A DNA-Based Nanocarrier for Efficient Gene Delivery and Combined

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A DNA-Based Nanocarrier for Efficient Gene Delivery and Combined Cancer Therapy Jianbing Liu, Linlin Song, Shaoli Liu, Qiao Jiang, Qing Liu, Na Li, Zhen-Gang Wang, and Baoquan Ding Nano Lett., Just Accepted Manuscript • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on April 30, 2018

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A DNA-Based Nanocarrier for Efficient Gene Delivery and Combined Cancer Therapy Jianbing Liu,†,# Linlin Song,†,‡,# Shaoli Liu,†,‡ Qiao Jiang,† Qing Liu,†,‡ Na Li,† Zhen-Gang Wang,† and Baoquan Ding*,†,‡ †

CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, CAS Center for Excellence

in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China. ‡

University of Chinese Academy of Sciences, Beijing 100049, China.

ABSTRACT: The efficient delivery of a therapeutic gene into target tissues has remained a major obstacle in realizing a viable gene-based medicine. Herein, we introduce a facile and universal strategy to construct a DNA nanostructure-based co-delivery system containing a linear tumor therapeutic gene (p53) and a chemotherapeutic drug (doxorubicin, DOX) for combined therapy of multi-drug resistant tumor (MCF-7R). This novel co-delivery system, which is structurally similar to a kite, is rationally designed to contain multiple functional groups for the targeted delivery and controlled release of the therapeutic cargoes. The self-assembled DNA nano-kite achieves efficient gene delivery and exhibits effective inhibition of tumor growth in vitro and in vivo without apparent systemic toxicity. These structurally and chemically welldefined co-delivery nano-vectors provide a new platform for the development of gene therapeutics for not only cancer but also a wide range of diseases.

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KEYWORDS: self-assembly, DNA nanotechnology, drug delivery, gene therapy, cancer therapy

Gene therapy is a promising therapeutic strategy to combat many serious gene related diseases. Gene therapy drugs based on viral vector, such as glybera for lipoprotein lipase deficiency (LPLD) and strimvelis for adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID), have been approved for clinical used.1-6 Another potential application of gene therapy is in the treatment of cancers by enhancing the expression of tumor suppressor genes in a desired time frame.7, 8 More than 50% of all types of human tumors harbor mutated p53, an important tumor suppressor gene, which has accordingly become an attractive target for cancer therapy.9-11 Importantly, as patients undergo long-term chemotherapy, the emergence of multidrug resistance increases.12, 13 Extensive researches demonstrate that p53 gene expression can enhance the sensitivity of multi-drug resistant tumors to chemotherapeutics.14 Much attention has therefore been paid to a combined cancer therapy with p53 gene expression and chemotherapy drugs to elicit an enhanced anti-tumor effect. The efficient co-delivery of p53 gene and chemotherapy drugs to the same cells is a great challenge. To address this problem, much effort has been devoted to the development of various co-delivery carriers based on safe non-viral vectors, such as liposomes, polymers, dendrimers, and inorganic nanoparticles.15-21 These delivery vehicles exhibit enhanced cell growth inhibition. However, the carried gene cargo in these systems is mostly a conventional p53 expression plasmid with numerous unnecessary unmethylated CpG motifs in the backbone and the obstinate bacterial remnants, such as lipopolysaccharides (LPS), which may induce an adverse immune response.22, 23 Because of the inherent obstacles of plasmids, linear gene expression cassettes with fewer unmethylated CpG


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motifs generated by polymerase chain reaction (PCR) amplification are attracting much interest.24-27 Owing to the smaller size and lower zeta potential than the conventional plasmids, linear gene expression cassettes are difficult to be encapsulated within classical nanocarriers.28, 29 Efficient delivery of a linear p53 gene and a chemotherapy drug to the target cells of tumor tissue is the first key step in the development of an effective combined cancer therapy.

Due to the addressability and programmability, DNA nanostructures, in particular DNA origami, can be rationally designed to assemble a variety of functional components with nanometer precision.30-37 Remarkably, well sequences designed DNA nanostructures show no obvious cytotoxicity and immunogenicity38-40 in the indicated dosage and have been widely applied in biomedical researches, such as bio-imaging, therapeutic diagnosis, and drug delivery.41-46 With controllable size and shape, DNA origami is a promising candidate for drugs co-delivery vehicles. To the best of our knowledge, targeted delivery of a long and flexible linear gene expression cassette using a DNA nanostructure carrier for combined cancer therapy has not been previously reported.

