Y-Shaped Circular Aptamer–DNAzyme Conjugates for Highly Efficient in Vivo Gene Silencing
Kaixiang Zhang, Yanan Li, Junjie Liu, Xue Yang, Yuanhong Xu, Jinjin Shi, Wei Liu, Jinghong Li
Abstract
Open AccessCCS ChemistryRESEARCH ARTICLE1 Dec 2020Y-Shaped Circular Aptamer–DNAzyme Conjugates for Highly Efficient in Vivo Gene Silencing Kaixiang Zhang†, Yanan Li†, Junjie Liu, Xue Yang, Yuanhong Xu, Jinjin Shi, Wei Liu and Jinghong Li Kaixiang Zhang† School of Pharmaceutical Sciences, Key laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Zhengzhou University, Zhengzhou 450001 Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084 , Yanan Li† School of Pharmaceutical Sciences, Key laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Zhengzhou University, Zhengzhou 450001 , Junjie Liu School of Pharmaceutical Sciences, Key laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Zhengzhou University, Zhengzhou 450001 , Xue Yang School of Pharmaceutical Sciences, Key laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Zhengzhou University, Zhengzhou 450001 , Yuanhong Xu College of Life Sciences, Qingdao University, Qingdao 266071. , Jinjin Shi *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] School of Pharmaceutical Sciences, Key laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Zhengzhou University, Zhengzhou 450001 , Wei Liu *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] School of Pharmaceutical Sciences, Key laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Zhengzhou University, Zhengzhou 450001 and Jinghong Li *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084 https://doi.org/10.31635/ccschem.020.202000170 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Oligonucleotide drugs have been used widely as therapeutic agents for gene therapy, while their instability in biological media and inefficiency for intracellular delivery remain major hurdles for practical in vivo applications. Herein, we report a circular Y-shaped aptamer–DNAzyme conjugate (cYAD) for highly efficient in vivo gene silencing via RNA cleavage, which can been employed in various disease treatments, including cancer, inflammation, as well as viral infections. Systematic studies revealed that cyclization of the DNA structure could improve the stability of oligonucleotide drugs in vivo. Besides, the bivalent aptamer motifs provided a specific and enhanced tumor cell targeting ability for accumulation and retention of the oligonucleotide drugs at the tumor site. As a proof of concept, a widely applicable Na+-dependent fluorescent sensor, NaA43 DNAzyme, was used to inhibit MET gene expression in mice tumor model tissues, which exhibited highly efficient gene silencing performance in vivo, which confirmed our findings with cYAD. This strategy provides a novel approach for the construction of oligonucleotide drugs for practical therapeutic applications. Download figure Download PowerPoint Introduction Oligonucleotide therapeutics are emerging as promising platforms for gene therapy.1–4 As a powerful method to manipulate the gene expression level in cells, multiple types of oligonucleotide drugs such as siRNA5, antisense oligonucleotides,6 and DNAzymes7 have been utilized for the treatment of diseases ranging from viral infections8 to hereditary disorders9 and cancers10. However, inefficient delivery of oligonucleotides to their intracellular disease sites for action remains a major hurdle for their practical applications.11 Ligand–oligonucleotide conjugation is regarded as a favorable strategy for enhancing the delivery of oligonucleotide drugs.12 This approach produces a well-defined, single-component molecular gene therapy drug by conjugating equal amounts of delivery molecules and oligonucleotides to achieve enhanced and targeted oligonucleotides delivery.13 For example, a fusion-oligopeptide gene carrier has been designed for targeting nonviral gene delivery to adipocytes for treatment of obesity and obesity-induced metabolic syndromes.14 In another case, a nucleoli-specific aptamer was conjugated with antisense oligonucleotides to modulate the RNA splicing process in different cancer cells.15 However, these conjugates were still susceptible to exonucleases in the blood plasma in vivo, which affected their in vivo applications