Beside carrying the code of life, DNA has been used as building blocks for creating two- and three-dimensional nanostructures. Through taking advantage of its highly programmable sequences and precise recognition moieties, the highly specific base-pairing interactions of DNA molecules not only allow for the building of static DNA nanostructures, but also enable the construction of smart dynamic structures capable of serving as smart platforms and active components in a myriad of bionanotechnological applications. Such DNA-based devices could be used in targeting cells and triggering the cellular actions in the biological environment. This project paper reviews DNA origami based nanostructures that can be used as devices for cell therapy and other biomedical applications.
Since the discovery of DNA in 19531, the focus of DNA research had been predominantly focus on its biological properties. Due to its unique chemical and structural composition DNA, DNA was introduced into the field of nanoscience as a new type of building block 2-6. DNA origami was used as a powerful tool to fabricate nanoscale materials 7-12. Due to its remarkable molecular recognition properties, mechanical rigidity, stability and easily custom synthesis, DNA origami technique allows the formation of various shape of nanostructures, ranging from two dimensional to three dimension complex structures. Many research group have recently focused their attention on construction of DNA-based materials for biomedical-applications such as drug delivery system13-16.
The delivery of drug molecules to specifically targeted site is a requirements to avoid side effects and increase the therapeutic efficacy of the drug. However, this requirement is still unmet due to the lack of effective drug delivery system. In the last several decades, a great number of organic and/or inorganic nanomaterials have been generated for this purpose17. Because DNA origami is biocompatible, easily controllable, and offer simple loading methods of biomolecules, DNA origami based systems provide many advantages over to the conventional drug delivery approaches such as polymer and liposomes18-22. In addition, the high drug loading efficiency and effective cellular intake render DNA origami a “smart” building block for more sophisticated drug carriers23,24. This project paper reviewed on the DNA-based nanostructures that can be used as nanocarriers of therapeutic molecules for specific and effective drug delivery system.
Switchable Drug Delivery from Origami Robots
DNA-origami based dynamic structure that are stimuli-responsive, for instance, change its structure in response to release a drug, is considered as promising platform for targeted drug delivery. In this section, several of these nanostructures will be discussed, including a 3-dimensional DNA-origami box with a sequence-specific opening lid 9 and a barrel-shaped structure that can carry and release cargos based on intracellular logic-gated aptamer binding events 25.
Andersen et al.9 created a DNA-origami box with a programmable lid that has the potential to both ‘sense and act’, responding to an externally supplied DNA “key” as shown in Figure 1. This dual lock key system functionss as an equivalent AND gate requiring two inputs to open. Temporary closing strands on the lid were shorter than the complementary strands on the box’s front face leaving a toehold binding site for the “key” strands; the key strands could then replace the closing strands via toehold-mediated DNA strand displacement 26 to open the box. To confirm this switching process, the DNA-origami box and key-lock system was functionalized with Cy3 (donor) and Cy5 (acceptor) fluorophores. The opening of the box was monitored in fluorescence kinetic experiments when DNA ‘keys’ were supplied.
Figure 1: Illustration of the controlled opening process of the DNA box9
One limitation of this system is that the lid opening process is not reversible, so Zadegan et al 27 further advanced this design by reducing its size, and incorporating a reversible locking system that would allow multiple switching events: opening and closing cycles of the boxes lid. The reversible locking system was achieved through attaching a single strand DNA to both the lid and the box face, and these strands form hairpins structure to close the box, leaving a loop region to function as a toehold for key strands. In absence of the key, the lid stays closed; when supplied with keys, they bind to the toehold region and unzip the lock to open the lid. These keys can be strand-displaced from the lid through the toehold binding region on themselves using a second input strand that is complementary to the key strand. Once the keys are displaced, the lock re-hybridized to close to lid. Unlike the original design by Andersen et al. 9, the switching of the lid is repeatable 27. This modification provides the promising potential for loading therapeutic molecules in the box, and selectively released the drugs on instruction.
