Efficient Intracellular Delivery of RNase A Using DNA Origami Carriers

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Efficient Intracellular Delivery of RNase A Using DNA Origami Carriers Shuai Zhao, Fangyuan Duan, Shaoli Liu, Tiantian Wu, Yingxu Shang, Run Tian, Jianbing Liu, Zhen-Gang Wang, Qiao Jiang, and Baoquan Ding ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21724 • Publication Date (Web): 15 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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ACS Applied Materials & Interfaces

Efficient Intracellular Delivery of RNase A Using DNA Origami Carriers Shuai Zhao,^ † ‡ § Fangyuan Duan, ^ † Shaoli Liu, † ‡ Tiantian Wu, † ‡ Yingxu Shang, † ‡ § Run Tian, † ‡ Jianbing Liu, † Zhen-Gang Wang, † Qiao Jiang,* † and Baoquan Ding*† ‡ # CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, 11 BeiYiTiao, ZhongGuanCun, Beijing 100190, China. ‡ University of Chinese Academy of Sciences, Beijing 100049, China § Sino-Danish College, Sino-Danish Center for Education and Research, University of Chinese Academy of Sciences, Beijing 100049, China # School of Materials Science and Engineering, Zhengzhou University, Zhengzhou 450001, China †

Abstract: Delivery of proteins to carry out desired biological functions is a direct approach for disease treatment. However, protein therapy is still facing challenges due to low delivery efficiency, poor targeting during trafficking, insufficient therapeutic efficacy, and possible toxicity induced by carriers. Here, we present a novel delivery platform based on DNA origami nanostructure that enables tumor cell transportation of active proteins for cancer therapy. In our design, cytotoxic protein ribonuclease (RNase) A molecules are organized on the rectangular DNA origami nanosheets, which work as nanovehicles to deliver RNase A molecules into the cytoplasm and execute their cell-killing function inside the tumor cells. Cancer cell-targeting aptamers are also integrated onto the DNA origami-based nanoplatform to enhance its targeting effect. This DNA origami-protein co-assembling strategy can be further developed to transport other functional proteins and therapeutic components simultaneously for synergistic

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effects and be adapted for integrated diagnostics and therapeutics. Keywords: DNA origami, RNase A, self-assembly, protein delivery, cancer therapy

Protein therapy, which delivers active proteins into cells and carry out desired biological functions, is a direct approach for treating diseases such as diabetes and cancer1-4. Although this approach has been explored for several decades, the majority of protein drug candidates in clinical application are still facing several challenges, including lack of delivery efficiency, poor targeting during trafficking and insufficient therapeutic effects4. For instance, only a small amount of bare protein molecules can pass through the diseased cell membrane by permeation or receptor-mediated endocytosis5-7. Even if a few cytotoxic protein drugs can be transported into cells, they usually accumulate in lysosomes and endosomes, hampering important therapeutic functions that are necessary for effective cell-proliferation inhibition5. Protein therapeutics can be modified to facilitate their cellular internalization5, 8. However, the synthesis or conjugation processes of modified proteins are usually costly and require tedious work. An alternative strategy is nanocarrier based protein delivery, which can incorporate coated shells and functional targeting moieties to address the aforementioned drawbacks9-14. Liposome-wrapped strategies can be used to enhance the stability of proteins in the cellular environment; however, the uptake efficiency is usually affected by the polymer shells10. Cationic lipid-like materials and cell-membrane-covering mesoporous silica nanoparticles have been utilized to carry cytotoxic proteins ribonuclease (RNase) A for


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cancer therapy, demonstrating effective anti-cancer performance11, 12. Rolling circle technique-based DNA flowers have been reported to work as protein nanocarriers13. DNA flowers can encapsulate bioactive proteins with high loading efficiency and enhanced stability. However, in the DNA-wrapping approach, it is difficult to quantify accurately the loaded protein molecules, which may hamper the evaluation of their therapeutic effects. The effective and safe delivery of protein-based therapeutics is still under development. DNA origami nanostructures, which are characteristic of rationally designed geometries, precise spatial addressability and marked biocompatibility, act as a promising candidate for drug delivery15-17. With the advances in chemical synthesis techniques and biotechnological production methods of DNA nanostructures18-20, DNA nanotechnology now allows scalable mass production of DNA nanostructures for applications such as therapeutics and drug-delivery systems. As versatile nanoplatforms, DNA origami templates can assemble multiple payloads to facilitate functional cargo delivery for disease treatment. Several tunable and biologically amenable DNA-based nanomaterials have been developed for cell or tumor-targeted delivery of molecular payloads17,

21-27.

