Versatile Catalytic Deoxyribozyme Vehicles for Multimodal Imaging

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Versatile Catalytic Deoxyribozyme Vehicles for Multimodal ImagingGuided Efficient Gene Regulation and Photothermal Therapy Jie Feng, Zhen Xu, Feng Liu, Yun Zhao, Wenqian Yu, Min Pan, Fuan Wang, and Xiaoqing Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b08101 • Publication Date (Web): 12 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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Versatile Catalytic Deoxyribozyme Vehicles for Multimodal Imaging-Guided Efficient Gene Regulation and Photothermal Therapy Jie Feng, Zhen Xu, Feng Liu, Yun Zhao, Wenqian Yu, Min Pan, Fuan Wang, Xiaoqing Liu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China Corresponding author email address: [email protected]

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ABSTRACT: Catalytic deoxyribozyme has great potential for gene regulation, but poor efficiency of the cleavage of mRNA and lack of versatile DNAzyme vehicles remain big challenges for potent gene therapy. By rational designing a diverse vehicle of polydopamine-Mn2+ nanoparticles (MnPDA), we demonstrate that MnPDA has integrated functions as an effective DNAzyme delivery vector, self-generation source of DNAzyme cofactor for catalytic mRNA cleavage, inherent therapeutic photothermal agent, as well as contrast agents for photoacoustic and magnetic resonance imaging. Specifically, the DNAzyme-MnPDA nanosystem protects catalytic deoxyribozyme from degradation and enhances cellular uptake efficiency. In the presence of intracellular glutathione, the nanoparticles are able to in-situ generate free Mn2+ as a cofactor of DNAzyme to effectively trigger catalytic cleavage of mRNA for gene silencing. In addition, the nanosystem shows high photothermal conversion efficiency and excellent stability against photothermal processing and degradation in complex environments. Unlike previous DNAzyme delivery vehicles, this vehicle exhibits diverse functionalities for potent gene regulation, allowing multimodal imaging-guided synergetic gene regulation and photothermal therapy both in vitro and in vivo.

KEYWORDS: deoxyribozyme; gene therapy; DNAzyme; photothermal therapy; multimodal imaging


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Gene therapeutics exploit various oligonucleotides that target pre-mRNA and mRNA to silence or inhibit disease-related gene expression.1-5 It has attracted increasing attention for the treatment of disease such as cancer, neurological disorder as well as infections.6,7 As one type of catalytic nucleic acids, DNAzyme is recognized as a promising therapeutic agent for selective modulate gene expression.8,9 DNAzymes are single-stranded DNA molecules generated via in vitro selection that can catalyze various reactions.10,11 The function of the catalytic activity is performed by recruiting metal ions as cofactors such as Pb2+, Zn2+, Mn2+ and Mg2+. Functioning as RNAcleaving oligodeoxyribozyme for suppression of gene expression, DNAzymes bind target mRNA by base pairing in a sequence-specific manner, and catalyze hydrolysis of target mRNA and removal of disease-causing gene product. A remarkable advantage of DNAzymes as gene inhibitors is their inherent catalytic property resulted from a reiterative process of targeted mRNA cleavage, as well as high specificity and low immunogenicity.12 Moreover, DNAzymes inhibit translation in a way independent of RNA interference. Unlike short interference RNA (siRNA) and antisense oligonucleotides, DNAzymes don’t require cellular enzymes (RNA-induced silencing complex) to recognize antisense compounds for RNA degradation.13 Another motivation of DNAzymes for gene regulation is the practical advantage of DNA relative to RNA, because DNAzyme is fairly short and relatively inexpensive, and they do not require expensive chemical modifications for in vivo use.8,9 In addition, DNAzyme has relatively high stability in serum, resistance to chemical and enzymatic degradation compared to RNA like siRNA, microRNA and ribozyme for gene


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inhibition.14 All the features make DNAzymes suitable and promising for gene regulation. Although DNAzymes have been employed to downregulate important genes, efficient in vivo catalytic cleavage remains the main issue to realize wide potential of DNAzyme-based therapeutics. Another key element restraints potent DNAzyme-based therapeutics is the lack of versatile DNAzyme vehicles with diverse functionalities besides delivery functionality, which is important towards precise cancer treatment with improved efficacy for broad clinical applications. First, while existing vehicles have been explored as a potential delivery system to transport DNAzyme,15-21 most of them suffer from effective delivery because of limited cellular uptake efficiency and lack of mechanisms to release sufficient DNAzyme, which hinders its catalytic results. Second, an insufficient amount of intracellular DNAzyme cofactors severely impedes in vivo catalytic cleavage for gene silencing, since the catalytic activity of DNAzyme is highly dependent on the DNAzyme cofactors of metal ions. It was reported that the most widely investigated 10-23 DNAzymes require at least 5-10 mM Mg2+ to form catalytic domain and enable efficient cleavage. Yet the concentration of free Mg2+ in mammalian cells is around 0.5 mM in living cells, or in the range from 0.2 to 2 mM, far below the level for efficient catalytic reaction.22 The DNAzyme cofactor of Zn2+ or Mn2+ can catalyze the reaction more efficiently, although free concentration of these ions is within picomolar to nanomolar range in living cells.14 It is clear that the lower cofactor content in living cells seriously limits in vivo cleavage efficiency of the DNAzyme. Third, the reported nanocarriers for DNAzyme delivery are inherent with


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no multi-functionality, to the best of our knowledge, while a diverse vehicle with integrated functions is critical for effective and precise cancer therapy. Therefore, overcoming these obstacles need new vehicles and approaches for improved performance of DNAzyme-based therapeutics. To address these challenges, we envisaged that an inherent therapeutic nanocarrier with DNAzyme-cofactor or multifunctional precursor as components can create potent DNAzyme vehicles for cancer therapy. Herein, we report on rational design of a diverse vehicle, polydopamine-Mn2+ nanoparticles (MnPDA), by incorporating multifunctional components through one-pot synthesis. The MnPDA facilities effective DNAzyme delivery, self-supplying DNAzyme factor and tumor microenvironment-stimulated release, for improved gene silencing and therapeutic efficacy both in vitro and in vivo. More importantly, the vehicle exhibits diverse functionalities besides gene regulation, enabling excellent synergetic gene-photothermal therapy and multimodal imagingguided therapy. Since precise cancer therapy with improved efficacy is the trends for cancer treatment,23-25 versatile DNAzyme vehicles with integrated functions are promising for potent gene regulation and would have broad clinical implications. RESULTS AND DISCUSSION Principle of the Nanosystem with Diverse Functionalities for Potent DNAzymeBased Therapeutics. As shown in Figure 1, the MnPDA was prepared via a facile onestep polymerization doping approach in aqueous solution, with dopamine as polymerization precursor and manganese (II) chloride as the Mn source. The