In 2012, Douglas et al.25 took the DNA origami based delivery system one step further. Douglas et al. designed a DNA nanorobot capable of targeting cells. This nanorobot was able to deliver molecular payloads to the targeted cells. In their design, a barrel-shaped nanostructure was fabricated, composed of two halves with ssDNA entropic springs on one end and two locks on the other end, similar to the aforementioned box design. The keys used in this study are equivalent AND gate made of aptamers, they work by responding to the input of cell surface antigen of either binding or not binding, and producing out of either closed or adapt a conformational change to release the payloads inside. In this design, the cargo – gold nanoparticles or fluorescently labeled Fab’ antibody fragments (to human HLA-A/B/C antigen) was loaded into the barrel by complementary hybridization of staples facing the inside of the barrel. Douglas et. al first demonstrated the ability of such nanorobots to selectively open according the lock sequence used, and tested the opening of the nanorobots when culturing with a different cancer cell lines including leukemia and lymphoma cells lines. Their results showed that these nanorobots were able detect cells from a mixed population down to a single cell level25. Finally, the authors described some therapeutic applications with the nanorobots. They showed upon recognition of cell-surface receptor on the leukemia cells, the barrel was opened to release Fab antibodies that binds to CD33 and CDw328, which lead to inhibition of growth in these cells. Also, they loaded antibodies to CD3ε and flagelin in the nanorobots, these nanorobot “scavenge” flagellin, and opened by T-cells and activated the T-cells through the CD3ε and flagellin binding. This nanorobots showed precise and targeted cell delivery of cargo molecules as well as sequestration of molecules to deliver to the targeted cells. The logic-gated locks with specific recognition of cell membrane marker were designed to improve the targeting specificity. These DNA origami nanorobots can be potentially used for simultaneous delivery of multiple therapeutics in a predefined manner.
Figure 2: Design of aptamer-gated nanorobot 25
One of the biggest challenge in an effective drug delivery system is to deliver the molecules across the cell membranes. Synthetic DNA-based nanochannel/nanopores that can span the lipid membranes of the cells can be used to direct molecules to deliver into the cells. To this end, the potential for functional synthetic DNA nanopores has been described by Burns et al.28 who designed synthetic pores can be inserted in cell membranes to control the flux of molecules across the cell membrane. After the molecules are delivered into the cell, they induce cytoxicity which lead to the apoptosis of the cells 28. The membrane insertion strategy here was to include a hydrophobic belt of charge neutral ethyl phosphorotioate groups. When these nanopores were cultured with cervical cancer cells, they inserted into the lipid membranes and therefore facilitated transport of the therapeutic molecule across the membrane to induce cytotoxicity.
Moreover, Langecker et al. developed a DNA-origami-based channel that can span the lipid bilayer of the cells 29. In this study, a DNA-origami-based structure was created, made of an inner structure which will go crossed the membrane, as well as an outer barrel structure that stayed external to the cell membrane. They designed a synthetic origami nanopore that consisted of a stem that was capable of penetrating and spanning into a lipid membrane. It also had a barrel-shaped cap that could adhere to the cis side of the membrane(Figure 3). The nanopores constructed out of DNA conducted electricity when a voltage was applied, and it displayed similar gating properties as natural protein nanopores due to the structure’s thermal fluctuations. Langecker et al. also demonstrated that the origami nanopore was useful for single-molecule analysis to investigate DNA hairpin unzipping and G-quadruplex unfolding. DNA origami nanopores could be incorporated into a lipid membrane either by using a large number of hydrophobic functionalization on their sides or by using streptavidin linkages between biotinylated nanopores and lipids. Given the large number of protein/peptide channels that span lipid bilayer membranes to control transport in a cell, the ability to insert synthetic channels in cell membranes may provide a new insight to facilitate the cellular intake of the therapeutic cargos.
Figure 3: Schematic of a DNA-origami-based synthetic nanopore29.
Chemotherapy, Photodynamic and Photothermal Therapy
Jiang et al. used a variety of DNA origami structures to delivery doxorubicin (Dox) to kill the cancer cells30. Because of the presence of abundant duplex structure, DNA origami is able to load a large amount of Dox by its affinity with the Dox molecules. The Origami-Dox complex system exhibited high cytotoxicity on breast adenocarcinoma cancer cells as well as Dox-resistant cancer cells. The complex increase the cellular intake of Dox presumably through inhibition of lysosomal acidification, which results in cellular redistribution of the drug to the nucleus.
Figure 4: DNA-origami-DOX complex for delivery of DOX31
After showing the in vitro function of the complex, the same group further evaluated the in vivo performance of the origami-Dox complex by tail vein injection of the complexes into a tumor mouse model and found that it exhibited prominent therapeutic efficacy without any detectable systemic toxicity 31. DNA origami structures offers high degree of customization of loading Dox into DNA origami structure through simple intercalating Dox to DNA duplexes, and therefore Högberg et al. demonstrated that the drug load and release profile can be tuned with different origami design32. By comparing the twisted and untwisted nanotube structure, Högberg et al. showed that the twisted DNA nanotube had a superior drug delivery rate for its loading efficiency and releasing rate. Besides the increased delivery efficacy, Halley et al found that the Dox-DNA origami complex can also circumvent the efflux pump-mediated drug resistance in leukemia cells to achieve clinically relevant drug concentrations 33.