Here, we describe a novel delivery platform

based on DNA origami nanostructure that enables tumor cell transportation of cytotoxic protein RNase A28, 29 for cancer therapy. In our study, DNA origami nanosheets served as nanovehicles to organize RNase A molecules on the surface with nanometer spatial control. The rectangular DNA origami carriers were modified with cancer cell-targeting aptamers on the edges to enhance the uptake efficiency of the origami-based



nanoplatforms. We demonstrated that the RNase A loaded DNA origami transported cytotoxic protein molecules into tumor cells and triggered intracellular RNA degradation-mediated killing of cancer cells. The fabrication of RNase A-origami hybrid structure is schematically illustrated in Figure 1. The rectangular DNA origami nanostructure served as the template, and 54 capture strands (blue) were extended on the surface of the origami sheet for the precise organization of cytotoxic protein RNase A molecules (Figure 1). The rectangular origami nanostructures containing capture strands were assembled according to Rothemund’s method30 with several modifications (see detailed structure design in Figure S1). The sample was prepared by slowly annealing the M13mp18 genome DNA strands (scaffold, grey in Figure 1), corresponding capture strands and staple strands (grey in Figure 1) in a molar ratio of 1:10:10 from 95 °C to room temperature. Folded rectangular origami structures were purified by filters (100 kDa MWCO, Amicon, Millipore) to remove the excessive short DNA strands. Atomic force microscopy (AFM) images showed that the sizes of the rectangular origami sheets were approximate of 90 nm × 60 nm × 2 nm (Figure 2A). Three groups of capture strands were extended on the surface of the origami template and functioned as binding sites to assemble DNA-modified RNase A via hybridization (Figure 1 and S1). The proteinDNA conjugates were synthesized by attaching thiolated polyT oligonucleotides to cytotoxic protein molecules through a crosslinker sulfo-SMCC (sulfosuccinimidyl-4[N-maleimidomethyl] cyclohexane-1-carboxylate) (Figure S2). The raw products were purified by 4% native polyacrylamide gel electrophoresis (PAGE) (Figure S3) followed


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by electro-elution. Purified protein-DNA conjugates were subsequently analyzed by sodium dodecyl sulphate (SDS)-PAGE. The gel image of DNA-modified RNase A (highlighted by a black arrow) is shown in Figure S4. We estimated the concentration and DNA labeling ratio of the purified DNA-conjugated RNase A by using a dyelabeling approach. Cy5-modified 5’-thiol polyT ssDNA strands were used to conjugate to RNase A molecules by sulfo-SMCC chemistry mentioned above. The concentration of Cy5-ssDNA on the protein-DNA conjugate was quantified by measuring the absorbance of Cy5 at 650 nm. Using bicinchoninic acid (BCA) assay for protein quantification, the average DNA-to-protein ratio was calculated to be 2.1 ± 0.3 (See Figure S5 for the detailed information).

Figure 1. Schematic illustration of the self-assembly of functionalized DNA nanostructure and its cellular fate. The long single-stranded M13mp18 genomic DNA



scaffold strands (grey) are folded into rectangular structures through the hybridization of rationally designed staple strands and capture strands. Capture strand groups (blue) extending from the binding sites hybridize with the complementary strands on modified RNase A molecules, resulting in the organization of proteins into linear patterns. Tumor targeting strands (red) containing MUC1 aptamer sequences are positioned at four edges of the rectangular origami. After bounded with protein drugs and targeting ligands, the functionalized DNA nanostructure delivery vessels were administered to MCF-7 cells. We next mixed the purified conjugates with the DNA origami nanostructures and annealed the mixture from 42 °C to 25 °C over the course of 2 h for 6 cycles. The annealed products were purified by filters (100 kDa MWCO, Amicon, Millipore) to remove the excessive protein-DNA conjugates. On the origami template, the extended capture strands with identical polyA sequences were used to assemble each polyT DNA-modified RNase A molecule. These capture strands were able to hybridize with the polyT on the conjugates, allowing the cytotoxic protein molecules to be precisely located on the surface of the DNA origami (Figure 2B). The capture strands were arranged to show linear patterns on the surface of the origami rectangle so that the fabricated protein-origami nanostructures can be clearly imaged by AFM (Figure 2B and S6). Naked RNase A is reported with dimensions of ~ 2.2 nm × 2.8 nm × 3.8 nm 13.

