DNA Nanotechnology-Enabled Drug Delivery Systems - Chemical

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Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

DNA Nanotechnology-Enabled Drug Delivery Systems Qinqin Hu,†,‡,⊥ Hua Li,§,∥,⊥ Lihua Wang,#,◊ Hongzhou Gu,*,†,‡,§ and Chunhai Fan*,#,◊ †

Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China Department of Systems Biology for Medicine, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China § Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China ∥ Research & Development Center, Shandong Buchang Pharmaceutical Company, Limited, Heze 274000, China # Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ◊ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China ‡

ABSTRACT: Over the past decade, we have seen rapid advances in applying nanotechnology in biomedical areas including bioimaging, biodetection, and drug delivery. As an emerging field, DNA nanotechnology offers simple yet powerful design techniques for self-assembly of nanostructures with unique advantages and high potential in enhancing drug targeting and reducing drug toxicity. Various sequence programming and optimization approaches have been developed to design DNA nanostructures with precisely engineered, controllable size, shape, surface chemistry, and function. Potent anticancer drug molecules, including Doxorubicin and CpG oligonucleotides, have been successfully loaded on DNA nanostructures to increase their cell uptake efficiency. These advances have implicated the bright future of DNA nanotechnology-enabled nanomedicine. In this review, we begin with the origin of DNA nanotechnology, followed by summarizing state-of-the-art strategies for the construction of DNA nanostructures and drug payloads delivered by DNA nanovehicles. Further, we discuss the cellular fates of DNA nanostructures as well as challenges and opportunities for DNA nanostructurebased drug delivery.

CONTENTS 1. Introduction 2. Structural DNA Nanotechnology 2.1. Origin of Structural DNA Nanotechnology 2.2. Tile-Based Bottom-Up Assembly 2.3. Origami Assembly 2.4. Single-Stranded Tile Assembly 2.5. Hybrid of Oligonucleotide Tile, Origami, and Single-Stranded Tile 2.6. Nanoparticle-Templated DNA Nanostructures 2.7. Other Strategies for the Construction of DNA Nanostructures 3. DNA Nanostructure-Based Drug Delivery 3.1. Primary Targeting―Getting to the Organ 3.2. Secondary Targeting―Getting to the Cell and Cellular Subunits 3.2.1. Gateways into the Cell 3.2.2. Cellular Targeting 3.2.3. Creating Artificial Channels with DNA Nanotechnology 3.2.4. Subcellular Targeting 3.3. Delivery of Drug Molecules by DNA Nanotechnology 3.3.1. Small Molecules 3.3.2. Oligonucleotides © XXXX American Chemical Society

3.3.3. Proteins 3.3.4. Inorganic Nanoparticles 4. Cellular Fate of DNA Nanostructures 5. Challenges and Outlook Author Information Corresponding Authors ORCID Author Contributions Notes Biographies Acknowledgments Abbreviations References

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1. INTRODUCTION Both chemical and biomolecular drugs (e.g., iRNA, antibody) have intrinsic issues in maximizing their drug functions in healthcare. The former often has poor solubility and tends to cause unwanted side effects and toxicity problems, whereas the latter is prone to enzymatic degradation and difficult to penetrate

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Special Issue: Nucleic Acid Nanotechnology

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Received: November 2, 2017

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DOI: 10.1021/acs.chemrev.7b00663 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. Origin and the principle of structural DNA nanotechnology. (A) Holliday junction in biology. (B) Holliday junction in DNA nanotechnology. (C) 2D DNA lattice self-assembled from four-armed junctions. (D) 3D DNA scaffold (purple) serves as a template to organize macromolecules (green). Reproduced with permission from ref 15. Copyright 1982 Elsevier.

toxic elements or residuals13 and are usually difficult to be degraded,14 resulting in safety concerns. As an emerging delivery system, DNA nanostructures exhibit great potential to fulfill the ultimate goal of drug delivery, reaching the maximal efficacy with the minimal toxicity. DNA is a genetic material in nature. Thus, it is inherently biocompatible and biodegradable. In biology, through A-T, G-C Watson−Crick base pairing (bp), DNA mainly adopts the B-form structure, which is a double-stranded helix with about 2 nm in diameter and 3.4 nm (or 10.4 bp) per helical turn. The persistence length of B-DNA is about 50 nm or 150 bp, which makes DNA a relatively stiff polymer in the nanoscale. Invented by Seeman in the early 1980s,15,16 DNA nanotechnology has grown exponentially in the past two decades. Owing to the intrinsic properties of DNA including predictable intermolecular interactions (Watson−Crick base pairing), convenient automated chemistry synthesis, convenient modifying enzymes, locally stiff polymer, externally readable code, high functional group density, and prototype for many derivatives, numerous DNA or DNA-based nanostructures,17−62 from simple to complex and from small to large, have been selfassembled with precisely controlled size and shape at one, two, or three dimensions, as well as with controllable surface chemistry and dynamic function. These characteristics make DNA nanostructures unique platforms with great delivery capacities and offer new prospects for the bottom-up construction of nanoscale drug carriers.63−74