In addition to delivery of therapeutics, DNA origami could also enable photothermal and photodynamic therapy 34,35. For example, a DNA origami-gold nanorod complex were able to generate heat with near-infrared red irradiation, and these heat was able to kill the MCF cancer cells36.They further advanced this technology to develop an optoacoustic imaging agent 35.The DNA origami-gold nanorod complex improved the imaging resolution of the mouse tumor tissue. Also, it performed photothermal therapy to inhibit tumor growth using near-infrared irradiation (NIR). Besides its application in photothermal therapy, DNA origami was also used as agents for intracellular photodynamic therapy. In a study by Zhuang et al, a photosensitive agent BMEPC, was loaded into the DNA origami through intercalation37. When the photosensitizer-loaded DNA origami complex was taken up by the tumor cells, BMEPC could produce free radicals to induce apoptosis of cancer cells upon light irradiation.
Figure 5: A DNA origami triangle and nanotube can be used to load anti-tumor drugs, gold nanorods, and photosensitizers for chemotherapy, photodynamic therapy and photothermal therapy34-36.
The development of DNA nanotechnology, essentially DNA origami based structures, demonstrated that DNA is not a genetic material of cells, but also can be considered as a powerful building blocks for nanoscale structures and materials. These DNA-origami based structures could be used as drug delivery agents for assisting more targeted and effective cellular therapies.
1 Watson, J. D. & Crick, F. H. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171, 737-738 (1953).
2 Liedl, T., Sobey, T. L. & Simmel, F. C. DNA-based nanodevices. Nano Today 2, 36-41, doi:10.1016/S1748-0132(07)70057-9 (2007).
3 Liu, H. & Liu, D. DNA nanomachines and their functional evolution. Chem. Commun., 2625-2636, doi:10.1039/b822719e (2009).
4 Teller, C. & Willner, I. Functional nucleic acid nanostructures and DNA machines. Curr. Opin. Biotechnol. 21, 376-391, doi:10.1016/j.copbio.2010.06.001 (2010).
5 Krishnan, Y. & Simmel, F. C. Nucleic acid based molecular devices. Angew. Chem. Int. Ed Engl. 50, 3124-3156, doi:10.1002/anie.200907223 (2011).
6 Willner, I. & Willner, B. Biomolecule-based nanomaterials and nanostructures. Nano Lett. 10, 3805-3815, doi:10.1021/nl102083j (2010).
7 Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297-302, doi:10.1038/nature04586 (2006).
8 He, Y. et al. Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra. Nature 452, 198-201, doi:10.1038/nature06597 (2008).
9 Andersen, E. S. et al. Self-assembly of a nanoscale DNA box with a controllable lid. Nature 459, 73-76, doi:10.1038/nature07971 (2009).
10 Dietz, H., Douglas, S. M. & Shih, W. M. Folding DNA into twisted and curved nanoscale shapes. Science 325, 725-730, doi:10.1126/science.1174251 (2009).
11 Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414-418, doi:10.1038/nature08016 (2009).
12 Han, D. et al. DNA origami with complex curvatures in three-dimensional space. Science 332, 342-346, doi:10.1126/science.1202998 (2011).
13 Li, J., Fan, C., Pei, H., Shi, J. & Huang, Q. Smart drug delivery nanocarriers with self-assembled DNA nanostructures. Adv. Mater. 25, 4386-4396 (2013).
14 Surana, S., Shenoy, A. R. & Krishnan, Y. Designing DNA nanodevices for compatibility with the immune system of higher organisms. Nat. Nanotechnol. 10, 741-747, doi:10.1038/nnano.2015.180 (2015).
15 Linko, V., Ora, A. & Kostiainen, M. A. DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices. Trends Biotechnol. 33, 586-594, doi:10.1016/j.tibtech.2015.08.001 (2015).
16 Chen, Y.-J., Groves, B., Muscat, R. A. & Seelig, G. DNA nanotechnology from the test tube to the cell. Nat. Nanotechnol. 10, 748-760, doi:10.1038/nnano.2015.195 (2015).