In the AFM images of Figure 2B, the three bright lines on the surface of origami

rectangles represented the patterns of the bounded RNase A molecules (Figure 2A-C). Additional large-scaled AFM images showed that almost 80% of the DNA rectangles


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contained linear patterns of protein cargoes (Figure S6 and S7). According to BCA assay, the average number of RNase A on each DNA origami rectangle was estimated to be 48±5, demonstrating high loading efficiency of protein (Figure S8). The purified Cy5-labelled protein-origami assemblies were next analyzed by agarose gel electrophoresis. As shown in Figure 2D, M13 DNA or DNA rectangular origami (SQ) showed sharp bands under UV illumination. After hybridization with Cy5-polyTRNase A conjugates, the band of DNA nanostructures was observed using the Cy5 channel. The merged gel images showed that the DNA origami co-migrated with Cy5polyT-RNase A, indicating successful assembling of protein molecules to the DNA origami templates (Figure 2D). Moreover, modification of proteins would affect their bioactivities to some extent. So we analyzed the activities of RNase A-DNA conjugates and RNase A-origami assemblies. Quantitative assays demonstrated that both DNAmodified RNase A conjugates and RNase A bound to the DNA origami nanostructures held >80% catalytic activity of unmodified RNase A. (Figure 2E-F). The stability of RNase A-origami assemblies against the enzymatic degradation was investigated (Figure S9). Though a partial degradation of the SQ-RA assemblies after incubation with 5000 or 10000 lysed cells was observed, the majority of nanostructures were not damaged and still could exert tumor cell-killing functions.



Figure 2. Characterization of protein-loaded DNA origami. Rectangular DNA origami A) and protein-loaded ones B) were examined by AFM and representative images are shown. The three bright lines displayed on the surface of the origami square represent the RNase A molecules. Scale bars, 100 nm. C) The heights of the bare origami nanostructures (SQ) and RNase A bounded origami (SQ-RA) were measured by AFM


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images of A) and B). D) Ethidium bromide-stained agarose gel image (merged UV and Cy5 channels) of M13 DNA, bare DNA origami nanosheet and Cy5-RNase A bounded origami structures. The target band is highlighted by a white wire frame. E) The kinetics of bare RNase A, RNaseA-ssDNA or RNase A-origami in absorbance at 520 nm was determined by analyzing their ability to cleave fluorescent-labeled RNase A substrate F)Quantitative analysis of the activities of bare RNase A, RNase A-ssDNA and RNaseorigami. Data represent the mean ± s.d. of three independent experiments. We systematically investigated the RNase A-origami assemblies with uptake efficiency and cellular trafficking assays using human breast adenocarcinoma cell line MCF-7, which selectively overexpresses MUC1, a glycoprotein on the cell surface. We engineered 32 anti-MUC1 DNA aptamer strands as targeting ligands on the origami sheet for enhancing the uptake efficiency of the nanovehicle. For attaching the targeting moieties, the staple strands of the origami rectangles on the four edges of the sheets were extended with aptamer sequences on the 5’ end (Figure S1). Cy5-tagged polyT DNA was prepared to label RNase A molecules or protein-origami assemblies to quantify the cell internalization. We first used flow cytometry to characterize the recognition and cell uptake of RNase A-DNA origami assemblies by MCF-7 cells (Figure 3 A-B). After incubation with malignant cells with 1h at 37 °C, a small amount of Cy5-polyT DNA modified RNase A molecules (red fluorescence) was observed inside MCF-7 cells (Figure 3A). RNase A-DNA origami sheets (SQ-RA) treated cells displayed significant fluorescence enhancement compared with RNase A-polyT DNA conjugates (SQ-RA vs RA, ***p < 0.001, Figure 3B), suggesting that DNA origami