severely. Recently, a nuclease-resistant circular bivalent aptamer system was developed to achieve high stability and high tumor cell targeting capabilities in vivo.16 Aptamers are single-stranded oligonucleotides that possess sequence-dependent binding ability for specific molecular targets, thereby, discriminate the molecular signatures between normal cells and cancer cells.17–21 Besides, aptamer molecules are nonimmunogenic and could be synthesized readily and modified.22 It has been demonstrated that by ligation of two linear aptamer-based oligonucleotide sequences, the resultant ligated circular bivalent aptamer showed improved thermal and physical stability in biological media and exhibited enhanced tumor cell targeting ability in vivo.23 However, the therapeutic potential of this circular aptamer structure for the delivery of oligonucleotide drugs for gene therapy has not yet been demonstrated. Among all the oligonucleotide drugs, DNAzymes, which are single-stranded DNA molecules, are able to catalyze the cleavage of mRNA, and have become powerful therapeutic agents for gene silencing.24 In comparison with siRNA and antisense oligonucleotide, DNAzymes hold strong enzymatic turnover properties and do not need to perform through the formation of the typical endogenic RNA-induced silencing complex (RISC).25 However, their low-efficiency cellular uptake, instability, and insufficient cofactor supply still limit the development of DNAzymes for clinical applications. In this work, we integrated a modified NaA43 DNAzyme sequence into two cell-specific aptamer-DNA structures (Sgc8), and ligated to construct a circular Y-shaped aptamer–DNAzyme conjugate (cYAD) to achieve high in vivo stability, highly tumor cell targeting ability, and high in vivo gene silencing efficiency, attributable to the following features: (1) The aptamer motif guaranteed tumor cell-specific targeting. (2) Since we used a Na+-dependent DNAzyme26 to construct the aptamer conjugate, and because there is always adequate concentration of cellular free Na+ ion in almost all cell-types, it facilitated the catalytic domain of NaA43 to perform efficiently, leading to high intracellular catalytic RNA cleavage activity in the targeted tumor. (3) The circularization of the conjugated aptamer to generate cYAD made the structure a more stable Na+-dependent circular aptamer-DNAzyme complex. Experimental Methods Experimental Methods are available in the Supporting Information. Results and Discussion The design of cYAD is shown in Scheme 1. Specifically, two cell-specific DNA aptamers, (2 X Sgc8), and one NaA43 DNAzyme were self-assembled with three linker sequences ( Supporting Infomation Table S1). All the sequences had 5′-phosphate group and 3′-OH group. After annealing and cooling down to room temperature, the sequences could hybridize with each other and self-assembled to form a Y-shaped structure with six nicks. Then we achieved the circular structure by using T4 DNA ligase to seal the six nicks. Thus each cYAD structure had two aptamer domains for tumor cell targeting and one DNAzyme domain for gene silencing. The cyclization of the oligonucleotide structure guaranteed stability of Sgc8 and DNAzyme in the biological system. When cYAD was injected into the tumor-bearing mice through tail vein, the bivalent Sgc8 aptamer structures in cYAD drove specific tumor cell targeting and PTK7 receptor-mediated endocytosis process. Then the DNAzyme structure boosted targeted gene silencing, aided by the adequate cytoplasmic Na+ ion supply to trigger tumor cell apoptosis, with the subsequent realization of gene therapy. Scheme 1 | Design of circular Y-shaped aptamer–DNAzyme conjugate (cYAD) for in vivo molecular gene silencing. (a) Preparation of cYAD. Two Sgc8 aptamers and one NaA43-Y DNAzyme were self-assembled with DNA linkers to form a Y-shaped conjugate. The circular structure of cYAD was prepared by sealing the six nicks in the conjugate with T4 DNA ligase to improve the stability of the DNA structure. (b) When injected into the tumor-bearing mice through tail vein, the cYAD underwent the following steps: (I) specific tumor cell targeting driven by bivalent Sgc8 aptamer, (II) PTK7 receptor-mediated endocytosis, (III) DNAzyme-based boost cleavage of target mRNA by virtue of the