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DNAzyme-MnPDA nanosystem was developed by adsorption of a 10-23 DNAzyme26 onto the surface of MnPDA and utilization of folate-labeled DNAzyme (fol-DNAzyme) for targeted recognition and gene modulation. The delivery vehicle of MnPDA protects DNAzyme from intracellular degradation, supplies cofactor source of Mn2+ for DNAzyme catalysis, and exhibits excellent photothermal transduction efficiency. When the catalytic deoxyribozyme-MnPDA nanoparticles are specifically recognized by cancer cell and delivered into the cytoplasm, the nanosystem is stimulated by intracellular glutathione, releasing DNAzyme and concentrated free Mn2+ for catalytic cleavage of intracellular mRNA. Thus, the nanosystem improves cellular uptake efficiency, ensures in vivo stability and improves catalytic efficiency for gene regulation. In addition, this vehicle is capable of near-infrared photothermal imaging (IR), photoacoustic imaging (PAI) and magnetic resonance imaging (MRI), enabling multimodal imaging-guided synergetic gene-photothermal therapy in vivo. Synthesis and Characterization of MnPDA. The MnPDA nanoparticles were synthesized by using manganese (II)-catalyzed the polymerization of dopamine through a facile one-step reaction. During the reaction, the solution quickly turned from colorless into green upon adding MnCl2, and then became black, indicating the formation of the nanoparticles. Scanning electron microscopy (SEM) showed that the as-prepared MnPDA had a spherical shape with a diameter of 300 nm (Figure 2A), consisted with the dynamic light scattering (DLS) result of 314 nm. The presence of Mn element in the MnPDA nanoparticles was proved by the scanning electron microscopy-energy dispersive X-ray (SEM-EDX) element mapping and spectroscopy


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analysis (Figure 2 A, B), which was also confirmed by inductively coupled plasma mass spectrometry (ICP-MS) analysis revealing the Mn content in MnPDA was about 6.72%. Besides, DLS analysis showed that the zeta potential of MnPDA nanoparticles increased from -35.2 to -22.7 mV compared with PDA nanoparticles (Table S1). The positive shift indicated the presence of positively charged manganese ions within the nanoparticles. In order to analyze the chemical states of manganese, X-ray photoelectron spectroscopy (XPS) was carried out. It was found four peaks at 284.79, 532.39, 399.87 and 641.24 eV in Figure S1, corresponding to the characteristic peaks of C 1s, N 1s, O 1s and Mn 2p, respectively. As shown in Figure 2C, there were four peaks in primary XPS spectrum of Mn 2P, in which the peaks at 641.2 and 653.4 eV belonged to Mn 2P2/3 and Mn 2P1/2 respectively, and the peaks at 645.5 eV and 657.4 eV were the corresponding shake-up peaks. In addition, XPS spectrum of Mn 3S split into two peaks at 83.4 eV and 89.4 eV (Figure 2D). These results indicated that the majority of Mn element was in the form of Mn (II) for MnPDA nanoparticles.27 The possible structure and chemical bond between Mn and PDA were shown in Figure S2. Stability and Stimulated Catalytic Cleavage of DNAzyme by DNAzymeMnPDA Nanosystem. For the application of MnPDA as a gene delivery material, 1023 DNAzyme was chosen as a model catalytic deoxyribozyme to construct DNAzymepolydopamine nanosystem for gene regulation. Fluorescence spectrum and DLS analysis were used to confirm the successful loading of fol-DNAzyme onto the surface of MnPDA. In detail, the fluorophore modified nucleic acid, FAM-fol-DNAzyme, was incubated with the MnPDA to obtain FAM-fol-DNAzyme-MnPDA nanoparticles. As


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expected, the fluorescent intensity of FAM-fol-DNAzyme was gradually quenched by MnPDA (Figure 3A and Figure S3). The amount of the absorbed DNAzyme on MnPDA was calculated to be 1 and 5 pmol/μg, according to respective fluorescence and absorbance measurements. Besides, after fol-DNAzyme absorption, the zeta potential for fol-DNAzyme-MnPDA was more negative than that of MnPDA (-27.8 mV versus -22.3 mV). We supposed that the main reason for the high DNAzymeloading capability of the nanosystem was due to the - interaction between oligonucleotides and PDA.28,

29

Stability of DNAzyme in biofluids is an essential

requirement for their clinical application, especially in gene therapy. Accordingly, we tested the stability of the catalytic deoxyribozyme by monitoring fluorescence intensity of FAM-fol-DNAzyme subjected to nuclease, phosphate-buffered saline (PBS) as well as cell culture medium (Figure 3B). Kinetics of the fluorescence intensity for folDNAzyme-MnPDA was recorded in the presence of DNase I, and no obvious fluorescence changes were observed. This showed that the DNAzyme absorbed on the surface of MnPDA could resist against degradation by the nuclease. At the same time, the fol-DNAzyme-MnPDA also exhibited excellent stability in PBS or DMEM containing 10% FBS. These results demonstrated that fol-DNAzyme-MnPDA had a promising potential in complex biological system for gene therapy. For efficient gene silencing, the stimuli-responsive release of DNAzyme and cofactors are preferred. We found that GSH, which was overexpressed in cancerous cells,30 could lead to the release of both FAM-fol-DNAzyme and Mn2+ ions from the MnPDA nanoparticles. As can be seen from Figure S4, the significant recovery of


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fluorescence was observed from FAM-fol-DNAzyme-MnPDA in the presence of GSH, compared with the nanosystem without GSH treatment. These results suggested desorption of the DNAzyme from surface of MnPDA. Moreover, it was found that upon addition of GSH into the fol-DNAzyme-MnPDA aqueous solution, there was a gradually enhanced released amount of Mn2+ ions measured by ICP-MS. Figure S5 showed that the elevated amount of Mn2+ ions were released with increasing concentration of fol-DNAzyme-MnPDA in the presence of GSH. The concentration of released Mn2+ ions content was calculated about 0.176 mM from 400 μg/mL of MnPDA pre-treated with GSH for 24 h, which was enough to act as the provider for intracellular cofactor. Then polyacrylamide gel electrophoresis was carried out to access the catalytic effect of the Mn2+ ions for RNA cleavage. As shown in Figure 3C, the Mn2+ ions released from the fol-DNAzyme-MnPDA nanosystem could catalyze DNAzyme to completely cleave the substrate mRNA at 37 C within 60 min. These data demonstrated that the MnPDA nanoparticles could provide efficient cofactor for 10-23 DNAzyme in cancerous environment, and the fol-DNAzyme-MnPDA was a promising nanovesicle for gene regulation. Magnetic Resonance, Photothermal and Photoacoustic Performance of the Nanosystem. The MnPDA nanoparticles comprised manganese as a component and exhibited intense absorbance band in the NIR region (Figure S6), this suggested that fol-DNAzyme-MnPDA could be a type of promising MR contrast as well as photothermal agent. Magnetization measurement of the longitudinal relaxation times (T1) was taken on a 7.0 T MRI scanner. As shown in Figure 4A, the T1-weighted MR