Herein, we describe a facile and universal strategy to construct DNA nanostructure-based drugs co-delivery system to realize combination of gene therapy and chemotherapy (Figure 1). A biocompatible triangle DNA origami (TO) (Scheme S1) was chosen to efficiently load the chemotherapeutic drug doxorubicin (DOX; a hydrophobic molecule, which intercalates into the base pairs of DNA duplex) and subsequently assemble the capped linear tumor suppressor gene p53. The resultant nanostructure, referred to as TODP, structurally resembles a kite (“nano-kite”). In consideration of the targeted delivery and controlled release, the MUC1 aptamer47-49 and


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disulfide linker are added on TODP, respectively. The multi-functionalized DNA nano-kite exhibits superior anticancer activity against multi-drug resistant tumor (MCF-7R) in vitro and in vivo. Our work presents the first example of DNA origami as a platform for the combined delivery of a chemotherapeutic drug and a precisely organized gene to circumvent the drug resistance in vivo. Our design realized precise control over the size and shape of the nanocarrier and spatial arrangement of active targeting groups and loaded genes.

We initially developed a novel strategy to construct the hybridizable and capped linear tumor suppressor gene LS20-p53-Cap (Figure 2A, lane 4) through classical PCR technology using a 5’ extended primer LS20-F40 and subsequent capping with eukaryotic tRNA D-loop for enhanced stability50-52 (the detailed synthetic method is shown in Figures S1 and S2 in the Supporting Information). This PCR process was based on a reconstructed p53 gene template without unnecessary sequences of p53 plasmid. We designed the triangle DNA origami (TO) to contain two reducible capture strands A16-LAS20 and A45-LAS20 (detailed sequences are provided in Table S1) on one side for the assembly of LS20-p53-Cap gene. The targeted DNA origami with the controlled-release capture strand was assembled in one step according to Rothemund’s method53 (Figure 2A, lane 2).

We subsequently optimized conditions of the hybridization process between the hybridizable gene (LS20-p53-Cap) and targeted DNA origami (TO). As shown in Figure S3A, a greater yield of triangle DNA origami with two copies of the p53 gene (TOP) was generated as the molar ratio of LS20-p53-Cap gene to triangle DNA origami increased. When the ratio reached 4:1, a clear band of assembled product was observed by agarose gel electrophoresis analysis (Figure 2A,


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lane 3). The self-assembly product TOP was then incubated with 5 mM glutathione (GSH, usually in the millimolar range inside cells) to investigate the effect of a reducing environment on the nano-kite. After incubating for 2 h, TOP was efficiently cleaved into two components: TO and the p53 gene (Figure S3B, lane 3). The serum stability of this reducible DNA nanostructure TOP was also evaluated. Gel electrophoresis analysis showed that more than 65% of triangle DNA origami remained after incubation with freshly prepared physiological buffer RPMI-1640 containing 10% FBS with ﬁnal Mg2+ concentration of about 0.6 mM for 24 h (Figure S4). Interestingly, the LS20-p53-Cap gene loaded in the TOP structure exhibited enhanced stability compared to the free linear LS20-p53-Cap gene. This protection may be partly due to a steric effect of triangle DNA origami, shielding the terminal of linear gene from digestion in serum (Figure S4).

We next loaded the targeted origami (TO) with the chemotherapeutic drug DOX. Briefly, 2.5 mM DOX was incubated with 20 nM TO under gentle shaking for 12 h. Assessment of the encapsulation efficiency (EE) of DNA origami to DOX revealed that almost half of the DOX (1.25 mM) was successfully loaded (Figure 2B). Next, we assembled the nanostructure containing both DOX and the p53 gene (TODP) by adding the hybridizable LS20-p53-Cap gene to the purified DOX-loaded DNA origami (TOD). Subsequently, the DOX release efficiency was also evaluated at pH 7.4 (approximate pH of the blood), pH 5.0 (approximate pH of acidic endosomes), and DNase I. The controlled-release ability was observed under pH 5.0 and DNase I incubation (Figure S5). After successfully constructing TODP, atomic force microscopic (AFM) characterization was performed to visualize the self-assembled nanostructures (Figures 2C and


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S6). The AFM image of TODP with compacted sides (indicating the DOX intercalation) and the captured p53 gene was clearly observed (Figures 2C and S6B, TODP panels).