17 Davis, M. E., Chen, Z. G. & Shin, D. M. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat. Rev. Drug Discov. 7, 771-782, doi:10.1038/nrd2614 (2008).
18 Duncan, R. The dawning era of polymer therapeutics. Nat. Rev. Drug Discov. 2, 347-360, doi:10.1038/nrd1088 (2003).
19 Tanner, P. et al. Polymeric vesicles: from drug carriers to nanoreactors and artificial organelles. Acc. Chem. Res. 44, 1039-1049, doi:10.1021/ar200036k (2011).
20 Kakizawa, Y. & Kataoka, K. Block copolymer micelles for delivery of gene and related compounds. Adv. Drug Deliv. Rev. 54, 203-222 (2002).
21 Ganta, S., Devalapally, H., Shahiwala, A. & Amiji, M. A review of stimuli-responsive nanocarriers for drug and gene delivery. J. Control. Release 126, 187-204, doi:10.1016/j.jconrel.2007.12.017 (2008).
22 Charoenphol, P. & Bermudez, H. Aptamer-targeted DNA nanostructures for therapeutic delivery. Mol. Pharm. 11, 1721-1725, doi:10.1021/mp500047b (2014).
23 Zhang, F., Nangreave, J., Liu, Y. & Yan, H. Structural DNA nanotechnology: state of the art and future perspective. J. Am. Chem. Soc. 136, 11198-11211, doi:10.1021/ja505101a (2014).
24 Chao, J. et al. Structural DNA nanotechnology for intelligent drug delivery. Small 10, 4626-4635, doi:10.1002/smll.201401309 (2014).
25 Douglas, S. M., Bachelet, I. & Church, G. M. A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831-834, doi:10.1126/science.1214081 (2012).
26 Yurke, B., Turberfield, A. J., Mills, A. P., Jr., Simmel, F. C. & Neumann, J. L. A DNA-fuelled molecular machine made of DNA. Nature 406, 605-608, doi:10.1038/35020524 (2000).
27 Zadegan, R. M. et al. Construction of a 4 zeptoliters switchable 3D DNA box origami. ACS Nano 6, 10050-10053, doi:10.1021/nn303767b (2012).
28 Burns, J. R., Seifert, A., Fertig, N. & Howorka, S. A biomimetic DNA-based channel for the ligand-controlled transport of charged molecular cargo across a biological membrane. Nat. Nanotechnol. 11, 152-156, doi:10.1038/nnano.2015.279 (2016).
29 Langecker, M. et al. Synthetic lipid membrane channels formed by designed DNA nanostructures. Science 338, 932-936, doi:10.1126/science.1225624 (2012).
30 Jiang, Q. et al. DNA origami as a carrier for circumvention of drug resistance. J. Am. Chem. Soc. 134, 13396-13403, doi:10.1021/ja304263n (2012).
31 Zhang, Q. et al. DNA origami as an in vivo drug delivery vehicle for cancer therapy. ACS Nano 8, 6633-6643, doi:10.1021/nn502058j (2014).
32 Zhao, Y.-X. et al. DNA origami delivery system for cancer therapy with tunable release properties. ACS Nano 6, 8684-8691, doi:10.1021/nn3022662 (2012).
33 Halley, P. D. et al. DNA Origami: Daunorubicin-Loaded DNA Origami Nanostructures Circumvent Drug-Resistance Mechanisms in a Leukemia Model (Small 3/2016). Small 12, 307-307, doi:10.1002/smll.201670014 (2016).
34 Kong, F. et al. Gold Nanorods, DNA Origami, and Porous Silicon Nanoparticle-functionalized Biocompatible Double Emulsion for Versatile Targeted Therapeutics and Antibody Combination Therapy. Adv. Mater. 28, 10195-10203, doi:10.1002/adma.201602763 (2016).
35 Du, Y. et al. DNA-Nanostructure-Gold-Nanorod Hybrids for Enhanced In Vivo Optoacoustic Imaging and Photothermal Therapy. Adv. Mater. 28, 10000-10007, doi:10.1002/adma.201601710 (2016).
36 Jiang, Q. et al. A Self-Assembled DNA Origami-Gold Nanorod Complex for Cancer Theranostics. Small 11, 5134-5141, doi:10.1002/smll.201501266 (2015).
37 Zhuang, X. et al. A Photosensitizer-Loaded DNA Origami Nanosystem for Photodynamic Therapy. ACS Nano 10, 3486-3495, doi:10.1021/acsnano.5b07671 (2016).