nanocarriers efficiently delivered protein cargoes. Integrated with DNA aptamers, the intensity of the fluorescence signal of MUC1-RNase-origami rectangles was even stronger (SQ-RA vs MUC1-SQ-RA, *** p < 0.001, Figure 3B), indicating the transporting ability of nanocarrier can be enhanced by aptamer decoration in 1h. Confocal imaging was performed on MCF-7 cells to visualize the cell targeting and trafficking process of protein loaded DNA origami nanovehicles. MCF-7 cells were incubated with Cy5-labelled RNase A-DNA origami assemblies for different time periods at 37 °C. Cy5-RNase A or the loaded DNA origami nanostructures were shown as red fluorescent dots in Figure 3C. In comparison with two DNA origami constructs, Cy5-RNase A-polyT DNA conjugates only showed weak cellular uptake inside MCF7 cells after 1h incubation. The cellular uptake results of confocal microscopy agreed well with that of flow cytometry analysis, indicating DNA origami vehicles were able to mediate effective cellular internalization of bounded RNase A. MUC1 aptamer ligands decoration can enhance the delivery capability of DNA origami, elevating the optimal cellular transportation of their molecular cargoes. The intracellular distribution of MUC1 decorated RNase A-DNA origami assemblies were also measured (Figure 3D and S10). Nucleus and lysosome of MCF-7 cells were stained by Hoechst 33342 (blue) and LysoTracker (green), respectively. MUC1 decorated RNase A-DNA origami assemblies were shown as red fluorescent dots, which merged with lysotracker green signals at 1h and partially separated after 24h incubation (Figure 3D and S10). The colocalization of fluorescence (highlighted by yellow arrows) indicated that protein-DNA origami assemblies were endocytosed and accumulated in lysosomes (Figure 3D and


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S10). The co-localization of fluorescence (Cy5 v.s. LysoTracker green) of images was quantified by the Pearson coefficients as a function of incubation time using ImageJ. Importantly, MUC1 modified RNase A-DNA origami constructs were observed to traffic from lysosome to cytoplasm within 24h (partially separated red fluorescent signals, highlighted by red arrows), which is significant to induce the cell-killing effect.

Figure 3. Cellular uptake of RNase A-origami constructs. A) Flow cytometry analysis of MCF-7 cells treated with vehicle solvent (blank), Cy5-DNA-RNase A conjugates (RA, 48nM), RNase A bounded DNA origami (SQ-RA, RNase A 48nM, and DNA origami 1nM) and MUC1 decorated RNase A-DNA origami assemblies (MUC1-SQ-



RA, RNase A 48nM and DNA origami 1nM). B) Quantification of the flow cytometry data in A. Data are shown as mean ± s.d. of three independent experiments. *** p < 0.001. C) Confocal images of the three groups of cells. Scale bars, 100μm. D) Confocal images showing the cellular location of MUC1 decorated RNase A-DNA origami assemblies to MCF-7 cells after incubation for 1 h or 24 h. Hoechst 33342 (25µg/ml) and LysoTracker green (5µM) were used for cell nuclei and lysosome labeling. Scale bars, 30μm. We next examined whether our RNase A bounded DNA origami assemblies could exhibit tumor cells killing activity. Figure 4A showed the viabilities of MCF-7 cells after exposure to increasing concentrations of native RNase A molecules (0, 1, 2, 4, 8 μg/mL, doses equivalent to the DNA origami-loading groups) and RNase A bounded DNA origami assemblies (with a final RNase A concentration from 0, 73, 146, 292, to 584 nM, DNA origami from 0, 1.52, 3.04, 6.08, to 12.16 nM) for 48 h at 37 °C. The results obtained with a cell counting kit-8 (cck-8) indicated escalating cytotoxicities induced by both RNase A-DNA origami constructs and MUC1 decorated constructs in dose-dependent manners, while bare DNA origami nanostructures and native RNase A molecules showed no inhibition of the tumor cells. For RNase A bounded groups with different concentrations, MUC1 decorated nanovehicles showed slightly enhanced cytotoxicity compared to non-targeted DNA origami/protein assemblies. After exposure to RNase A-DNA origami constructs at a maximum concentration of cytotoxic proteins (8 μg/mL, equals to 584 nM), the cell viability decreased to ~ 21% (Figure 4A), demonstrating their prominent anti-tumor performance. The differences