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images of the nanoparticles in aqueous solution became brighter as the concentrations increased. The value of relaxivity (γ1) was calculated to be 4.96 mM-1s-1 according to the linear correlation curve of MR signal intensity to the MnPDA concentration, which was comparable to that of commercial contrast agent Gd-DTPA (4.8 mM-1s-1).31 These findings indicated excellent T1 contrast ability of fol-DNAzyme-MnPDA and the potential of fol-DNAzyme-MnPDA with enhancing MRI contrast. Photothermal performance of the fol-DNAzyme-MnPDA was investigated by monitoring temperature change of various aqueous concentrations of the nanoparticles under 808 nm laser irradiation. Figure 4B showed that as the concentration of fol-DNAzymeMnPDA increased, the temperature increased subsequently. The fol-DNAzymeMnPDA could rapidly increase by 45 C at a concentration of 400 μg/mL in 5 min, while pure water showed negligible temperature change. The photothermal conversion efficiency was calculated to be 46% according to the method reported in the literature (Figure S7).32 This value was higher than that of some current reported photothermal agents, such as Au nanorods (21%), Cu2-xSe (22%) and Cu9S5 (25.7%).33, 34 Furthermore, kinetics of the temperature change kept consistent upon five cycles of laser exposure (Figure 4C), revealed that the nanosystem had a good photothermal stability. These results demonstrated that fol-DNAzyme-MnPDA was an excellent photothermal agent for photothermal imaging and therapy. Meanwhile, based on the excellent photothermal transduction efficiency and thermal expansion under NIR irradiation, the folDNAzyme-MnPDA nanoparticles could be used as a potential contrast agent for PA imaging. Figure 4D demonstrated the photoacoustic signals from different aqueous


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concentrations of fol-DNAzyme-MnPDA. A higher photoacoustic signal was accompanied with increased concentration of the nanoparticles, indicating the capacity of the fol-DNAzyme-MnPDA as PA contrast agent. Cellular Uptake of fol-DNAzyme-MnPDA. In order to apply the fol-DNAzymeMnPDA nanoparticles for gene therapy and multimodal imaging, cellular uptake of the nanosystem and stimuli-responsive release of therapeutic component were studied by confocal laser scanning microscopy (CLSM) and flow cytometry. With different levels of folate receptor expression, MCF-7 cancer cells and MCF-10A normal cells were used as the target and control cells, respectively.35 Figure 5A and 5B showed fluorescence changes of the FAM-fol-DNAzyme-MnPDA incubated with MCF-7 and MCF-10A for different times. The green fluorescence increased gradually over extended incubation time for MCF-7 cells. In contrast, very weak green fluorescence was observed from the lower folate receptor-expressed MCF-10A cells cultured with the nanoparticles. The significant difference of fluorescent intensities between cancer cells and normal cells were caused by the folic acid of fol-DNAzyme-MnPDA, as folic acid could specifically recognize MCF-7 cells with over-expressed folate acceptor on the membrane and resulted in the high uptake efficiency. Furthermore, the MCF-7 cells were separated into three groups with different treatments (Figure S8). The first group was treated with FAM-fol-DNAzyme-MnPDA for 4 h. The second group was preincubated with free folic acid for 0.5 h to block the folate acceptor, and then incubated with FAM-fol-DNAzyme-MnPDA under the same condition. The third group utilized DNAzyme without folic acid conjugation, FAM-DNAzyme-MnPDA, for MCF-7 cells


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incubation. The negligible fluorescence signals were observed from the last two groups compared with the group treated with fol-DNAzyme-MnPDA under CLSM. These results clearly verified the ability of the folate moiety for targeting. In addition, the flow cytometry analysis further confirmed the enhanced cellular uptake efficiency of folDNAzyme-MnPDA (Figure 5C). To understand the subcellular localization of the folDNAzyme-MnPDA nanoparticles, MCF-7 cells were incubated with the nanoparticles and stained with LysoTracker Blue and 4’, 6-diamidino-2-phenylindole (DAPI) for CLSM imaging. It was found that FAM-fol-DNAzyme-MnPDA colocalized with lysosomes after 4 h incubation (Figure S9). Since the DNAzyme-MnPDA nanosystem could release DNAzyme upon GSH activation in aqueous solution, as illustrated in Figure S4, the ability of GSH-stimulated release of DNAzyme in MCF-7 cells was then investigated by CLSM, as GSH was overexpressed in cancer cells. As shown in Figure 5D, there was a strong green emission from cytoplasm of MCF-7 cells treated with FAM-fol-DNAzyme-MnPDA. However, MCF-7 cells pre-treated with a GSH scavenger, N-ethylmaleimide (NEM) greatly decreased the fluorescence signal, while the cells pre-treated with a GSH synthesis enhancer, α-lipoic acid (LPA), showed stronger green fluorescence. Taken together, these results demonstrated the folDNAzyme-MnPDA nanoparticles exhibited a stimuli-responsive release of catalytic deoxyribozyme in cancer cells. In Vitro Gene Silencing Efficacy of fol-DNAzyme-MnPDA. Encouraged by the specific and efficient cellular uptake performance of the DNAzyme-MnPDA nanosystem, we further applied the nanoparticles for gene silencing with cancer cells.


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Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was firstly carried out to evaluate the cleavage of Egr-1 mRNA in MCF-7 cells cultured with different materials including MnPDA conjugated with a folic-acid-modified random DNA sequence as control, fol-random DNA-MnPDA. As shown in Figure 6A, folDNAzyme, MnPDA and fol-random DNA-MnPDA reduced mRNA level of Egr-1 by 22%, 4% and 12%, respectively, while fol-DNAzyme-MnPDA successfully cleaved Egr-1 mRNA by 80%. The remarkably improved mRNA-cleavage ability of folDNAzyme-MnPDA may be attributed to its high cellular uptake efficiency and high specificity to target mRNA. Additionally, western blot analysis demonstrated that DNAzyme, MnPDA or fol-random DNA-MnPDA have negligible any influence on Egr-1 protein expression of MCF-7 cells. In contrast, the inhibitory effect of Egr-1 protein expression of MCF-7 cells treated with fol-DNAzyme-MnPDA increased about 5-folds (Figure 6B, C). Immunofluorescence assay was also carried out to investigate the ability of fol-DNAzyme-MnPDA for gene silencing. The MCF-7 cells were cultured with different agents, and then treated with Egr-1 antibody and DAPI for Egr-1 conjugation and nucleus staining, respectively. Figure 6D showed weaker red emissions from the cells treated with fol-DNAzyme-MnPDA than those treated with free folDNAzyme, MnPDA or fol-random DNA-MnPDA, suggesting that fol-DNAzymeMnPDA could inhibit the expression of Egr-1 protein to a great extent. Taken together, the significantly reduced mRNA level and Egr-1 protein content revealed by qRT-PCR, western blot and immunofluorescence assay showed that fol-DNAzyme-MnPDA