supply of adequate Na+ inside cells, (IV) induction of tumor cell apoptosis by gene silencing. Download figure Download PowerPoint The synthesized cYAD was characterized initially by agarose gel electrophoresis (Figure 1). By carefully evaluating the annealing conditions and lengths of the complementary sequences, we achieved highly efficient and controlled preparation of cYAD.27 As shown in Figure 1b, different component sequences of cYAD (Linker 1, 2, 3, Sgc8, and NaA43-Y) were added step by step, and a gradual increase in the molecular weight was observed. Notably, the size of the final assembled structure was as expected (∼ 160 bp), suggesting that cYAD was assembled as design. By semiquantifying the intensity of the main band and other residual bands of the gel, the percentage of assembled cYAD was estimated to be ∼ 91%. As a comparison to demonstrate the bivalent binding property of the Y-shaped conjugates, we synthesized a dumbbell-like structure (cDAD) in parallel (Figure 1a), where the Sgc8 sequence was linked directly to the NaA43-D sequence, and the self-assembly process was also characterized by agarose gel electrophoresis (Figure 1c). Figure 1 | Design, construction, and characterization of cYAD and cDAD. (a) Design of cDAD and cYAD. Detailed sequence information is provided in Supporting Information Table S1. (b) and (c) 3% Agarose gel electrophoresis analysis of the self-assembly process of (b) cYAD and (c) cDAD. (d) and (e) Stability analysis of Sgc8, NaA43-Y, (d) cYAD, and (e) cDAD after incubation with or w/o 0.25 U/μL exonuclease I (Exo I) for 1 h, determined by 3% agarose gel electrophoresis . The stability of DNA sequence gets significantly improved by forming a closed circular structure. (f) Stability analysis of Sgc8, cDAD, and cYAD after incubation with DMEM-10% FBS at different times, analyzed by 3% agarose gel electrophoresis. (g) Normalized semiquantitative analysis of (f). Download figure Download PowerPoint The six nicks held by the self-assembled cYAD needed nick-closing reaction to from the circular structure. For this cyclization reaction, T4 DNA ligase was chosen to achieve a highly efficient nick closing.28 Under optimized conditions, the efficiency of each ligation reaction was ∼ 95%.29 In particular, for the cYAD synthesis, since there were six nicks to be sealed, the theoretical ligation efficiency was ∼ 73%. However, by analyzing the band intensity in Figure 1d, the estimated synthesis efficiency of cYAD was ∼ 51%. Subsequently, we investigated the effects of cyclization on the stability of cYAD in biological medium (Dulbecco's Modified Eagle Medium; DMEM). As shown in Figures 1d and 1e, the Sgc8 or the DNAzyme sequences (NaA43-D for cDAD and NaA43-Y for cYAD) could be degraded readily after incubation with 0.25 U/μL exonuclease I for 1 h. However, the cDAD and cYAD structures resisted the cleavage of exonuclease I and maintained their sequence integrity under the same reaction conditions. Also, we tested the stability of both structures in DMEM containing 10% fetal bovine serum (FBS). As shown in Figure 1f, the band of the linear aptamer, Sgc8, quickly disappeared after incubation with DMEM-10% FBS for 1 h. However, for cDAD and cYAD, even after incubation in DMEM-10% FBS for 24 h, the integrity of each conjugate was still evident. By semiquantification of each band intensity (Figure 1g), it was apparent that the stability of cDAD and cYAD was significantly improved in the biological media when compared with the counterpart linear sequences. To further demonstrate that the improved stability was due to the formation of the circular structure but not the self-assembly process, we performed another experiment to compare the stability of cYAD and cDAD with their corresponding noncircular control fabrications (ncYAD and ncDAD) in which the oligonucleotide sequences were self-assembled to form Y-shaped or dumbbell-like structure, but no ligation reaction was performed to obtain circular complexes. As shown in Supporting Information Figure S1, ncYAD and ncDAD showed much less stability than cYAD and cDAD, further demonstrating that the stability stemmed mainly from the cyclization effect. To integrate the NaA43 DNAzyme sequence into cDAD and cYAD, we had to modify the NaA43 DNAzyme sequence from its