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of cytotoxicity after 48 h treatment by SQ-RA and MUC 1-SQ-RA were not as obvious as cellular uptake results of 1 h treatment. Both SQ-RA and MUC 1-SQ-RA could be transported into cells after long incubation period (Figure S11). The therapeutic effects were confirmed by dead cell staining assay using SYTOX Green. The unloaded RNase A molecules were inefficient to kill MCF-7 cells after 48 h incubation (Figure 4B). Only slight red fluorescence was detected from Cy5-RNase A-DNA treated cells, which also showed no significant morphological changes or green fluorescence compared with control groups. In contrast, enhanced SYTOX green signals were observed from cells treated with RNase A bounded DNA origami for 48 h (Figure 4B). In addition, the MUC1 modified RNase A loaded DNA origami nanostructure showed the highest efficiency in killing MCF-7 cells among all the tested groups, which is in good accordance with cell viability results. The RT-qPCR results indicated that the amount of GAPDH mRNA of MUC 1-SQ-RA treated sample was significantly decreased compared to that of whole medium-treated control (Figure S12). This is in accordance with previous report that RNase A can catalyze the cleavage of cytosolic RNA and lead to cell death28-29.

Figure 4. Cytotoxicity induced by RNase A-DNA origami constructs. A) Cell viability



of MCF-7 cells after administration with equal concentrations of bare DNA origami nanosheets, native RNase A, RNase A bounded DNA origami and MUC1 decorated RNase A-DNA origami assemblies for 48 hours. Data are shown as mean ± s.d. of three independent experiments. ** p < 0.01. B) Bright field and fluorescence images of SYTOX Green dye (Invitrogen) stained MCF-7 cells treated with bare DNA origami nanosheets (12.16 nM), Cy5-DNA-RNase A conjugates (584 nM), RNase A bounded DNA origami (12.16 nM, containing 584 nM RNase A) and MUC1 decorated RNase A-DNA origami assemblies (12.16 nM, containing 584 nM RNase A). Scale bars, 100μm. To conclude, we have demonstrated a novel and effective protein delivery strategy based on self-assembled DNA origami nanostructures. Chemically well-defined DNA origami/protein complexes were constructed and used for inhibition of cancer cells proliferation. Our results indicate that the efficient delivery of cytotoxic protein-DNA origami constructs has the potential to trigger potent anti-tumor effects. The DNA origami-based nanocarrier also enables the facile functionalization required for future application of additional tumor cells types. DNA origami-protein delivery systems could be developed to transport additional biologically active payloads, facilitating biomedical applications in which precise and efficient delivery is required.

ASSOCIATED CONTENT


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Supporting Information. Additional experimental data, the design and the additional characterization of the RNase A-DNA origami constructs. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author *Qiao Jiang: [email protected]; *Baoquan Ding: [email protected] Author Contributions ^

These authors contributed equally to this work.

ACKNOWLEDGMENT This work was supported by the National Basic Research Programs of China (2016YFA0201601, 2018YFA0208900), the National Natural Science Foundations of China (21573051, 31700871, 21708004, 51761145044), the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (21721002), Beijing Municipal Science & Technology Commission (No. Z161100000116036), Key Research Program of Frontier Sciences, CAS, Grant No. QYZDB-SSW-SLH029, CAS Interdisciplinary Innovation Team and K. C. Wong Education Foundation. REFERENCES (1) Leader, B.; Baca, Q. J.; Golan, D. E., Protein Therapeutics: a Summary and Pharmacological Classification. Nat. Rev. Drug Discov. 2008, 7, 21-39. (2) Goeddel, D. V.; Kleid, D. G.; Bolivar, F.; Heyneker, H. L.; Yansura, D. G.; Crea, R.; Hirose, T.; Kraszewski, A.; Itakura, K.; Riggs, A. D., Expression in Escherichia coli of Chemically Synthesized Genes for Human Insulin. Proc. Natl. Acad. Sci. 1979, 76, 106-110. (3) Scott, A. M.; Wolchok, J. D.; Old, L. J., Antibody Therapy of Cancer. Nat. Rev. Cancer 2012, 12, 278-287. (4) Yan, M.; Du, J.; Gu, Z.; Liang, M.; Hu, Y.; Zhang, W.; Priceman, S.; Wu, L.; Zhou,



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Efficient Intracellular Delivery of RNase A Using DNA Origami Carriers

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