To investigate the cellular uptake efficiency of the co-delivery nano-vectors, the DOX-resistant human MCF-7 breast cancer cells (MCF-7R) was screened and treated with TODP. Confocal images revealed that the targeted DNA origami (labeled with Cy5) with or without the p53 gene, can efficiently penetrate the cell membrane (Figure 3A). Importantly, the accumulation of DOX in the TOD and TODP groups was much higher than the free DOX group (Figures 3A and S7). This result was corroborated by the data of flow cytometry analysis (Figure 3B). Moreover, a higher DOX uptake efficiency was observed in the MUC1 aptamer modified triangle DNA origami with DOX than in the non-targeted group (Figure S7A and B). Additionally, intracellular release of Cy3-labeled p53 gene from the DNA origami was also visualized (Figure S7C).

To study the gene delivery efficiency, we replace the p53 gene with an EGFP gene. As shown in Figure 3C, the percentage of EGFP positive cells after incubation with EGFP gene loaded triangle DNA origami (TOE) was similar to Lipo transfected group. Next, a p53 gene was loaded on the DNA nano-kite and the expression level was assessed by western blot analysis. As shown in Figures 3D and S8, a much greater p53 expression level was present in the TOP and TODP groups, compared with the control group. It has been rigorously demonstrated that DNA damage caused by chemotherapeutic drugs, such as DOX, can induce the up-regulation of the endogenous p53 expression.54, 55 In accordance with this, we found that the p53 levels in the DOX and TOD groups (lacking p53 gene) was also higher than in the control group. This is likely due to the DNA damage resulting from the cumulative DOX uptake.


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Encouraged by the efficient cellular uptake and high p53 expression level elicited by the codelivery nano-vectors, we subsequently investigated their ability to inhibit proliferation of MCF7 breast cancer cells. We first evaluated the DOX-resistance of screened MCF-7R cells using the MTT assay. We found that the IC50 value of free DOX for MCF-7R cells (> 40 μM) was much greater than that of regular MCF-7 breast cancer cells (MCF-7S) (< 5 μM; Figure S9A). After confirming the high DOX resistance of MCF-7R cells, we found no obvious inhibition by coincubation of the cells with free DOX, p53 plasmid, and unstructured DNA strands (mixture of scaffold, staple and aptamer) for 48 hours (Figure S9B). In contrast, TODP exhibited potent inhibition with less than 20% cell viability at a 0.64 nM (Figure 3E). Treatment with the free carrier (TO) did not cause any obvious cytotoxicity at the same concentration, while TOP and TOD resulted in cell viabilities of ~75% and 35%, respectively. Even at 24 h, TODP elicited a remarkable antitumor effect with ~ 40% cell viability remaining (Figure S9C). These results show that our rationally designed co-delivery system can circumvent the drug resistance of MCF-7R.

The in vitro cell proliferation inhibition results demonstrate that DNA origami can work as an efficient carrier for the co-delivery of therapeutic genes and chemotherapeutic drugs with enhanced antitumor effects. To further evaluate the in vivo therapeutic potential against a multidrug resistant tumor, a MCF-7R tumor xenograft model was generated in BALB/c nude mice. The biodistribution of these delivery vectors was initially evaluated in an animal imaging system utilizing the fluorescent signal of Cy5.5-labeled DNA origami. As shown in Figure 4A, TOPCy5.5 with MUC1 aptamer assembly (prepared by assembling DNA origami with multiple Cy5.5 conjugated DNA strands) accumulated at the tumor region in as little as 6 h after tail vein


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injection. After continuous dynamic imaging for 24 h, the mice were sacrificed to collect the tumor and major organs (heart, liver, spleen, lung, and kidney) for ex vivo fluorescence imaging. Quantitative analysis of the fluorescence also indicated that MUC1 aptamer assembled DNA nanostructures located mainly in the tumor tissue with substantially higher intensity than nontargeting group (Figure 4B). Cryosection of tumor tissues (with blood vessels stained by CD31 antibody) after administration of DOX, TOD, or TODP for 24 h were prepared to assess the targeted delivery of DOX. As shown in Figure 4C, the DOX of TOD and TODP was mainly distributed surrounding the blood vessels of the tumor tissues. The early immunogenic response of these co-delivery carriers was also monitored by evaluating the TNF-α, IFN-α and IL-6 level in blood samples from the mice (normal BALB/c and C57/BL6). No significant difference was observed with any of these treatments in the indicated dosage (Figure 4D and S10).