original design (NaA43-D for cDAD and NaA43-Y for cYAD), as listed in Supporting Information Table S1, followed by performing experiments to establish that the DNAzyme cleavage activity of cYAD was still maintained for gene silencing application. We performed a strategic fluorescent assay to investigate the DNAzyme cleavage efficiency (Figure 2a).30 The principle was that when the DNAzyme was activated, it would cleave the substrate to generate fluorescent signal. On the other hand, when the DNAzyme remained inactivated, the uncleaved substrate would bring the fluorophore- and quencher-labeled sequence of the molecule together, and consequently, quench the fluorescence. Thus, the assay could distinguish between the performances of our testing cYAD and the control cDAD. Favorably, the cYAD design achieved separation of the cleavage reaction and the succeeding fluorescence detection process, thus leading to ultralow background and enhanced sensitivity.30 We used this assay initially to test the cleavage kinetics of the prototype DNAzyme (NaA43). As shown in Figure 2b, 200 nM DNAzyme was able to cleave 200 nM substrate in the presence of 10 mM Na+ ion in 15 min, consistent with a previous report.26 Denaturing polyacrylamide gel electrophoresis (PAGE) analysis also confirmed that after 15 min incubation, almost all the substrates were cleaved, while the DNAzyme sequences still maintained its structure (Figure 2c). Figure 2 | Analysis of the mRNA cleavage ability of NaA43 DNAzyme, cYAD, and cDAD. (a) Schematic diagram of a fluorescence test for analyzing DNAzyme cleavage activity. Four strands were included in this test: DNAzyme strand, unlabeled Sub M, and 2 single-labeled substrates, Sub F and Sub Q. Uncleaved Sub M would result in fluorescence quenching. If the Sub M is cleaved, there won't be quenching effect and a fluorescent signal would be monitored. (b) Analysis of the cleavage kinetics of NaA43 DNAzyme. 200 nM NaA43 DNAzyme was incubated with 200 nM Sub M. The Na+ concentration was adjusted to 10 mM. (c) Denaturing PAGE analysis of NaA43 cleavage. After 15 or 30 min incubation of DNAzyme and Sub M, the Sub M band disappeared and only the DNAzyme band was left. (d) Fluorescence-based assay for analyzing the mRNA cleavage activity of NaA43, NaA43-Y, cYAD, NaA43-D, and cDAD. Data are presented as mean ± SD (n = 3). Statistical analysis: ***p < 0.001. (e) Denaturing PAGE analysis of the mRNA cleavage ability of cYAD, NaA43-Y, cDAD, NaA43-D, and NaA43. Download figure Download PowerPoint Then we used this developed fluorescent assay to evaluate the cleavage activity of our fabricated circular DNAzymes and their linear counterparts. As shown in Figure 2d, 200 nM of NaA43 was able to cleave ∼ 50% of 2 μM substrate in the presence of 10 mM Na+ ion in 60 min, while the modified NaA43-Y DNA showed significantly higher cleavage activity. The conjugated circularized cYAD DNAzyme sequence was able to catalyze a substrate cleavage up to ∼ 95%. Although the DNAzyme designed sequence for cDAD (NaA43-D) showed similar activity as the original DNAzyme (NaA43), interestingly, when sealed to generate circular cDAD, the cleavage activity was significantly higher than before ligation (Figure 2d). We inferred that the modification to the circular structure kept the sequence in a more confined conformation, which led to the higher cleavage activity. Denaturing PAGE gel electrophoresis was used to confirm the cleavage of substrates using DNAzymes in different forms (Figure 2e). According to the results, the modified DNAzyme sequences in cYAD or cDAD maintained their high mRNA cleavage activity, and thus, could be applied in efficient gene silencing reactions. Then the cell-binding affinity, endocytosis process, and lysosome escape property of cYAD and cDAD were tested using cervical cancer, HeLa cells. It has been characterized that HeLa cells had high protein tyrosine kinase-7 (PTK-7) expression for Sgc8 aptamer binding.31 Specifically, linear Sgc8 aptamer, NaA43 DNAzyme, cYAD, and cDAD were modified with the fluorescein amidites (FAM) dye in the middle of their sequences on the thymine base (T), essentially, for visualization (see Supporting Information Table S1). Then the modified aptamers and their linear counterparts were each added to HeLa cells, seeded