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efficiently cleaved Egr-1 mRNA and inhibited expression of the protein in MCF-7 cells. All these results clearly demonstrated efficient gene regulation of the nanosystem. In Vitro Anti-Tumor Efficacy of fol-DNAzyme-MnPDA. Considering the promising photothermal conversion efficiency and gene silencing capacity of folDNAzyme-MnPDA, we tested in vitro anti-tumor effect of the nanoparticles by 3-(4, 5-dimethylthialzol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and live and dead staining. First of all, cytotoxicity of MnPDA was measured at various concentrations with or without irradiation for 5 min (Figure 7A). Results of MTT assay suggested that MnPDA nanoparticles were biocompatible, since no significant cellular toxicity was observed in the absence of irradiation. Irradiation exposure resulted in decreased viability of MCF-7 cells, suggesting that MnPDA worked well for photothermal ablation of cancer cells. Second, in vitro gene-silencing efficacy was estimated by comparing the viability of MCF-7 cells treated with free DNAzyme, MnPDA, DNAzyme-MnPDA and fol-DNAzyme-MnPDA at dark (Figure 7B). It can be seen that the groups of free DNAzyme and MnPDA had negligible cell death, while the groups of DNAzyme-MnPDA and fol-DNAzyme-MnPDA displayed decreased cell survival ratio, and the fol-DNAzyme-MnPDA group had the lowest viability. The difference indicated that the delivered DNAzyme had good gene-silencing efficacy and the nanosystem exhibited efficient gene regulation in cancer cells. Third, synergistic therapy by using fol-DNAzyme-MnPDA was investigated from the following two aspects (Figure 7B). Under 808 nm laser irradiation, cell viability of MCF-7 cells treated with DNAzyme-MnPDA was remarkably decreased compared with that of free


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DNAzyme and MnPDA. In addition, the cell viability under irradiation was lower than that without irradiation for both the DNAzyme-MnPDA and fol-DNAzyme-MnPDA groups. All the results indicated significantly synergistic therapeutic effect between photothermal ablation and gene regulation. Meanwhile, major cell death was observed from the fol-DNAzyme-MnPDA group other than DNAzyme-MnPDA group, further validating an additional boost due to folate-mediated cellular uptake and thereby improved therapeutic effect. To visually evaluate the therapeutic effect of the agents in vitro, live/dead assays were carried out by incubating MCF-7 cells with different treatments, and then costaining live and dead cells with calcein AM (green fluorescence) and propidium iodide, PI (red fluorescence), respectively (Figure 7C). Incubation of MCF-7 cells with MnPDA could not kill the cancer cells as strong green fluorescence signal was observed, but upon illumination, dead cells appeared, accompanied with red fluorescence signal due to the photothermal ablation of cancer cells. Similarly, only green fluorescence were observed from the group treated with DNAzyme. In contrast, red fluorescence appeared for the cells treated with MnPDA-delivered DNAzyme in the absence of irradiation, indicating more dead cells existed because of gene therapy. Additionally, the cancer cells cannot be completely eliminated by monotherapy, while nearly all of the cells incubated with fol-DNAzyme-MnPDA were killed and stained with red fluorescence subjected to combination therapy, again supported anti-tumor efficacy of this method.


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Besides in vitro assay by MTT and live/dead staining, flow cytometry assay was performed to assess the potential of fol-DNAzyme-MnPDA as a delivery system for anti-tumor therapy. For cell apoptosis and cycle distribution investigations, MCF-7 cells with different treatments were divided into six groups: cancer cells only (1), under irradiation (2), incubated with MnPDA (3), incubated with fol-DNAzyme-MnPDA (4), incubated with MnPDA under irradiation, MnPDA + PTT (5) incubated with folDNAzyme-MnPDA under irradiation, fol-DNAzyme-MnPDA + PTT (6). As shown in Figure 7D and E, the apoptotic ratios of cells treated with MnPDA, MnPDA + PTT, fol-DNAzyme-MnPDA and fol-DNAzyme-MnPDA + PTT were 3.37%, 13.85%, 11.57% and 25.14%, respectively. Such difference showed a clear boost of apoptosis, indicating effective gene silencing and photothermal effect, as well as improvement of the combination therapy. Correspondingly, cell cycle assays were then carried out to access mechanism of the therapeutic effect (Figure 7F). For the group of (2) and (3), the ratio of the cells in G2/M, G1 and S phase is similar to the cell cycle distribution of the control group (1). In contrast, treating MCF-7 cells with fol-DNAzyme-MnPDA (4) or MnPDA + PTT (5) resulted in increased S population and decreased G2/M population. Moreover, more cells arrested at S phase and less at G2/M phase upon irradiation, when comparing (5) and (6) to (3) and (4) respectively. Specifically, the G2/M population decreased nearly 3-fold upon entry into S phase for the cells treated with fol-DNAzyme-MnPDA + PTT (6) compared to the control group, and thus greatly induced cell cycle arrest. The cell cycle redistribution implied that synergistic therapy could inhibit cell proliferation significantly.


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Multimodal Imaging and In Vivo Antitumor Efficacy of fol-DNAzyme-MnPDA. An efficient nanovesicle is expected to show high anti-tumor efficacy, preferably with multimodal imaging capability, for comprehensive learning of tumor location, morphology and edge detection, therapeutic response, biodistribution of the nanoparticles.36 On the basis of the satisfying performance of fol-DNAzyme-MnPDA as a photothermal, PA and MR contrast agent in the solution and the good in vitro antitumor efficacy, we investigated in vivo imaging capability of fol-DNAzyme-MnPDA to evaluate accumulation of nanoparticles and the location or distribution in the tumor. The multimodal images were captured using tumor-bearing nude mice treated with folDNAzyme-MnPDA. Thermal images were recorded with an infrared thermal camera to monitor temperature changes of the tumor site under laser exposure (Figure 8A). It was clear that under 808 nm irradiation, the increase of temperature in the tumor sites was higher for the mice treated with the nanoparticles than that with PBS. The temperature could reach 45 C, which was sufficient to ablate tumor cells (Figure S10). PA imaging is supposed to have satisfying imaging depth and spatial resolution, which is useful for understanding distribution of nanodrugs inside tumors. Correspondingly, PA image resulted from thermal expansion of the fol-DNAzyme-MnPDA under NIR irradiation was investigated in vivo. Figure 8B showed the PA images of the tumorbearing mice before and after intravenous injection of fol-DNAzyme-MnPDA. The latter showed a stronger PA signal at the tumor sites, indicating the promising future of fol-DNAzyme-MnPDA as an imaging agent to evaluate tumor distribution. We subsequently examined performance of fol-DNAzyme-MnPDA for in vivo MR


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imaging, since the MR technique was able to provide superior three-dimensional details and tomographic information of soft tissue with good spatial resolution. The T1weighted MR imaging of tumor-bearing mice exhibited that the tumor area had higher MR signal intensity treated with fol-DNAzyme-MnPDA for 24 h post-injection (Figure 8C). All the results showed strong tumor accumulation of the nanomedicine and indicated the potential of fol-DNAzyme-MnPDA as a multimodal contrast agent for cancer therapy. Under the guidance of different imaging techniques, the multimodal imaging would benefit precise cancer diagnosis and treatment. In order to evaluate the antitumor efficacy of fol-DNAzyme-MnPDA, the MCF-7 tumor-bearing nude mice were intravenously injected with different materials. The tumor-bearing mice were randomly divided into six groups with five mice in each group, and each group was separately treated with PBS only (1), MnPDA (2), MnPDA + PTT (3), fol-DNAzyme-MnPDA (4), DNAzyme-MnPDA + PTT (5), fol-DNAzymeMnPDA + PTT (6). During the treatment, the body weight and tumor volume were recorded every 2 days. Stability of the body weight for all the mice indicated that irradiation or nanomaterials had no negative influence on the mice (Figure 9A). The tumor volume and size of mice for group (1) and (2) gradually grew larger as times going on, but a relative slowly growth was observed for group (3), which indicated considerable photothermal efficiency of MnPDA (Figure 9B and C). However, the tumor volume and size was greatly suppressed for group (4) and (5), resulted from targeted gene silencing and photothermal ablation of the respective treatment. Specifically, a combination of the gene therapy and photothermal therapy represented