To test whether the dual-therapeutic nanocarrier can inhibit multi-drug resistant tumor growth in vivo, we administered saline, TO, TOP, DOX, TOD, or TODP by tail vein injection at a dosage of 4.0 mg/kg DOX and 0.3 mg/kg p53 gene every 6 days for 3 treatments. As shown in Figures 5A and 5B, free carrier (TO) did not inhibit tumor growth over the saline control in 24 days. In contrast, a marked and consistent suppression of tumor growth was observed with TODP treatment. Meanwhile, the TOP and TOD groups both exhibited moderate inhibition and the level of inhibition elicited by free DOX treatment was between that of the TOP and TOD. After treatments, we examined the relative p53 mRNA levels in the tumor tissues by qRT-PCR. In comparison with the control group, an almost 20-fold higher level of p53 mRNA was observed with TOP and TODP (Figure 5C).


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A noticeable loss of body weight was observed in animals in the DOX treatment group, indicating a high level of systemic toxicity (Figure 5D). Conventional histopathological examination of the major organs (heart, liver, spleen, lung, and kidney) was carried out to study the general toxicity of the nanocarriers in the MCF-7R tumor-bearing nude mice (Figures 5E and S11). No obvious morphological differences were observed in the TODP treated group compared to the saline control, while the group treated with free DOX showed a remarkable disorganization in heart and liver tissues (Figure 5E, labeled by the arrows). All these in vivo data indicate that our rationally designed co-delivery DNA nano-vectors can efficiently and simultaneously deliver a linear tumor therapeutic gene (p53) and a chemotherapeutic drug (DOX) to the targeted tumor region with an enhanced antitumor effect.

In summary, we have reported a novel strategy to construct the DNA nanostructure-based nano-vectors for the co-delivery of a linear gene expression cassette together with a chemotherapeutic drug. This precisely self-assembled DNA nano-kite exhibits several desirable features of a co-delivery vehicle. Firstly, the DNA nanostructures can be easily manufactured by a self-assembly process with reliable high yields. Secondly, the addressable DNA nanostructures are tailor-made for loading with a linear tumor therapeutic gene and a chemotherapeutic drug. Finally, the biocompatible DNA nanostructures can be rationally functionalized with active targeting and controlled-release elements. Using this structurally and chemically well-defined DNA nano-kite, a significantly enhanced tumor inhibition in multi-drug resistant tumor (MCF7R) was obtained in vitro and in vivo. Furthermore, additional functional groups such as RNAbased drugs, gene editing systems, antibody-based drugs, and imaging diagnosis components, may be also introduced into this co-delivery system for synergistic theranostics. With these


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attractive properties, we believe that the co-delivery DNA nano-kite is a promising platform for the development of a new generation of therapeutics for the treatment of both cancer and a range of diseases.

Figure 1. Schematic illustration showing the DNA nanostructure-based combination of gene therapy and chemotherapy against multi-drug resistant (MDR) tumor (MCF-7R) in vitro and in vivo (TO: Triangle DNA Origami, TOD: Triangle DNA Origami with DOX, TODP: Triangle DNA Origami with DOX and the p53 gene).


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Figure 2. Preparation and characterization of DNA-based nanocarrier. A) 1% agarose gel electrophoresis analysis of the self-assembled triangle DNA origami with p53 gene (TOP); lanes 1-4: M13 scaffold, triangle DNA origami (TO), p53 gene loaded triangle DNA origami (TOP), and LS20-p53-Cap gene. B) Time-dependence of encapsulation efficiency (EE) of DNA origami (20 nM) to DOX (2.5 mM). The error bars represent the standard error of the mean of three independent experiments. C) AFM characterization of the sequential constructs in the generation of the targeted DNA origami with DOX and p53 gene (TODP). The insets show enlarged images.