the cell membrane. To overcome these barricades it is necessary to develop active and targeted systems for delivery of drug molecules. An ideal drug delivery system would be capable of preserving the activity of drug molecules by protecting them against degradation, improving the solubility, and mitigating toxicity and other biological side effects of drug molecules, penetrating in vivo barriers like epithelium and cell membrane, specifically targeting cells and controlling drug release. Many different drug delivery systems have been developed so far,1,2 including natural systems such as viruses,3 biomimetic systems such as red blood cell mimics,4 synthetic organic systems such as liposomes5,6 and cationic dendric polymers,7 and synthetic inorganic systems such as gold nanoparticles8 and carbon nanomaterials.9 These natural, bioinspired, or synthetic platforms bypass the physiochemical limitation and barriers of drug molecules to increase the drug loading efficacy, the body circulation time, and the cellular uptake. Many of them have been in clinical trials and several approved for clinical therapeutics by the Food and Drug Administration (FDA). Despite the advances in the development of drug delivery systems, each of the current drug carriers has some limitation. For examples, viruses can only deliver short DNAs into the cells. There are risks of random insertion sites, cytophathic effects, and mutagenesis.10 The cationic surface charges of dendric polymers are inherently cytotoxic,11 and the heterogeneous distribution in size not only affects the drug loading capacity but also increases the immune toxicity.12 Many inorganic nanomaterials contain B


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Figure 2. Oligonucleotide tiles and tile-based 3D DNA nanostructures. (A) From left to right, DX,26 TX,75 PX,29 and JX229 tiles. Adapted with permissions from refs 26 and 75. Copyright 1993 and 2000 American Chemical Society. Adapted with permission from ref 29. Copyright 2010 Nature Publishing Group. (B) 3D DNA crystal assembled from the tensegrity triangle tile. Adapted with permission from ref 61. Copyright 2009 Nature Publishing Group. (C) DNA polyhedron including cube,20 tetrahedron,89 and octahedron.90 Adapted with permissions from refs 20, 89, and 90. Copyright 1991 Nature Publishing Group, 2005 American Association for the Advancement of Science, and 1994 American Chemical Society. (D) DNA tetrahedron, dodecahedron, and buckyball assembled from three-point star tiles and their corresponding cryo-EM images. Adapted with permission from ref 92. Copyright 2008 Nature Publishing Group. (E) DNA octahedron and icosahedron assembled from four-point and five-point star tiles, respectively, and their corresponding cryo-EM images. Adapted with permissions from refs 93 and 94. Copyright 2008 National Academy of Sciences and 2009 American Chemical Society.

In this review, we will discuss DNA nanostructure-based drug delivery systems. We will start with the origin of DNA nanotechnology, introducing the immobile Holliday junction in DNA nanotechnology, the key element for the construction of DNA nanostructures. We will then move to the design milestones in the development of DNA nanotechnology, including Seeman’s oligonucleotide tile-based bottom-up approach, Rothemund’s DNA origami approach, as well as Yin’s single-stranded tile approach. We will also discuss the hybrid of the above three techniques and some others, including design of the nanoparticle, the metal−organic, and the rollingcircle amplification (RCA) templated DNA nanostructures.

Following this will be a brief description of primary and secondary targeting for drug delivery. Then we will discuss drug molecules that have been loaded and delivered by DNA nanostructures, including CpG oligos, aptamers, siRNA, CRISPR-Cas9, nanoparticles, and so on. In addition, we will discuss the cellular fate of certain DNA nanostructures. Finally, we will touch on RNA nanotechnology, a derivative of DNA nanotechnology, and finish with challenges and our thoughts on the future direction of DNA nanostructure-based drug delivery. C


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2. STRUCTURAL DNA NANOTECHNOLOGY

of a designed, self-assembled, 3D crystal based on the DNA tensegrity triangle.61 This strongly supports Seeman’s vision on DNA nanotechnology in 1982. Besides the assembly of 1D, 2D, and even 3D lattices from individual DNA tiles, numerous DNA nano-objects have also been built with Seeman’s tile-based bottom-up strategy. Following the initial immobile 4-arm junction, junctions with less or more arms including 3-arm,86 5-arm,87 6-arm,87 8-arm,88 and even 12-arm88 were created. On the basis of these building blocks, polyhedra of different sizes and shapes such as cubes,20 tetrahedron,89 and octahedron,90 were assembled (Figure 2C). Through multistep ligations, Seeman’s group constructed a covalently closed cube-like molecular complex containing 12 equal-length double-helical edges arranged about eight vertices. Each of the six faces of this object is a single-stranded cyclic DNA, doubly catenated to four neighboring strands, and each vertex is connected by an edge to three others. However, to synthesize such a cube, laborious work was required and the yield was not high. Later, Turberfield’s group reported a facile way to build a DNA tetrahedron with four synthetic DNA strands. By a quick annealing step (95 to 4 °C in 30 s), tetrahedrons assembled with a yield of around 95%. The shape of the tetrahedron can be visualized under an atomic force microscope (AFM). Bundling more DNA duplexes together can increase the tile’s rigidity, which has been demonstrated during the assembly of periodic arrays with DNA tiles. This principle also applies for the construction of robust DNA nano-objects self-assembled from DNA tiles. Yan and LaBean reported an improved second generation of the four-arm junction tile, which contains four fourarm DNA branched junctions pointing in four directions (Figure 2E).91 This point star-like tile is composed of nine strands, with one strand participating in all four junctions. Bulged T loops were placed at each of the four corners inside the tile core to decrease the probability of stacking interactions between adjacent fourarm junctions and to allow the arms to point to four different directions. Comparing to Seeman’s original four-arm junction, the new generation contains two DNA duplexes bundled in each arm instead of one. Hence, such a tile is more rigid and robust for various bottom-up assemblies. Mao’s group further extended the design of point star tiles.92−95 They exploited sequence symmetry to design a series of DNA tiles, including three-point, four-point, and five-point star tiles (Figure 2D and 2E). Each arm of the point star tile is more rigid due to the bundling of two duplexes. The adjacent arms are linked by the bulged loops to provide appropriate bending angles. With longer bulged loops, the point star tiles bend more from their original geometric plane. For the threepoint star tiles, Mao’s group reported that tetrahedrons selfassembled from the tiles with a loop length of five bases and a low concentration of 75 nM, while the tiles with a three-base loop and a 50 nM concentration yielded dodecahedrons and a three-base loop at a 500 nM concentration generated buckyball structures. Each of the above DNA nano-objects is around 14 nm in diameter. Similarly, by tuning the flexibility of four- and fivepoint star tiles, octahedrons and icosahedrons were successfully assembled, respectively (Figure 2E). The robustness of the polyhedrons was further confirmed by cryo-EM 3D reconstruction. In the first two decades after the origin of DNA nanotechnology, Seeman’s oligonucleotide-assembled tiles and such tile-based nanoarrays and nanostructures were the major research scope and topic in this nascent field. By exploiting sequence symmetry, a couple of synthetic DNA strands in a one-

