK (Yu) 2018-0004, approved by the Laboratory Animal Center of Henan Province (China). Construction of pH-RE The DNA TP framework was prepared according to the literature reports.39,40 In short, equimolar molar ratios of S1, S2, and S3 were first mixed in 1× TAE-Mg buffer (20 mM Tris-Ac, 2 mM ethylenediaminetetraacetic acid (EDTA), and 12.5 mM MgAc2, pH 7.4) and heated at 95 °C for 5 min. The mixture was then slowly cooled down to 4 °C to form DNA TP scaffold. The two recognition modules (i-motif/cF and sgc8c/cS) were modified on the DNA TP by DNA hybridization at 25 °C for 2 h. The report module (R/S/F) was assembled on the scaffold via Watson–Crick hybridization at 4 °C for 4 h. The final product (DNA nanorobot, pH-RE) was stored at 4 °C for further use. Gel electrophoresis analysis To characterize the preparation process of pH-RE, 5% native polyacrylamide gel electrophoresis (N-PAGE) was performed. Briefly, 10 μL of different samples were mingled with 6× loading buffer (2 μL) and SYBR Gold (2 μL). The gel was then run in 1× TAE-Mg buffer at 80 V for 1 h in an ice bath. Next, the gel was photographed and analyzed via a Molecular Imager Gel Doc XR system (Bio-Rad Laboratories, Hercules, CA). To investigate the stability of pH-RE in serum, 1 μM pH-RE was mixed with FBS [10% (v/v)], reduced glutathione (GSH), Cys, H2O2, and ClO− in 1× TAE-Mg buffer and incubated for different periods (0, 1, 2, 3, 4, 5, 6, 7, and 8 h) at 37 °C. Finally, 5% N-PAGE analysis was carried out. Fluorescence measurement in vitro In this system, in the presence of pHe and PTK7 receptors of the CCRF-CEM cell, the cF and cS strands were released from corresponding recognition modules and then acted as secondary-inputs for logic computation. Therefore, the feasibility of the logic gate design was first investigated in buffer via mixing free cS and free cF with pH-RE. The mixtures were kept at 4 °C for 60 min to study the response of the logic computation. All fluorescence measurements were performed on a fluorescence spectrophotometer (Hitachi F-7100, Tokyo, Japan), and the fluorescence emission spectra were recorded from 655 to 750 nm at an excitation wavelength of 633 nm. All experiments were repeated at least three times. Flow cytometry assay Generally, 2 × 105 CCRF-CEM or Ramos cells were incubated with pH-RE (250 nM) in 200 μL of binding buffer (pH 7.4, 6.5, or 5.0) on ice for 30 min in the dark. The cells were then washed three times with washing buffer and resuspended in 200 μL of binding buffer (pH 7.4, 6.5, or 5.0). Finally, all samples were subjected to flow cytometric analysis using a flow cytometer (Gallios, Beckman Coulter, Brea, CA). To validate the precise recognition of CEM from cell mixtures, CEM cells (1 × 105) were first prestained with 5 μg/mL of Hoechst-33342 for 5 min and then were washed three times with 500 μL of washing buffer. Next, the pretreated CEM cells were mixed with an equal amount of Ramos cells in 200 μL of binding buffer (pH 7.4, 6.5, or 5.0). The flow cytometry assay was then carried out according to the above-described method. The target cell-recognition accuracy was calculated as Q2/(Q1 + Q2). Cell imaging In total, 5 × 103 cells were plated in a 20-mm confocal dish and incubated with 250 nM pH-RE in binding buffer with different pH values (pH 7.4, 6.5, or 5.0) on ice for 30 min in the dark. After being washed three times with washing buffer, the cell images were acquired on a Leica TCS SP8 confocal laser scanning fluorescence microscope (Wetzlar, Germany) with a 63× oil immersion objective. Cy5 fluorescence was collected in the red channel with 633-nm excitation, and the Hoechst-33342 fluorescence image was collected in the blue channel with 405-nm excitation. In vivo and ex vivo fluorescence imaging The BALB/c-nu mice received a subcutaneous injection of 5 × 106 CEM cells (or Ramos cells) in their right shoulders. When the tumors grew to 0.8–1.2 cm in diameter, the mice were intravenously administrated 200 μL of 1× TAE-Mg buffer containing 0.5 nmol of pH-RE and 5 nmol of random oligonucleotides. Then, time-lapse fluorescence imaging was monitored on an IVIS Lumina II in vivo imaging system (Caliper Life Science, Waltham, MA). A 640 nm bandpass filter was chosen as the excitation filter, and a 680 nm bandpass filter was used as the emission filter. For ex vivo tissue fluorescence imaging, the mice of different groups were euthanized 30 min after injection. Then tumors and normal tissues were harvested and imaged by the IVIS Lumina II in vivo imaging system. Results and Discussion Design and characterization of pH-RE To ensure that the biocomputing procedure of biomarker-recognition/data-process/output-engendering could be actualized within single-step behavior, pH-RE was assembled by controlled organizations of a pHe-recognition module (i-motif/cF), a receptor-recognition module (sgc8c/cS), and a report module (R/S/F) on a DNA TP scaffold (Figure 1a). As the skeleton of pH-RE, DNA TP was first constructed via hybridization among 96-base strands of S1, S2, and S3 (lane 7, Figure 1b), which was verified by gradually slow migration of one single band following the addition