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Figure 3. Cellular level evaluation of DNA nanostructure-based drugs co-delivery system. A) Confocal images and B) flow cytometry analysis of DOX-resistant human MCF-7 (MCF-7R) breast cancer cells after incubation with DOX, TOD, TOP or TODP for 12 h, (excitation wavelength: 488 nm for DOX, pseudocolor red; 633 nm for Cy5-labeled triangle DNA origami, pseudocolor green; scale bars: 20 μm). C) Flow cytometry analysis of MCF-7R cells after incubation with free linear EGFP gene, linear EGFP gene transfected by lipo, or triangle DNA origami with EGFP gene (TOE) for 48 h. The error bars represent the standard error of the mean of three independent experiments. D) Western blot analysis of the protein expression level of p53 gene in MCF-7R breast cancer cells after incubation with PBS, TOD, TOP or TODP for 48 h, respectively (β-actin serves as an internal reference). E) Cell viability of MCF-7R cell after


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treatment with TO, TOP, TOD or TODP for 48 h. The error bars represent the standard error of the mean of three independent experiments (* P < 0.05, *** P < 0.001).


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Figure 4. Biodistribution and immune response of constructed drugs co-delivery system. A) Dynamic biodistribution of Cy5.5-labeled single strand DNA, TOP without aptamer or TOP with aptamer assembly in MCF-7R tumor-bearing nude mice over 24 h (circles indicate the location of tumor). B) Fluorescence imaging and average fluorescence signal of the tumor (T) and major organs (heart: H, liver: Li, spleen: S, lung: Lu, and kidney: K) excised at 24 h post tail vein injection with the indicated treatments The error bars represent the standard error of the mean of three independent experiments. C) Immunohistochemistry analysis of co-localization relationship between the tumor blood vessels (detected by CD31 antibody) and DOX at 24 h post tail vein injection with saline, DOX, TOD, or TODP, respectively. The green fluorescence of CD31 and red fluorescence of DOX of tumor tissues were captured under the same slide view (scale bars: 50 μm, white arrows indicate the DOX distributed surrounding the blood vessels of tumor tissues). D) Analysis of immune response of normal BALB/c mice treated with saline, TO, TOP, DOX, TOD, or TODP by measuring the cytokine levels including TNF-α, IFN-α and IL-6


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in blood sample, respectively. The error bars represent the standard error of the mean of three independent experiments.


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Figure 5. In vivo combined therapy against multi-drug resistant tumor (MCF-7R). A) Tumor volume of MCF-7R tumor-bearing mice after the indicated treatments. The error bars represent the standard error of the mean of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001). B) Photographs of excised tumors from mice treated with saline, TO, TOP, DOX, TOD, or TODP after 24 days. C) Analysis of relative p53 mRNA level of tumor tissues


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after the indicated treatments through qRT-PCR method. The error bars represent the standard error of the mean of three independent experiments (*** P < 0.001). D) Body weight of MCF-7R tumor-bearing mice after the indicated treatments. The error bars represent the standard error of the mean of three independent experiments (** P < 0.01). E) Images of heart, liver, spleen, lung, and kidney sections of MCF-7R tumor-bearing mice treated with saline, DOX, or TODP after H&E staining (arrows indicate the affected areas of organs). Scale bar: 100 μm.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Experimental section, extra figures, and DNA sequences (PDF). AUTHOR INFORMATION Corresponding Author *Baoquan Ding, E-mail: [email protected]. Author Contributions #

These authors contributed equally.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (21573051, 21708004, 21721002), the National Basic Research Program of China (2016YFA0201601), Beijing Municipal Science & Technology Commission (Z161100000116036), Key Research Program of Frontier Sciences, CAS, Grant QYZDB-SSW-SLH029, CAS Interdisciplinary Innovation Team and K. C. Wong Education Foundation. REFERENCES (1) Kotterman, M. A.; Schaffer, D. V. Nat. Rev. Genet. 2014, 15, 445-451. (2) Morrison, C. Nat. Biotechnol. 2015, 33, 217.


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A DNA-Based Nanocarrier for Efficient Gene Delivery and Combined

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