2.1. Origin of Structural DNA Nanotechnology

DNA is a linear polymer that carries genetic information in biology. Occasionally nonlinear DNA occurs in cells. For example, during genetic recombination, homologous DNAs exchange genetic information through a Holliday junction, which is a branched nucleic acid structure that contains four doublestranded arms joined together (Figure 1A). Because of the sequence symmetry, Holliday junctions are mobile intermediates. The four individual arms can slide through the junction back and forth. In 1982, Seeman proposed that by breaking the sequence symmetry of the four strands that make up the Holliday junction, one should be able to lock the junction in a stable state (Figure 1B).15 Using four synthetic DNA strands with programmed sequences to avoid symmetry, Seeman demonstrated that such an immobile Holliday junction indeed formed.16 By adding cohesive ends or single-stranded overhangs to the four-arm branched junction, Seeman proposed this junction could act as tiles to self-assemble into infinite twodimensional lattices (Figure 1C). The number of branched arms in a junction might not stop at four. With six synthetic strands, a six-arm junction might be created and act as a tile to further assemble in the three dimensions (3D) to generate a 3D DNA lattice (Figure 1D). Seeman pointed out that such a 3D DNA lattice could serve as a 3D template to organize and orient macromolecules, such as proteins through DNA−protein interactions, in a 3D crystalline fashion to enable their structure determination. The immobile Holliday junction together with the proposal of using 3D DNA scaffolds for structural biology is considered as the origin of “structural DNA nanotechnology”. Along the development trajectory of the field are several breakthroughs in the design technique of DNA nanostructures. Also, branched from its initial goal, DNA nanotechnology has shown promising applications in many different aspects including biosensing, bioimaging, diagnosis, and drug delivery. 2.2. Tile-Based Bottom-Up Assembly

The immobile Holliday junction was the first tile built in DNA nanotechnology. It can be considered as one simple crossover between two DNA duplexes. Later, more robust DNA tiles, such as double crossover (DX),26 triple crossover (TX),75 paranemic crossover (PX),29,56 paranemic crossover with two juxtaposed sites (JX2),29,56 six-helix bundles,76 etc., were constructed by Seeman’s group using multiple junctions to bundle two or more DNA duplexes together (Figure 2A). By doing so, the rigidity of those tiles is greatly enhanced. For example, the DX molecules were characterized twice as stiff as linear DNA duplex,77,78 and the estimated persistence length for six-helix bundle was 1−5 μm, about 20−100-fold stiffer than the DNA duplex.79 All of the tiles mentioned above have shown the ability to self-assemble into large 1D and 2D arrays,80−84 including one-dimensional tubes and ribbons and two-dimensional arrays with stripes, triangle, quadrilateral, and hexagon patterns, within micrometer scales through tile−tile interactions via cohesive ends. Besides 1D and 2D DNA arrays, efforts to fabricate 3D DNA lattices have never been stopped. In 2004, Mao’s group reported a triangular motif called a tensegrity triangle (Figure 2B).85 It is a rigid DNA motif with 3-fold rotational symmetry, consisting of three helices that are directed along linearly independent vectors. Their helix axis directions do not share the same plane. Hence, the tensegrity triangle has the potential to extend in the three dimensions to self-assemble into 3D lattices. In 2009, Seeman and Mao’s group reported the crystal structure at 4 Å resolution D


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Figure 3. DNA origami. (A) Rothemund’s 2D origami. Adapted with permission from ref 45. Copyright 2006 Nature Publishing Group. (B) Shih’s 3D origami with honeycomb lattice. Adapted with permission from ref 24. Copyright 2009 Nature Publishing Group. (C) Shih’s 3D origami with twist and curvature. Adapted with permission from ref 21. Copyright 2009 American Association for the Advancement of Science. (D) Yan’s origami with curvature. Adapted with permission from ref 101. Copyright 2011 American Association for the Advancement of Science.