in 96-well culture plate (5 × 104 cells/well) grouped into 1–5, incubated in DMEM at 37 °C for 4 h) and prepared for fluorescent-based cell-binding affinity assays by confocal microscopy and flow cytometry to confirm the specificity of our conjugated aptamer construct ( Supporting Information Figure S2). The experimental set up groups were as follows: Group 1: FBS-free DMEM, used as control, Group 2: FAM-labeled Sgc8, Group 3: FAM-labeled NaA43 DNAzyme, Group 4: FAM-labeled cDAD, Group 5: FAM-labeled cYAD. We observed that when the HeLa cells were incubated with serum-free DMEM, FAM-labeled Sgc8, cDAD, and cYAD, these four groups demonstrated similar cell-binding affinity on HeLa cells, while the FAM-labeled NaA43 DNAzyme did not show obvious fluorescent signals, demonstrating that the oligonucleotide drug without a delivery strategy could not be taken up efficiently by the cells, contrary to the purpose of the gene silencing application. However, when incubated with complete DMEM-10% FBS cell medium, cYAD and cDAD showed high cell-binding affinity to HeLa cells after 36 h incubation, while the linear Sgc8 aptamer sequence showed diminishing signal strength over time (Figures 3a and 3b). This might be because the linear Sgc8 aptamer sequence had less stability in 10% FBS. Besides, the bivalent aptamer structure in cYAD slightly enhanced the binding affinity of the DNA conjugates to HeLa cells than cDAD in DMEM-10% FBS (Figure 3b). This phenomenon was consistent with the literature report that a bivalent aptamer was able to induce enhanced intracellular cargo delivery than monovalent aptamer.23 Figure 3 | Cellular uptake and lysosome escape processes of cYAD and cDAD in HeLa cells. (a) Confocal laser scanning microscopy (CLSM) imaging of HeLa cells treated with 200 nM FAM-labeled Sgc8 aptamer, cDAD, and cYAD in cell culture media containing 10% FBS for 4 h. Scale bar: 25 μm. (b) Flow cytometry assay of HeLa cells treated with 200 nM FAM-labeled Sgc8, cDAD, and cYAD for different time. (c) Intracellular localization analysis of cDAD and cYAD in HeLa cells. LysoTracker was used to stain the lysosomes (Red). The spatial colocalizations of cDAD and cYAD were statically analyzed by Image J. Scale bar: 10 μm Download figure Download PowerPoint Further, we performed endocytotic study using a dynamic fluorescence microscopy with the FAM-labeled cYAD above, using HeLa cells and a cell-type control, human lung carcinoma A549 cells, which had low PTK7 expression.32 Compared with the HeLa cells, when the FAM-labeled cYAD was incubated with the A549 cells (with low PTK-7 expression), there was no observable endocytosis of the cYAD ( Supporting Information Figure S4); however, a profound endocytotic process was observed with the HeLa cells, indicating the role of the Sgc8 moiety of cYAD in targeting and binding to expressed PTK7 in the HeLa cells. To further study the internalization process of cYAD and cDAD and investigate whether they can escape the lysosome, we labeled the lysosomes in HeLa cells with LysoTracker and incubated with cYAD and cDAD to monitor the internalization process at different time intervals. As shown in Figure 3c, the LysoTracker signal (red) was strong at the beginning and became weak after 4 h or 8 h incubation, indicating that the lysosome structure might have been degraded during the incubation process. Interestingly, we observed that, after 8 h incubation, FAM-labeled cDAD (green) still colocalized well with the LysoTracker signal (red), giving yellow color in the cytoplasm. While after the same 8 h incubation, the cYAD green signal spread widely in the cytoplasm and did not show much colocalization with the red signal, suggesting that the Y-shaped conjugates were more capable of escaping easily from the lysosome and entering the cytosol (Figure 3c). Then we tested the gene silencing ability of cYAD and cDAD for the inhibition of MET gene expression.33MET gene is an important proto-oncogene, encoding tyrosine kinase MET, which is a receptor for hepatocyte growth factor/scatter factor (HGF/SF).34 Aberrant MET gene activation occurs in many types of cancers, affecting lots of intracellular signaling cascades, including PI3K-AKT, RAC1, RAP1, and RAS-MAPK pathways.35,36 Clinical studies have confirmed that inhibition