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by group (6) could completely ablate tumor. The tumor disappeared in 4 days and had not recurred compared with other groups. In addition, hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed to evaluate the tumor inhibition effect of different treatment. After 14-day treatment, all the mice of the six groups were sacrificed and tumors were collected for H&E staining and immunohistochemical evaluation. The histological images in Figure 9D showed distinct nuclear deficiencies in morphology for the groups with gene therapy and photothermal therapy. Comparison of fluorescence images for TUNEL assay in Figure 9E exhibited a most drastic apoptosis in the tumor tissues of group (6) after treatment with fol-DNAzyme-MnPDA and NIR light, suggesting the best tumor regression activity of the combination therapy. Overall, efficient gene regulation with the catalytic deoxyribozyme was clearly confirmed by comparison of groups (4) and (5) with (2) and (3), respectively, while effective photothermal ablation effect was proved by comparison of the groups (3) and (6) with (2) and (4), respectively, however, the combinational therapy in group (6) achieved the best therapy efficiency revealed by the in vivo results shown in Figure 9B, C, D and E. For possible concern of in vivo biosafety of fol-DNAzyme-MnPDA, hemolysis tests, biochemical indexes, pathology change of organs, biodistribution and blood circulation time were evaluated. After treatment, all mice were sacrificed and the blood and main organs were obtained for analysis. Figure S11 showed that no hemolysis of the red blood cells was detected, which indicated satisfying blood compatibility of the materials. Importantly, the biochemical indexes of the serum kept normal (Figure S12), and the slices of main


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organs didn’t exhibit any histological changes (Figure S13). The biodistribution and pharmacokinetics of fol-DNAzyme-MnPDA were also investigated by ICP-MS to avoid possible toxicity by long-time accumulation. A major accumulation of the folDNAzyme-MnPDA was observed in tumor sites instead of other tissues except liver, which was involved in foreign particle excretion and clearance (Figure S14). In addition, the circulation half-life time for distribution and elimination phase were calculated to be 0.6 ± 0.1 and 9.7 ± 0.9 h, respectively (Figure S15). This indicated that treatment of the mice with the nanodrug avoided possible toxicity by accumulation and ensured excretion of the nanoparticles in the body. All results revealed that the nanomaterials had admirable compatibility and bio-safety for therapeutic treatment. CONCLUSIONS In summary, rationally designed DNAzyme vehicle with diverse functionalities is presented for potent gene regulation via a facile one-step polymerization. This vehicle ensures DNAzyme with good stability, biocompatibility and enhanced cellular uptake efficiency. More importantly, the vehicle is able to self-generate free Mn2+ in cancer cells and supplies the essential cofactor of DNAzyme for RNA cleavage, and thus effective cleaves target mRNA and greatly suppresses gene expression both in vitro and in vivo. In addition, the nanosystem performs well for NIR thermal imaging, PAI and MRI and provides multimodal imaging-guided gene-photothermal therapy without noticeable in vivo toxicity. This design principle provides a way developing versatile DNAzyme vehicles to realize wide potential of DNAzyme-based therapeutics. For


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example, supramolecular assemblies or nanoparticles for potent gene therapeutics can be prepared by incorporating different therapeutic and functional precursors such as photothermal agents for photothermal therapy, photosensitizers for photodynamic therapy, and fluorophores or quantum dots for optical imaging. MATERIALS AND METHODS Materials. Trizma base, trizma hydrochloride, sodium phosphate dibasic, sodium phosphate monobasic and sodium acetate were obtained from Sigma-Aldrich. Dopamine hydrochloride was obtained from Aladdin. Pen Strep (100, Pan 10000 U/mL, Str 10000 μg/mL, PBS 0.01 M), trypsin-EDTA (0.25%), thiazolyl blue, phosphate buffer saline (0.0067 M), DMEM with high glucose, paraformaldehyde (4%) and fetal bovine serum were obtained from GIBCO. Annexin V-FITC apoptosis detection kit was obtained from Best Bio Science Co., Ltd. Calcein-AM/PI double stain kit was purchased from Shanghai Yeasen Biotechnology Co., Ltd. 2-(4Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was obtained from Beyotime Biotechnology Co., Ltd. Other reagents were of analytical grade and used without further purification. Characterization. Fluorescent, absorption and infrared spectra were obtained with fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies, USA), UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan) and FTIR spectrometer (Nicolet iS10, Thermo Fisher Scientific, USA), respectively. Powder crystal structure was analyzed using X-ray diffractometer (Rigaku Miniflex600, Japan). Morphology of the


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nanoparticles was investigated using field emission scanning electron microscopy (Zeiss Merlin Compact, Germany) and transmission electron microscopy (HT-7700, Hitachi, Japan). Dynamic light scattering analysis was carried out on Zetasizer (NanoZS90, Malvern, UK). The valence state of the element was determined through X-ray photoelectron spectrometer (ESCALAB250Xi, Thermo Fisher Scientific, USA). Cell imaging was performed with spinning-disk confocal microscope (Revolution XD, Andor, UK). Cellular uptake assays were performed on flow cytometer (BD FACS Verse, USA). Cell viability was measured using Multiskan GO (Thermo Scientific). Photothermal imaging was obtained with IR thermal camera (Fotric, China) and 808 nm-laser irradiation (Hi-Tech Optoelectronics Co., Ltd. China). Magnetic resonance signal was obtained with 7.0 T Small Animal Magnetic Resonance Imaging System. PA photoacoustic images were recorded by a multispectral optoacoustic tomographic (MSOT) imaging system (iThera Medical GmbH, Neuherberg). Synthesis of MnPDA and Catalytic Deoxyribozyme-Modified MnPDA. For the synthesis of MnPDA, 7.6 mg of dopamine was quickly added into 6.25 mL of Tris buffer (10 mM, pH = 8.5) containing 1.25 mL of ethanol under vigorous stirring at 37 °C for 15 min. To this solution, 7.916 mg of MnCl2·4H2O was added with stirring for 3 h. The MnPDA was collected by centrifugation at 13000 rpm for 10 min and washed with deionized water three times. For the synthesis of catalytic deoxyribozymemodified MnPDA, various concentrations of MnPDA were incubated with 100 nM of the nucleic acid at room temperature with gentle stirring for about 10 min, and the product was purified by centrifugation.