pot annealing can generate nano-objects, periodic arrays, and even 3D crystals. The elegance and powerfulness of this design approach are obvious. On the other hand, there are also some constraints for this approach. To design a tile, the sequences must be optimized via computer-aided programs. Also, the formation of a tile often requires exact stoichiometric and purity

control of the individual oligonucleotide strands, which are time consuming. The complexity of nanostructures is limited due to relatively simple geometric shapes or repetition of basic tiles. Furthermore, the addressable surface area and diameter of the self-assembled DNA nanoarrays and nano-objects are also limited by the small size of the individual tiles. In the next E


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closely stacked 3D object. Due to the multiple packing, 3D DNA objects made by this strategy are often very rigid. A series of 3D nanostructures such as mololith, square nut, railed bridge, slotted cross, and stacked cross with precisely controlled dimensions ranging from 10 to 100 nm were constructed on the honeycomb lattice and demonstrated under transmission electron microscope (TEM) (Figure 3B). Later, Shih’s group reported that multilayer 3D DNA origami can be packed not only on a honeycomb lattice but also on a square lattice.98 The square lattice provides a more natural framework for designing rectangular structures and an option for a more densely packed architecture, as well as the ability to create surfaces which are more flat than that given by the honeycomb lattice. Using the rigid 3D DNA origami elements as building blocks, Shih’s group hierarchically assembled a rigid heterotrimeric wireframe icosahedron.24 By connecting rigid DNA origami helix-bundle units into 3D shapes with multiple segments of unpaired scaffold, Shih’s group created tensegrity 3D DNA origami structures with very high strength-to-weight ratios and great resilience.99 Furthermore, in the same year (2009) of the invention of 3D DNA origami, Shih also expanded the design space of accessible 3D DNA origami shapes to include a rich diversity of nanostructures with designed twist and curvature.21 Shih and co-workers found that targeted insertions and deletions of base pairs in a 3D DNA origami caused the DNA bundles to develop a twist of either handedness or to curve (Figure 3C). The degree of curvature could be quantitatively controlled, and a radius of curvature as tight as 6 nm was achieved. Shih and coworkers used this strategy to build several multilayer 3D nanostructures with precisely controlled curvatures, such as square-toothed gears. Yan’s lab further developed the twist-and-curvature design approach. By precisely tuning the bending and twisting of a 2D DNA origami, they were able to make a Mobius strip.100 Later, they trained the double-helical DNA to bend following the rounded contours of the target object. Concentric rings of DNA were used to generate in-plane curvature, which is maintained and stabilized by rationally designed geometries and crossover networks (Figure 3D).101 Combining in-plane and out-of-plane curvatures, they assembled a series of single-layered DNA nanostructures with high curvature, such as 2D concentric rings and 3D spherical shells, ellipsoidal shells, and a nanoflask. Besides the 3D structures with high curvature, single-layered container-like 3D origami objects have also been constructed by folding and joining multiple single-layered 2D origami sheets.102,103 For example, Andersen et al.102 reported that by folding six 2D DNA origami sheets along the M13mp18 genomic DNA and by adding the staple strands to bridge the edges of the sheets that make the six faces of the box, a cuboid structure of external size 42 × 36 × 36 nm3 was generated. Similarly, Yan’s group constructed a tetrahedron origami container with the length of the edge around 54 nm by joining four 2D triangular origami sheets into an enclosed configuration.103 These hollow 3D DNA objects could be potential drug carriers with large capacity to encapsulate substantial molecules inside them, which will be discussed later. New progress on the origami design technique keeps coming out. Recently, Högberg’s team developed a general method to fold arbitrary DNA polygonal digital meshes including bottle, helix rod, and waving stickman.104 Using a routing algorithm based on graph theory and a relaxation simulation that traces scaffold strands through the target structures, the team made the design process highly automated. Similarly, Bathe’s group

section we will talk about another design approach that resolves most if not all of the above issues. 2.3. Origami Assembly