of MET gene expression had major therapeutic value for different solid human tumors.37 Therefore, we hypothesized that by silencing the expression of the MET gene, the tumor cell viability would decrease significantly. Thus to demonstrate the MET gene silencing ability of cDAD and cYAD in cells, plated HeLa cells were divided into three groups and each was incubated with the free NaA43 DNAzyme, cDAD, or cYAD, respectively, and the MET and protein were analyzed by (Figure As shown in Figure the free DNAzyme did not gene silencing mainly because the oligonucleotide drugs could taken up by the However, for cYAD and cDAD, which the Sgc8 aptamer sequence for tumor cell the MET gene silencing ability was compared with the control Besides, we that the gene silencing efficiency of cYAD was significantly higher than that of cDAD. According to the previous cellular uptake experiments (Figure this might be because cYAD was able to the cells and escape from the lysosome more The semiquantitative analysis (Figures and and the reaction (Figure showed similar indicating that cYAD efficient MET gene silencing Figure 4 | of the MET gene silencing of cDAD and cYAD in cells. (a) analysis of MET and expression in HeLa cells treated with nM NaA43 DNAzyme, cDAD, and cYAD (b) and (c) analysis of the (d) analysis of the expression of MET mRNA in HeLa cells treated with nM NaA43 DNAzyme, cDAD, and cYAD. (e) viability analysis of cells, A549 cells, and HeLa cells treated with different of NaA43, cDAD, and cYAD. (f) apoptosis analysis of HeLa cells incubated with nM NaA43, cDAD, and cYAD for h. and were used to stain the cells and flow cytometry was used for Data are presented as mean ± SD (n = 3). Statistical analysis: ***p < 0.001. Download figure Download PowerPoint Then the of cYAD and cDAD to induce tumor cell apoptosis was types of cell human normal and were used in the Specifically, cells have high PTK7 but low MET A549 cells have low PTK7 but high MET HeLa cells both PTK7 and The cellular uptake efficiency of cDAD and cYAD for and HeLa cells was investigated by flow cytometry ( Supporting Information Figure with the PTK7 expression cYAD and cDAD had high cell-binding affinity to and HeLa cells, but not with A549 cells. Notably, because of the inefficient cellular The gene silencing of cYAD and cDAD to A549 cells was also low ( Supporting Information Figure demonstrating a cell receptor specificity for gene silencing. Further, we performed cell viability studies with the and HeLa cell treated with free NaA43 DNAzyme, cDAD, and cYAD respectively, using the experimental set as The cell were by the cell As shown in Figure compared with cells with low MET expression or A549 cells with low PTK7 cYAD showed higher to HeLa cells with an value of ∼ Interestingly, cYAD, which showed even high cell-binding affinity to cells, was without to this normal cell mainly because the MET gene did not a role in the of the normal cells. This phenomenon demonstrated a specificity of cYAD for tumor therapy. the A549 cells could not up cYAD efficiently, there was still cell inhibition growth because of the MET gene silencing, leading to value of cYAD to A549 Subsequently, the cell apoptosis processes were analyzed by flow cytometry using the same experimental set up (Figure We that nM cYAD was able to induce ∼ HeLa cell apoptosis cells, which was much higher than the for and A549 cells ( Supporting Information Figure these demonstrated that cYAD had both cell receptor specificity and specificity for gene therapy, able to induce tumor cell apoptosis in a We tested the in vivo of cYAD and cDAD by the and of these DNA conjugates in tumor-bearing mice using an in vivo imaging system. We used this imaging to the of (1) cYAD and (2) cDAD, via of each mice in 1 and 2 (n = (Figure (3) free Sgc8 aptamer, free NaA43 DNAzyme, ncYAD and ncDAD were treated the same and used as We that cYAD and cDAD showed much signal in the tumor than the (Figure Figure | In vivo of cDAD and cYAD. (a) In vivo imaging for the analysis of dynamic of Sgc8, NaA43, cDAD, and cYAD in tumor-bearing mice over 24 h. (b) imaging of tumor and major and after with (c) analysis of fluorescence signal of (b) using (d) imaging of tumor with (e) analysis of fluorescence signal of (d) using Scale bar: μm Download figure