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In Vitro Photothermal Effect. Different concentrations of fol-DNAzyme-MnPDA (0, 25, 50, 100, 150, 200, 250, 300 and 400 μg/mL) were exposed to 808 nm laser irradiation for 5 min (3 W/cm2). Photothermal stability of fol-DNAzyme-MnPDA was detected by repeated irradiating fol-DNAzyme-MnPDA solution to reach a steady-state temperature followed by naturally cooling to room temperature. The photothermal conversion efficiency of MnPDA was calculated by the following equation 1: η=

ℎ𝑆(𝑇𝑚𝑎𝑥 ― 𝑇𝑠𝑢𝑟𝑟) ― 𝑄𝑑𝑖𝑠 𝐼(1 ― 10

―𝐴808

)

(1)

Where Tmax is the maximum steady-state temperature, Tsurr is the ambient temperature, Qdis is the heat dissipated from light absorbed by the container itself, I is incident laser power density, A808 represents the absorbance of MnPDA at 808 nm, h represents heat transfer coefficient, and S means surface area of the container. The value of hS is determined according to the equation 2: τs =

𝑚𝐷𝐶𝐷 ℎ𝑆

(2)

Where mD and CD mean mass (1 g) and heat capacity (4.2 J/g) of H2O, respectively, and τs represents the sample system time constant. Gel Electrophoresis. A 12% native polyacrylamide gel (PAGE) was utilized to assess the cleavage efficiency of fol-DNAzyme-MnPDA. The electrophoresis was carried out at 120 V in 1× TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.3) for 60 min. The gel was finally stained with GelRed for 20 min, and imaged with FluorChem FC3 (ProteinSimple, USA). Western Blot Assay. MCF-7 cells (5 × 105 per/well) were seeded into 6-well plates and grown overnight in DMEM medium supplemented with 10% FBS at 37 °C in 5%


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CO2 incubator. The medium was replaced by different materials dissolved with fresh medium, and incubated with MCF-7 cells for 72 h. Then the culture medium and materials were removed, and the cells were washed three times using PBS and lysed with cell lysis buffer. Clarified lysates were collected by microcentrifugation, and the concentration of the extracted protein was measured using Nanodrop. The proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Subsequently, the membrane was incubated with primary antibody overnight and secondary antibody for another 2 h, follow by washing with TBST. The signal was read out using BIORAD ChemiDocTM Touch imaging system. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). MCF-7 cells (5 × 105 per/well) were seeded into 6-well plates and grown overnight in DMEM. The cells were then treated with nanoparticles for 48 h at 37 °C in 5% CO2 incubator, followed by washing with PBS three times. Subsequently, RNA Extraction Kit was used to extract total RNA from the cells, and 2 μg of the obtained RNA was transcribed into cDNA with PrimeScript First Strand cDNA Synthesis Kit according to the manufacture’s protocol. Real-time PCR runs were performed in 6-well plates by adding 20 L of the obtained cDNA, forward and reverse primers, and SYBR Premix Ex Taq (Takara, Dalian, China). The amplified product was monitored with CFX ConnectTM Real-Time System (BIO-RAD, Singapore). Cell Viability Analysis. MCF-7 cells (2 × 105 per/well) were seeded in 96-well plates at 37 °C in CO2 (5%) atmosphere for 24 h, and incubated with different concentrations of MnPDA or different nanoparticles for another 24 h. The medium was


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then removed and cells were washed with PBS three times. The cells with fresh DMEM were then irradiated with 808 nm laser at 3 W/cm2 for 5 min, while cells received no irradiation were used as controls. After 24 h, 100 μL of 3-(4, 5-dimethylthiazol-2-yl)2, 5 diphenyl tetrazolium bromide (MTT) was added into the above wells and incubated for another 4 h at 37 °C. Subsequently, 150 μL DMSO was used to dissolve the formed formazan crystals. Cell viability was analyzed by measuring absorbance of the solution at the wavelength of 570 nm using microplate reader. Confocal Laser Scanning Microscopy. For cellular uptake investigation, MCF-7 and MCF-10A cells were separately seeded into confocal dish overnight. Then the cells were incubated with fol-DNAzyme-MnPDA (50 μg/mL) dispersed in DMEM for 1, 2 and 4 h. Finally, the cells were washed three times with PBS and imaged with confocal laser scanning microscope (CLSM) using 488 nm excitation. For CLSM imaging of MCF-7 cells incubated with nanoparticles upon different GSH stimulation, the method was similar expect that the cells were first incubated with NEM (10 μM) for 20 min or LPA (50 μM) for 24 h. Then the medium was replaced, and cells were incubated with fol-DNAzyme-MnPDA dissolved in DMEM (50 μg/mL) for 4 h. For live/dead staining, MCF-7 cells with different treatments were stained with calcein-AM (2 μM) and PI (4.5 μM) at 37 °C. After 20 min, cells were washed with PBS and imaged by CLSM. Flow Cytometric Assay. For apoptosis analysis, MCF-7 cells (1 × 106 per/well) were seeded in 6-well plates and cultured overnight. Then cells were incubated with different samples and further irradiated with 808 nm laser for 5 min. After incubation for another 24 h, the cells were centrifuged at 1500 rpm for 5 min and resuspended in


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Annexin V binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1mM MgCl2, 1.8 mM CaCl2). Then cells were stained with Annexin V-FITC for 15 min, and PI for 5 min at 4 °C. Finally, cells were filtered through a 300-mesh filter and analyzed on flow cytometer. For cell cycle assay, the cells were seeded in 6-well plate culture dish and incubated with different samples for 48 h. Then trypsin-treated cells were fixed with 70% ethanol at -20 °C overnight. In the end, the cells were stained with PI at 4 °C for another 5 min in dark and detected on flow cytometer. Establishment of Xenograft Tumor Model. MCF-7 cells (1 × 106 per/well) were subcutaneously injected into selected position of BABL/c nude mice (4-5 weeks). The MCF-7 tumor-bearing mice were randomly divided into different groups. Treatment began when tumor volumes reached 100 mm3 after cell implantation. The tumor volume (V, mm3) as calculated by the following equation: V = 𝐿 × 𝑤2/2, where L and W were the largest longitudinal and the shortest transverse diameter of the selected tumor, respectively. Different materials (50 μL, 800 μg/mL) were injected into the mice through tail vein injection. The animal studies were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals. The animal protocol was approved by The Institutional Animal Use and Care Committee of Wuhan University. In Vivo Photothermal, Magnetic Resonance and Photoacoustic Imaging. For in vivo photothermal imaging (IR) of the MCF-7 tumor-bearing mice, the tumor sites were irradiated with 808 nm laser at the power density of 1 W/cm2 for 5 min at 24 h postinjection of the nanoparticles or PBS. Temperature changes of the tumor site were


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recorded by IR thermal camera. For magnetic resonance imaging (MRI), the photos of the mice pre-injected and 24 h post-injected with fol-DNAzyme-MnPDA were obtained by MRI scanner for small animal imaging. For in vivo photoacoustic imaging (PAI), MCF-7 tumor-bearing mice were injected by tail vein with 100 μL of fol-DNAzymeMnPDA, and the PA images of the mice were recorded by MSOT imaging system. A 128-element concave transducer array spanning a circular arc of 270° were used to obtain PA signals. Biosafety Investigation. MCF-7 tumor-bearing mice were intravenously injected with fol-DNAzyme-MnPDA (150 μL, 2 mg/mL), and the bio-distribution and blood circulation time were investigated by measuring content of Mn in the body with ICPMS. For bio-distribution assay, the mice were sacrificed after 24 h treatment, and then tissues were taken out and weighed. Then the mixture of HNO3 and H2O2 (volume ratio = 1:2) were used to digest the tissues at 100 °C to obtain clear solutions. The content of Mn in the solutions was then detected. The amount of fol-DNAzyme-MnPDA was normalized to the percentage of injected dose per gram of tissue (% ID/g). For blood circulation time measurements, blood (10 μL) was collected from the tail veins of the Balb/c nude mice at certain time points after intravenous injection of fol-DNAzymeMnPDA. Each blood sample was weighted and dissolved in the digesting solution for ICP-MS measurement using the similar procedure for bio-distribution assay. For hemolysis analysis, blood cells were obtained from the mice and incubated with different concentrations of fol-DNAzyme-MnPDA for a certain time. The PBS and pure water were used as negative and positive controls, respectively. The samples were then