In 2004, Shih and co-workers designed and synthesized a 1669nucleotide, single-stranded DNA molecule through clonal production. With the help of five 40-mer synthetic oligo DNAs, the long DNA strand folded into an octahedron structure by a simple denaturation−renaturation procedure.50 This work laid the foundation of the following DNA origami technique. In 2006, Rothemund described a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes.45 To design a desired shape, a 7-kilobase M13mp18 genomic DNA was raster filled into the shape as a scaffold, and over 200 short oligonucleotide staple strands were carefully chosen to hold the scaffold in place (Figure 3A). After being mixed in a pot, the scaffold and staple strands self-assembled in a single step. Various shapes such as squares, triangles, smiley faces, and five-pointed stars were created and visualized under AFM with a spatial resolution of 6 nm. Structures made by this socalled “DNA origami” approach are roughly 100 nm in diameter in Rothemund’s work. The invention of DNA origami is a milestone in the development of DNA nanotechnology. There are several advantages for this new design technique. First, the shape of an origami structure is usually well defined, with a size about an order of magnitude larger than the previously reported DNA nano-objects self-assembled in the tile-based bottom-up way. Second, to design a desired DNA origami, sequence optimization is generally not required. In fact, since most of the origami shapes are built on M13mp18 genomic DNA, sequences of the staple strands are actually predetermined once the folding pathway is defined. Currently, a few modeling software programs have been developed to assist users for the design of origami structures.96,97 By setting up the scaffold pathway and its starting point, sequences of hundreds of staple strands can be generated automatically and exported in a single file, ready for the following chemical synthesis. The DNA origami technique enables convenient design and assembly, especially for beginners and those that are interested in the field of DNA nanotechnology. Third, unlike the tile-based bottom-up approach which often requires a strict stoichiometric ratio and purity control of the involved oligonucleotide strands, DNA origami can be assembled with raw, unpurified staple strands. The molar ratio of the staple strands to the M13mp18 long strand is usually 10 to 1 or even lower. Also, the yield of a desire shape usually reaches above 90%. All of these properties make DNA origami a simple yet powerful design technique, which stimulates and accelerates the development of DNA nanotechnology. However, it should be noted that the basic concepts or the principles behind DNA origami remain unchanged, that is, using crossovers to connect or bundle DNA duplexes together to form nanostructures. In the origami strategy, multiple crossovers are generated at every possible position within an area defined by M13mp18 scaffold. Perhaps it is the high density of crossovers and the packing of multiple duplex domains at once that makes the origami shapes well defined. Rothemund’s original origami strategy is suitable for the creation of 2D flat shapes. In 2009, Shih’s group extended and improved the origami strategy to building custom 3D shapes formed as pleated layers of helices constrained to a honeycomb lattice (Figure 3B).24 Shih’s 3D origami strategy can be simply considered as packing multiple single-layer 2D origamis into a F


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Figure 4. Single-stranded tile-based assembly. (A) 2D assembly with single-stranded tiles. Adapted with permission from ref 55. Copyright 2012 Nature Publishing Group. (B) 3D assembly with single-stranded tiles. Adapted with permission from ref 31. Copyright 2012 American Association for the Advancement of Science.

2.4. Single-Stranded Tile Assembly

reported a general, top-down strategy to design nearly arbitrary DNA architectures autonomously based only on target shape.105 Unlike conventional origami designs built from close-packed helices, structures assembled with the above two methods generally have a more open conformation with one or two helices per edge and are therefore more stable under the low-salt biological condition. From Seeman’s oligonucleotide tile to Rothemund’s origami, DNA nanotechnology underwent a bottom-up to top-down transition in design techniques. The origami approach makes the design versatile, and computer-aided automation makes the design handy. However, there are also certain drawbacks for origami. Generally, hundreds of synthetic staple strands are required for an origami construction, setting the total price relatively high. Purifying and accumulating the intact origami structures to certain concentrations for biotechnological applications require tedious work and perhaps large amounts of sample. The size of an origami object is restricted by the long single-stranded scaffold, which is mainly defined by the 7kilobase M13mp18 genomic DNA. Overcoming these drawbacks would make DNA origami a more powerful design technique for various applications.

In 2012, Yin’s group pushed Seeman’s tile-based assembly to the extreme of simplicity.31,55 Unlike Seeman’s tile-based assembly, where a few oligonucleotide strands form tiles and through tile− tile interactions generates large assembly, Yin and co-workers designed single-stranded tiles (SST) consisting of 42-base DNA strands (Figure 4A).55 These strands can be divided into four domains, each acting as sticky ends to bind with four local neighbors during self-assembly. To simplify the design process and minimize the cost for assembling many different shapes, Yin and co-workers treated a desired shape as a molecular canvas. Each of the constituent SST strands acts as a pixel and is folded into a 3 nm by 7 nm tile and attached to four neighboring tiles. The shape is produced by one-pot annealing of all of those strands that correspond to pixels covered by the target shape. Over 100 distinct 2D shapes, including the 26 capital letters of the Latin alphabet, 10 arabic numerals, 23 punctuation marks and other standard keyboard symbols, 10 emoticons, 9 astrological symbols, 6 Chinese characters, and various miscellaneous symbols, were constructed using this simple strategy. Later, Yin’s group extended this strategy to 3D design.31 They used 32-base short synthetic DNA strands as DNA bricks (Figure 4B). Similar to the 2D design, each brick is a modular component and binds to four local neighbors. A master brick collection G


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Figure 5. Hybridized assembly of oligonucleotide tile, origami, and single-stranded tile. (A) Origami seed guiding the algorithmic assembly of DNA tiles. Adapted with permission from ref 107. Copyright 2009 National Academy of Sciences. (B) Origami 2D lattice assembled from origami tiles. Adapted with permission from ref 108. Copyright 2011 Wiley-VCH. (C) 2D crystals assembled from single-stranded tiles. Adapted with permission from ref 109. Copyright 2014 Nature Publishing Group.