of different strands. Then, three functional modules were loaded onto DNA TP by Watson–Crick hybridization to achieve pH-RE. The successful construction of pH-RE was demonstrated by 5% PAGE imaging analysis, with another five different nanostructures for reference (lane 6, Figure 1c). Dynamic light scattering (DLS) analysis showed that the hydration radius increased from 19.2 to 25.9 nm after the assembly of the three modules to the DNA scaffold (Figure 1d). Moreover, the atomic force microscopy (AFM) image indicated that pH-RE appeared to be monodisperse with a height of about 2.86 nm in the dried state, further demonstrating the successful preparation of pH-RE (Figure 1e). Figure 1 | Construction and characterization of pH-RE. (a) Structural diagram of pH-RE. (b) 5% PAGE imaging analysis of stepwise assembly of DNA TP scaffold. (c) 5% PAGE imaging analysis of pH-RE. (d) DLS analysis of DNA TP and pH-RE. (e) Characterization of pH-RE by AFM. Line profile of a cross section the straight lines drawn in the AFM imaging. Download figure Download PowerPoint Performance of pH-RE in vitro Next, we investigated whether i-motif/cF and sgc8c/cS modules could recognize their targets, since the hybridization between FNAs and their complementary DNAs (cDNAs) might impact their response abilities. To explore the pHe-response of i-motif/cF module (2.5 μM), circular dichroism (CD) spectra were documented, as presented in Figure 2a. According to previous literature,41,42 the CD spectral profile characterized with a positive band centered at ∼285 nm and a negative band centered at ∼253 nm indicates the formation of i-motif structures. As illustrated in Figure 2c, the i-motif/cF module keeps steady as a double-helix structure at physiological pH with the lower ellipticity at 285 nm. Upon pH changing from 7.4 to 5.0, an indisputable red shift of both negative and positive bands and increased ellipticity were observed for i-motif/cF module, which was consistent with the free i-motif ssDNA ( Supporting Information Figure S1). This result implies that the pHe-recognition module (i-motif/cF) can be sensitively activated by shifting the initial equilibrium to pH-dependent displacement of cF strand. Figure 2 | (a) Schematic illustration of pH-dependent displacement of cF strand from i-motif/cF module. (b) Schematic illustration of PTK7-activated release of cS-BHQ1 from sgc8c-FAM to allow fluorescence reinstatement. (c) CD spectra of pHe-recognition module (2.5 μM) at different pH values. (d) The identification ability of receptor-recognition module (250 nM) for PTK7. (e) Fluorescence changes of logic computation of pH-RE. (f) Stability analysis of pH-RE and sgc8c ssDNA. Download figure Download PowerPoint Then, to test the receptor-activatable ability of the sgc8c/cS module (250 nM), the flow cytometry analysis was carried out by employing fluorescein (FAM)-labeled sgc8c aptamer and black hole quencher-1 (BHQ1)-labeled cS strand (Figure 2b). The switch-on performance of the receptor-recognition module was investigated using a PTK7-positive cell line, human acute lymphoblastic leukemia CCRF-CEM cells, while human Burkitt’s lymphoma Ramos cells barely expressed the protein used as negative controls. Even incubated with Ramos cells, the stable hybridization between sgc8c and cS brought the FAM and BHQ1 into close proximity, effectively quenching FAM fluorescence ( Supporting Information Figure S2). When CCRF-CEM cells were introduced, the receptor-recognition module transformed its conformation from sgc8c/cS conjugation to sgc8c/PTK7 complex, followed by cS strand releasing and fluorescence reinstating (Figure 2d). These results indicated that the FNAs maintained their functions after forming i-motif/cF and sgc8c/cS complexes. Furthermore, the performance of the AND logic computation of pH-RE in vitro was investigated by directly mixing free cF and cS strands with pH-RE, since cDNAs released from recognition modules act as secondary-inputs to interact with report module for signal output. As shown in Figure 2e, obvious gated fluorescence is observed, which operates in a classic “dual-keys-and-locks” manner. In addition, the stability of pH-RE was explored before being used in complexed systems, since the ideal cancer diagnosis and therapy system can resist nuclease degradation and protein interference during blood circulation. First, DLS analysis was performed to investigate the stability of pH-RE. As shown in Supporting Information Figure S3, pH-RE showed excellent stability in 1× TAE-Mg (pH 7.4) at 25 °C for Then, the gel electrophoresis analysis was carried out. with the sgc8c a and single band of pH-RE indicated its stability after 8 h in 10% FBS (Figure μM ( Supporting Information Figure 200 μM ( Supporting Information Figure μM ( Supporting Information Figure and μM ClO− ( Supporting Information Figure a similar result was also observed by fluorescence assay ( Supporting Information Figure The of the nanorobot was also the cell assay results ( Supporting Information Figure DNA-based pH-RE of in CCRF-CEM cells, which might be to the of by these we that pH-RE possesses the precise logic operation