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centrifuged and the supernatant were used for absorbance measurement. For biochemical indexes analysis, blood from the mice was collected at the end of the treatment. The blood was allowed to stand for 4 h at room temperature, followed by centrifugation at 4000 rpm for 5 min. Then the upper solution of serum was collected for evaluation of blood biochemical indexes. For H&E assay, the major organs were collected at the end of the treatment and fixed in 10% paraformaldehyde solution. Then the slices were prepared in process routinely and stained with hematoxylin and eosin. The final slice was observed by confocal fluorescence laser microscope. ASSOCIATED CONTENT Supporting Information Available: Supplemental figures are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Xiaoqing Liu: 0000-0002-1309-5454 Fuan Wang: 0000-0002-3063-2485 Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT This work is supported by National Natural Science Foundation of China (21503151, 81602610, and 81472735), Fundamental Research Funds for the Central Univers ities (No. 2042018kf1006), Jiangsu Provincial Natural Science Foundation of China ( BK20161248, BK20160381), and 1000 Young Talent (F.W. and X.L.). REFERENCES (1) Naldini, L. Gene Therapy Returns to Centre Stage. Nature 2015, 526, 351-360. (2) Collins, F. S.; Gottlieb, S. The Next Phase of Human Gene-Therapy Oversight. New. Engl. J. Med. 2018, 379, 1393-1395. (3) Zhou, J.; Rossi, J. Aptamers As Targeted Therapeutics: Current Potential and Challenges. Nat. Rev. Drug Discovery, 2016, 16, 181-202. (4) Sun, M. Z.; Xu, L. G.; Qu, A. H.; Zhao, P.; Hao, T. T.; Ma, W.; Hao, C. L.; Wen, X. D.; Colombari, F. M.; de Moura, A. F.; Kotov, N. A.; Xu, C. L.; Kuang, H. SiteSelective Photoinduced Cleavage and Profiling of DNA by Chiral Semiconductor Nanoparticles. Nat. Chem. 2018, 10, 821-830. (5) Xu, L. G.; Gao, Y. F.; Kuang, H.; Liz‐Marzán, M.; Xu, C. L. MicroRNA-Directed Intracellular Self‐Assembly of Chiral Nanorod Dimers. Angew. Chem. Int. Ed. 2018, 57, 10544-10548. (6) Evans, C. H.; Ghivizzani, S. C.; Robbins, P. D. Arthritis Gene Therapy Is Becoming A Reality. Nat. Rev. Rheumatol. 2018, 14, 381-381. (7) Dunbar, C. E.; High, K. A.; Joung, J. K.; Kohn, D. B.; Ozawa, K.; Sadelain, M.


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Gene Therapy Comes of Age. Science, 2018, 359, eaan4672. (8) Fokina, A. A.; Stetsenko, D. A.; Francois, J.-C. DNA Enzymes As Potential Therapeutics: Towards Clinical Application of 10-23 DNAzymes. Expert Opin. Biol. Ther. 2015, 15, 689-711. (9) Silverman, S. K. Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes. Trends Biochem. Sci. 2016, 41, 595-609. (10) Santoro, S. W.; Joyce, G. F. A General Purpose RNA Cleaving DNA Enzyme. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4262-4266. (11) Liu, M.; Chang, D.; Li, Y. Discovery and Biosensing Applications of Diverse RNA-Cleaving DNAzymes. Accounts. Chem. Res. 2017, 50, 2273-2283. (12) Zhou, W.; Ding J.; Liu, J. Theranostic DNAzymes. Theranostics. 2017, 7, 10101025. (13) Kole, R.; Krainer, A.R.; Altman, S. RNA Therapeutics: Beyond RNA Interference and Antisense Oligonucleotides. Nat. Rev. Drug Discov. 2012, 11, 125-140. (14) Wang, F.; Saran, R; Liu, J. Tandem DNAzymes for mRNA Cleavage: Choice of Enzyme, Metal Ions and the Antisense Effect. Bioorg. Med. Chem. Lett. 2015, 25, 14601463. (15) Chen, F.; Bai, M.; Cao, K.; Zhao, Y.; Wei, J.; Zhao,Y. X. Fabricating MnO2 Nanozymes As Intracellular Catalytic DNA Circuit Generators for Versatile Imaging of Base-Excision Repair in Living Cells. Adv. Funct. Mater. 2017, 27, 1702748. (16) Yehl, K.; Joshi, J. P.; Greene, B. L.; Dyer, R. B.; Nahta, R.; Salaita, K. Catalytic Deoxyribozyme-Modified Nanoparticles for RNAi-Independent Gene Regulation. ACS


Page 30 of 45

Page 31 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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nano 2012, 6, 9150-9157. (17) Fan, H.; Zhao, Z.; Yan, G.; Zhang, X.; Yang, C.; Meng, H.; Chen, Z.; Liu, H.; Tan, W. A Smart DNAzyme-MnO2 Nanosystem for Efficient Gene Silencing. Angew. Chem. Int. Ed. 2015, 54, 4801-4805. (18) He, D.; Hai, L.; He, X.; Yang, X.; Li, H. W. Glutathione Activatable and O2/Mn2+Evolving Nanocomposite for Highly Efficient and Selective Photodynamic and GeneSilencing Dual Therapy. Adv. Funct. Mater. 2017, 27, 1704089. (19) Somasuntharam, I.; Yehl, K.; Carroll, S. L.; Maxwell, J. T.; Martinez, M. D.; Che, P. L.; Brown, M. E.; Salaita, K.; Davis, M. E. Knockdown of TNF-alpha by DNAzyme Gold Nanoparticles As an Anti-Inflammatory Therapy for Myocardial Infarction. Biomaterials. 2016, 83, 12-22. (20) Yehl, K.; Joshi, J. P.; Greene, B. L.; Dyer, R. B.; Nahta, R.; Salaita, K. Catalytic Deoxyribozyme-Modified Nanoparticles for RNAi-Independent Gene Regulation. ACS Nano 2012, 6, 9150-9157. (21) Li, N.; Li, Y. L.; Gao, X. N.; Yu, Z. Z.; Pan, W.; Tang, B. Multiplexed Gene Silencing in Living Cells and in Vivo Using a DNAzymes-CoOOH Nanocomposite. Chem. Commun. 2017, 53, 4962-4965. (22) Ward, W. L.; Plakos, K.; DeRose, V. J. Nucleic Acid Catalysis: Metals, Nucleobases, and Other Cofactors. Chem. Rev. 2014, 114, 4318-4342. (23) Gai, S.; Yang, G.; Yang, P.; He, F.; Lin, J.; Jin, D.; Xing, B. Recent Advances in Functional Nanomaterials for Light-Triggered Cancer Therapy. Nano Today 2018, 19, 146-187.