defines a 3D molecular canvas with dimensions of 10 × 10 × 10 voxels. Each 8-base pair interaction between neighboring bricks defines a voxel with dimensions of 2.5 × 2.5 × 2.7 nm3. By choosing subsets of bricks from the canvas, over 100 distinct 3D objects were created, including shapes with sophisticated surface features and intricate interior cavities and tunnels. Single-stranded tile-based assembly represents another milestone of design technique in DNA nanotechnology. Comparing to the DNA origami approach, the lack of a long single-stranded scaffold DNA in SST-based assembly removes the size restriction of a desired shape, which is previously defined by the limited

length of the scaffold DNA. Unlike Seeman’s oligonucleotide tile-based assembly, purification and exact stoichiometry of DNA components are not required in SST-based assembly. Prescribed 2D and 3D shapes can form by choosing the right pixel- or voxelcorresponding SSTs for one-pot annealing. Despite these advantages, there are still some drawbacks for the SST approach. As the size of the desired shape gets bigger, more synthetic SSTs are needed, meaning the increase in cost and complication in sequence design and optimization. Also, the assembly yield of desired shapes is inversely proportional to the total number of SSTs in the reaction mixture. H


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Figure 6. Nanoparticle-templated DNA nanostructures. (A) Macroscopic aggregates by isotropic AuNPs-assisted assembly of DNA. (B) Core−satellite clusters by anisotropic nanoparticles-assisted assembly of DNA. Adapted with permission from ref 70. Copyright 2015 American Association for the Advancement of Science.

2.5. Hybrid of Oligonucleotide Tile, Origami, and Single-Stranded Tile

stoichiometric quantities and treated as oligonucleotide tiles for bottom-up assembly, yielding a 2D origami array with 1 μm diameter. Ke and co-workers assembled DNA brick crystals with prescribed depths (Figure 5C).109 Single-stranded brick tiles and strands that connect each crystal unit (also considered as singlestranded bricks) were mixed together for one-step, nonhierarchically annealing. DNA crystals were obtained and grew to micrometer size in the lateral dimensions with depths up to 80 nm (Figure 5C). Sophisticated 3D features such as cavities and tubular crystals were displayed in the report. It is not surprising to see that these approaches can be mixed together very well. Regardless of tile- or origami-based designs, the central concepts of the above design approaches are the same, which are bundling or packing DNA helices via the immobile Holliday junction crossovers (half crossovers in SST approach)

In Seeman and Yin’s tile strategies, DNA nanostructures are designed and self-assembled in a bottom-up way. For Rothemund’s origami strategy, DNA nanostructures are designed in a top-down fashion. Nevertheless, the tile and origami approaches are not against each other. In fact, they complement each other very well in many aspects. For example, Winfree and co-workers demonstrated the use of origami as seeds to guide the algorithmic self-assembly of DNA tiles (Figure 5A).106,107 A complex binary counting pattern was generated using the origami seed to specify the initial value for the counter. Seeman’s team explored the 2D crystallization of origami tiles (Figure 5B).108 Two cross-shaped origamis were first assembled with the origami strategy. Then they were mixed together in I


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Figure 7. Other strategies for construction of DNA nanostructures. (A) Metal-assisted assembly of DNA nanotubes. Adapted with permission from ref 139. Copyright 2011 Wiley-VCH. (B) RCA-assisted formation of AuNP-linked DNA nanoribbons. Adapted with permission from ref 149. Copyright 2015 Wiley-VCH. (C) RCA-assisted formation of DNA nanoclew. Adapted with permission from ref 147. Copyright 2014 American Chemical Society.

to allow the structural expansion in the directions perpendicular to the DNA helical axis as well as interacting through the complementarity of Watson−Crick binding to extend the DNA nanostructure along the direction of the DNA helical axis. There are alternative ways to Watson−Crick base-pairing-based elongation. For example, Rothermund and co-workers showed

that they could harness the geometric arrangement of blunt-end stacking interactions to create diverse bonds to connect different origami shapes,110 which may guide strategies for molecular recognition in systems beyond DNA nanostructures. In addition, besides tile, origami, and hybrid approaches, there are some other mature methods to create DNA-based nanosystems, such as J


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analogs to common molecules, including linear CO2, triangular BF3, and tetrahedral CH4, were also assembled using a similar strategy (Figure 6B, bottom).131,132

nanoparticle-templated DNA nanostructures, which will be discussed next. 2.6. Nanoparticle-Templated DNA Nanostructures