and stability in living systems as as excellent to the for improved accuracy in the of tumor imaging in pH-RE for in vitro precise target tumor cell identification We pH-RE could accurate tumor ability in CCRF-CEM and Ramos cells in buffers with different pH were used to the logic operation of pH-RE. The flow cytometry analysis showed that Ramos cells with pH-RE at pH 7.4, 6.5, or were with fluorescence (Figure and Supporting Information Figure In CCRF-CEM cells at pH and were sensitively and by pH-RE according to an obvious Cy5 while significant fluorescence signal was observed for at pH 7.4 (Figure and Supporting Information Figure These results that pH-RE can the binding to cells that protein receptors at pH Moreover, a of control were designed by changing the of recognition modules to further the operation mechanism of pH-RE ( Supporting Information Figure As the first i-motif in pH-RE was with to The showed on target cells, the of i-motif for extracellular milieu response (Figure As the second some of the aptamer was to random The resulting target tumor cells, that the membrane receptor-activatable ability for pH-RE was to the sgc8c aptamer (Figure As the two recognition modules were also to target cells since the structure (Figure Furthermore, the confocal microscopy images also red fluorescence on the membrane of target tumor cells with pH-RE, which was CEM in milieu (Figure Moreover, in experiments, a emission for target CEM cancer cells with control was observed ( Supporting Information Figure flow and cell imaging results that pH-RE has the potential to recognize target tumor cells with Figure | Flow cytometry assay of of pH-RE for (a) Ramos and (b) CCRF-CEM at different pH values. (c) The of pH-RE and control for CCRF-CEM cells at pH (d) images of two different cell lines with pH-RE at different buffers (pH 7.4, 6.5, and 5.0). 10 Download figure Download PowerPoint Next, we whether pH-RE could recognize target tumor cells in mixed cell a that cell was chosen to CEM cells. After the prestained CEM cells were mixed with an equal amount of Ramos cells in buffers with different pH values for the recognition test ( Supporting Information Figure As presented in the CEM cells in the buffer showed Cy5 fluorescence signal that in another the buffer. The recognition accuracy was calculated by the of cells with fluorescence both from Cy5 and over the recognition accuracy was to be for CEM in an that for CEM at physiological pH was only The high for pH-dependent of target cells was also by laser scanning confocal microscopy (Figure these results the high and of pH-RE for identification and of target tumor in cell Figure 4 | of the accuracy of pH-RE for target cells from similar cell mixtures at (a) pH 5.0, (b) pH 6.5, and (c) pH The target tumor cell-recognition accuracy was as Q2/(Q1 + Q2). Download figure Download PowerPoint Accurate in vivo tumor imaging by pH-RE by the performance of pH-RE in a of cells, the potential application of pH-RE in vivo was using a PTK7-positive CCRF-CEM CCRF-CEM mice were first with pH-RE by injection at the tumor site and normal As shown in Supporting Information Figure a fluorescence signal was observed at the tumor site min and the imaging signal was obtained at min with tumor and normal tissues of In fluorescence was observed at the site of normal tissue with pH-RE. These results that pH-RE can be activated by the decreased pHe and membrane receptors on CEM tumors in Figure 5 | fluorescence images of the mixture of CCRF-CEM and Ramos cells at different pH 6.5, and 7.4) after with pH-RE. 10 Download figure Download PowerPoint We then the ability of the system to target tumors in vivo pH-RE was and was used as a control In addition, Ramos mice were as the negative control After 10 min via the pH-RE was activated in the CEM tumor the of pH-RE was observed 30 min after injection with a high fluorescence of and fluorescence signal with lower was in corresponding tumor for both control groups and These results that pH-RE can be activated to target tumors in vivo for cancer Moreover, according to the fluorescence signal of blood the of pH-RE in is min ( Supporting Information Figure Figure | (a) In vivo time-lapse fluorescence imaging of different mice after injection with different tumor are indicated by red CEM tumor + pH-RE. CEM tumor + Ramos tumor + pH-RE. (b) fluorescence as a of after (c) vivo fluorescence images of and (d) images of the corresponding tumor from mice of different pH-RE and in 25 Download figure Download PowerPoint To the of pH-RE, mice were euthanized 30 min after and the tumors and were harvested for ex vivo imaging As shown in Figure a fluorescence in the CEM tumor of the mice with pH-RE was observed with the two other control further demonstrating to target cancer in The was also observed by confocal images of corresponding tumor (Figure the results that pH-RE can in vivo cancer diagnosis with high accuracy by logic of extracellular milieu and membrane To further investigate the of the nanorobot, the and of the mice were monitored after being with 0.5 nmol of pH-RE. As

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