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(24) Xu, J. T.; Han, W.; Yang, P. P.; Jia, T.; Dong, S. M.; Bi, H. T.; Gulzar, A.; Yang, D.; Gai, S. L.; He, F.; Lin, J.; Li, C. X. Tumor Microenvironment‐Responsive Mesoporous MnO2-Coated Upconversion Nanoplatform for Self-Enhanced Tumor Theranostics. Adv. Funct. Mater. 2018, 28, 1803804. (25) Sun, M. Z.; Qu. A. H.; Hao, C. L.; Wu, X. L.; Xu. L. G.; Xu, C. L.; Kuang, H. Chiral Upconversion Heterodimers for Quantitative Analysis and Bioimaging of Antibiotic-Resistant Bacteria in Vivo. Adv. Mater. 2018, 1804241. (26) Mitchell, A.; Dass, C. R.; Sun, L. Q.; Khachigian, L. M. Inhibition of Human Breast Carcinoma Proliferation, Migration, Chemoinvasion and Solid Tumour Growth by DNAzymes Targeting the Zinc Finger Transcription Factor EGR-1. Nucleic Acids Res. 2004, 32, 3065-3069. (27) Biesinger, M. C.; Payne, B. P.; Grosvenor, A. P.; Lau, L. W. M.; Gerson, A. R.; Smart, R. S. C. Resolving Surface Chemical States in XPS Analysis of First Row Transition Metals, Oxides and Hydroxides: Cr, Mn, Fe, Co and Ni. Appl. Surf. Sci. 2011, 257, 2717-2730. (28) Liu, Y.; Ai, K.; Lu, L. Polydopamine and its Derivative Materials: Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chem. Rev. 2014, 114, 5057-5115. (29) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Mussel-Inspired Surface Chemistry for Multifunctional Coatings. Science 2007, 318, 426-430. (30) Perry, R. R.; Mazetta, J. A.; Levin, M.; Barranco, S. C. Glutathione Levels and Variability in Breast Tumors and Normal Tissue. Cancer 1993, 72, 783-787.


Page 32 of 45

Page 33 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(31) Wang, G.; Zhang, X.; Skallberg, A.; Liu, Y.; Hu, Z.; Mei, X.; Uvdal, K. One-Step Synthesis of Water-Dispersible Ultra-Small Fe3O4 Nanoparticles As Contrast Agents for T1 and T2 Magnetic Resonance Imaging. Nanoscale 2014, 6, 2953-2963. (32) Roper, D. K.; Ahn, W.; Hoepfner, M. Microscale Heat Transfer Transduced by Surface Plasmon Resonant Gold Nanoparticles. J. Phys. Chem. C. 2007, 111, 36363641. (33) Li, J.; Rao, J.; Pu, K. Recent Progress on Semiconducting Polymer Nanoparticles for Molecular Imaging and Cancer Phototherapy. Biomaterials 2018, 155, 217-235. (34) Ding, F.; Zhan, Y.; Lu, X.; Sun, Y. Recent Advances in Near-Infrared II Fluorophores for Multifunctional Biomedical Imaging. Chem. Sci. 2018, 9, 4370-4380. (35) Sudimack, J.; Lee, R. J. Targeted Drug Delivery via the Folate Receptor. Adv. Drug Deliv. Rev. 2000, 41, 147-162. (36) Meng, X. D.; Liu, Z. Q.; Cao, Y.; Dai, W. H.; Zhang, K.; Dong, H. F.; Feng, X. Y.; Zhang, X. J. Fabricating Aptamer-Conjugated PEGylated-MoS2/Cu1.8S Theranostic Nanoplatform for Multiplexed Imaging Diagnosis and Chemo-Photothermal Therapy of Cancer. Adv. Funct. Mater. 2017, 27, 1605592.


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Figure 1. Illustration of fol-DNAzyme-MnPDA nanoplatform as a versatile vehicle for multimodal imaging-guided therapy.


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Figure 2. Characterization of MnPDA. (A) SEM image and corresponding elemental mapping images of C, N, O and Mn. (B) Energy dispersive X-ray spectroscopy (EDX) of MnPDA. (C) XPS spectrum of Mn 2P1/2 and Mn 2P3/2. (D) XPS spectrum of Mn 3S.


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Figure 3. Evaluation of loading capability, stability and GSH-stimulated catalytic cleavage of DNAzyme with the nanosystem. (A) Fluorescence spectra of FAM-folDNAzyme in the presence of different concentrations of MnPDA. From top to bottom: 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, 125, 150 and 200 μg/mL. Inset: Normalized fluorescence as a function of MnPDA concentration. (B) Stability of fol-DNAzymeMnPDA in DNase I (1), PBS (2) and DMEM with 10% FBS (3). (C) Cleavage efficiency of fol-DNAzyme-MnPDA in the presence of 10 mM GSH by polyacrylamide gel electrophoresis. (1): 1 μM substrate, (2): 250 nM DNAzyme, (3-8): 1 μM substrate and 250 nM DNAzyme incubated with 0 (3), 12.5 (4), 25 (5), 50 (6), 100 (7) and 200 μg/mL (8) of MnPDA.


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Figure 4. MR imaging, photothermal property and PA imaging of fol-DNAzymeMnPDA in vitro. (A) T1 relaxation rate (1/T1) of fol-DNAzyme-MnPDA as a function of Mn concentration for fol-DNAzyme-MnPDA. Inset: T1-weighted MR images of various concentrations of fol-DNAzyme-MnPDA (0, 25, 50, 100, 200 and 400 μg/mL). (B) Photothermal heating curves of fol-DNAzyme-MnPDA aqueous solution with different concentrations upon 808 nm laser irradiation at a power density of 3 W/cm2. (C) Recycling heating profile of fol-DNAzyme-MnPDA aqueous solution (400 μg /mL) under 808 nm laser irradiation at a power density of 3 W/cm2 for five laser on/off cycles. (D) PA signal intensity of fol-DNAzyme-MnPDA as a function of fol-DNAzymeMnPDA concentration using a MSOT imaging system. Inset: PA images of fol-


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DNAzyme-MnPDA aqueous solutions with different concentrations (0, 25, 50, 100, 200 and 400 μg/mL).


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Figure 5. Cellular uptake of fol-DNAzyme-MnPDA. (A) MCF-7 cells and (B) MCF10 A cells for real-time intracellular imaging of FAM-fol-DNAzyme-MnPDA. (C) Mean fluorescence intensity of intracellular FAM by flow cytometry. The samples include MCF-7 cells only (1), treated with MnPDA-DNAzyme (2), and incubated with fol-DNAzyme-MnPDA in the absence (3) and presence (4) of FA. (***P

Versatile Catalytic Deoxyribozyme Vehicles for Multimodal Imaging

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