2.7. Other Strategies for the Construction of DNA Nanostructures

In the 1990s, at the early stage of DNA nanotechnology, the rigidity of DNA nanostructures was a problem due to the not-sorobust mechanical property of a single-DNA duplex. Seeman figured out that the rigidity could be enhanced by packing multiple DNA helices via a series of immobile Holliday junctions, leading to the creation of conformationally restricted and structurally robust DX, TX, PX, JX2, and 6-helix bundle tiles. Alternatively, Mirkin chose to blend in a rigid inorganic or organic nanoparticle such as gold nanoparticle (AuNP) and made it act as a core to template and organize DNA in a surfacenormal orientation to add rigidity to the assembly.111 The success of Mirkin’s approach leads to the establishment of the nanoparticle-templated DNA nanotechnology.70 To date, nanoparticle-templated DNA nanostructures have been used in a number of biological applications such as gene regulation112 and molecular probing.113,114 In 1996, Mirkin and co-workers reported a method for assembling colloidal gold nanoparticles rationally and reversibly into macroscopic aggregates.111 With terminal thiol modifications, single-stranded DNA oligonucleotides bound to the surfaces of two batches of 13 nm colloidal AuNPs. By adding an oligonucleotide duplex (linker) with “sticky ends” that were complementary to the two grafted sequences, the AuNPs selfassembled into aggregates. Mirkin’s initial work focused on exploring the linker DNA to hybridize the ssDNA−AuNP conjugates into macroscopic networks.115 Mirkin and co-workers found that the interparticle distances in the assemblies were proportional to the length of the DNA linkers,116,117 and significant correlation between the particle positions was only clear when the DNA linkers were double stranded.117 These results indicate that the rigid core and multiple duplexed linkers (DNA bonds) together maintain the configuration of the nanoparticle-templated DNA assemblies. Using this strategy, well-ordered super lattices with considerable long-range periodicity were created with AuNPs (Figure 6A).118−120 Since the only requirement for building up the DNA bonds is that they are attached to a solid core, a variety of inorganic nanoparticles, including catalytic noble metals, magnetic oxides, and semiconductors, were subsequently conjugated with DNA and assembled by the same strategy.121−123 Moreover, with a molecule that provides an initiation site for silica growth on a glass surface, AuNP−DNA lattices have been grown in a preferred crystallographic direction on the silica support (Figure 6A, bottom).124,125 The simplicity of the protocol allowed DNA−nanoparticle super lattices amenable to become solid-state materials, thus expanding their potential use in many aspects. Later, Mirkin’s group and others put a lot of efforts on making asymmetric DNA functionality conjugated on the surface of nanoparticles.126−130 One example is to use anisotropic nanoparticles that present regions of greater chemical reactivity that can be selectively functionalized with DNA (Figure 6B). This has been done at regions of high curvature, like tips and edges, where the native surfactant coating required for the synthesis of such nanoparticles is less dense and thought to be replaced by thiolmodified DNA strands more readily. Anisotropic core−satellite clusters were created by selective incorporation of DNA at the tips and edges of plate-like triangular nanoprisms128 or at the tips of gold nanorods (Figure 6B, top).130 In addition, colloidal

Other than the tile-based, origami-based, and nanoparticletemplated construction strategies, transition metal ions are also frequently used to build metal−DNA junctions and assemblies. The plan for this strategy is to obtain DNA−metal hybridized nanostructures in which DNA duplex serves as a nanoscale rigid molecular arm to spatially position addressable transition metals that are usually coordinated by certain organic molecular pocket and form vertices. In 2004, Sleiman and co-workers synthesized a branched ruthenium(II)−DNA complex,133 in which two parallel DNA strands are linked to a relatively rigid RuII tris(bipyridine) center. By designing the sequence of DNA to allow complementary hybridization, they were able to selfassemble a discrete metal−DNA cyclic nanostructure. Later, Sleiman’s team explored more transition metal ions for DNA− metal nanostructures.134−140 For example, Sleiman and coworkers used a DNA-templated method to form a chiral (righthanded) metal−DNA junction, which contains a single copper(I)−bisphenanthroline unit at its central point and four singlestranded DNA arms of different sequences (Figure 7A).139 The copper-binding ligand diphenylphenanthroline (dpp) was chemically synthesized in the middle of two DNA strands. The two strands were then brought together by a linker DNA to achieve close proximity of the two dpp ligands. Also, the addition of copper ions resulted in the formation of a highly stable Cu(dpp)2 complex that even resisted PAGE denaturation. Using these DNA−metal junctions as building blocks, Sleiman’s team generated DNA−metal nanotubular assemblies.139 Besides metals, the same team also demonstrated that hydrophobic− hydrophilic copolymers can be covalently linked with DNA and introduced into DNA nanostructures for dynamic functions.141 Ultrathin 2D nanosheets of layered transition metal dichalcogenides (TMDs) such as MoS2 are emerging as a class of key materials in chemistry and electronics due to their unconventional properties. Very recently, in 2017, Li et al.142 harnessed the S-atom defect sites on the exfoliated surface of MoS2 to incorporate S atom-terminated (thiolated) DNA anchor molecules into the otherwise inert TMDs. The DNA anchors were further programmed to form interspacers to assemble multiple layered MoS2 TMDs into superstructures of stacked nanosheets. Rolling-circle amplification (RCA) has also been demonstrated to be an efficient way for producing DNA nanostructures. During RCA, usually the Phi29 DNA polymerase goes around circular DNA templates many times to produce long singlestranded concatemeric DNAs with each repeated unit having sequences complementary to the template. Using the origami strategy, the long DNA produced from RCA can be directly folded into the scaffold for origami construction.143,144 Also, RCA has been used for the production of staple strands required by origami.145 Thus far a series of nanoshapes, including nanotubes,146 nanoribbons,143 nanoclews,147 and nanoballs,148 has been created in the RCA fashion. In 2013, Ouyang et al. used only three 32-base staple strands to fold each 96-base periodic unit of a RCA product into a rectangle and simultaneously connect the adjacent rectangle units to form nanoribbons.143 These RCA-based nanoribbons are easily internalized by cells. Later, Yan et al. developed a novel 3D K


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Figure 8. General considerations for nanoparticle delivery to specific organs (top) and a wide range of 3D DNA nanostructures with potential for carrying drug loads (bottom). Adapted with permissions from refs 150 and 179. Copyright 2012 Nature Publishing Group and 2014 American Association for the Advancement of Science.

integrated into the nanoclew to enhance the loading of the anticancer drug doxorubicin. Unlike the tile-, the origami-, or even the metal-based assembly strategies, RCA usually produces nanostructures with undefined size. However, RCA-based nanostructures generally have large loading capacity due to the repeated units. In addition, RCA not only allows for the creation of nanostructures with unique shapes but also can integrate multiple functional groups, such as cancerspecific targeting molecules, into the nanostructure assemblies. Thus, it is also frequently used as a drug carrier for targeted delivery. We herein have reviewed the history of structural DNA nanotechnology and summarized most if not all of the current strategies to construct DNA nanostructures, including tile-based, origami-based, hybridized, nanoparticle-templated, metal-assis-

superstructure based on the growth and origami folding of DNA on AuNPs (Figure 7B).149 The 3D superstructure contains a nanoparticle core and dozens of 2D DNA nanoribbons (belts) folded from long single-stranded DNAs grown in situ on the nanoparticle by RCA. Multiple functional groups such as drugs and targeting agents can be loaded onto the 3D superstructures by either a merging or an intercalating mechanism for theranostic purposes (Figure 7B). In 2014, Gu’s group developed a bioinspired cocoon-like anticancer drug delivery system consisting of a DNasedegradable DNA nanoclew, which was embedded with an acidresponsive DNase nanocapsule (Figure 7C).147 The DNA nanoclew was assembled from a long-chain single-stranded DNA synthesized by RCA. Multiple GC-pair sequences were L


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nanoparticles with a size less than 80 nm were trafficked to lymph nodes via subcutaneous administration.172,173 It is known that lipid and lipid-like materials tend to accumulate in the liver. In addition, particle size also plays an important role for liver targeting (Figure 8). Nanoparticles with a size less than 100 nm can cross liver fenestrae and target hepatocytes, while intravenously administered nanoparticles with a size more than 100 nm are more likely taken up by activated Kupffer cells that reside within and near the liver vasculature and so do not reach the hepatocytes.174,175 Through inhalation or intravenous administration, nanoparticles can be delivered to the lungs. Inhaled particles with low density (5 mm) are more likely retained in the lungs for prolonged periods of time (Figure 8).176 The route of intravenous administration was reported to be more effective using nanoparticles with sizes larger than 200 nm, because they are generally trapped in lung capillaries.177 Moreover, nanoparticles with positive surface charge are more likely to accumulate and reside in the lungs.178 DNA nanotechnology enables the precise assembly of welldefined nanostructures with sizes ranging from a few nanometers to over 100 nm,179 a range that can meet the size requirements for targeted delivery to most organs, including the brain, lung, liver, and lymph nodes (Figure 8). Negative charges on the surface may favor targeted delivery of DNA nanoparticles to organs like lymph nodes but disfavor organs like the lung and brain (Figure 8). The latter situation could be resolved by taking advantage of the maturation of DNA chemical synthesis, which allows for hundreds of modifications on DNA. By modifying only a few component strands, the surface property of a DNA nanostructure can be completely changed. For example, both Shih’ group and Lin’s group reported that thiol modification on DNA enabled covalent linking of DNA and lipid molecules, which turned the hydrophilic surface of DNA nanostructures to be hydrophobic.180,181 Alternative to chemical modifications, synthesized cationic molecules with strong affinity to DNA could potentially turn the surface charge of a DNA nanostructure from negative to positive.182,183 In principle, DNA nanotechnologyenabled nanostructures can potentially meet almost all necessary aspects including particle size and shape, surface charge and properties, material composition, and so on, for specific organ targeting. Once in the organ, nanoparticles need to be further directed to the diseased cells or even subcellular compartments. Before discussing DNA nanotechnology-associated cellular targeting, we will first review the known gateways and mechanisms for cell entry.

ted, and RCA-directed assembly approaches. In the next section, we will discuss the principles for targeted drug delivery and how DNA nanostructures can enter cells as well as what molecules have been delivered into cells by which DNA nanostructures.

3. DNA NANOSTRUCTURE-BASED DRUG DELIVERY Currently, the major work on DNA nanostructure-based drug delivery targets cancer cells. In fact, before reaching cancer cells, nanoparticles must be able to first accumulate or localize in the specific organ or organs where the cancel cells reside. This step is called primary targeting. Once in the organ or organs, drug molecules carried on the nanoparticles need to be directed to the cancer cells and potentially to a specific subcellular location within a cancer cell, a step called secondary targeting.150 In sections 3.1 and 3.2, we will review our current knowledge on primary and secondary targeting as well as discuss the pros and cons of using DNA nanostructures for targeting. In section 3.3, we will review the drug molecules that have been delivered via the aid of DNA nanotechnology. 3.1. Primary Targeting―Getting to the Organ

Most life-threatening cancers occur in the brain, lung, liver, lymph, and bone. Particle size and shape, surface charge and properties, material composition, and the route of administration, all affect the localization of nanoparticles to specific organs (Figure 8). Due to the existence of the blood−brain barrier, only certain materials can cross from the circulation into the cerebrospinal fluid. Thus, the brain is always considered as the most challenging organ to target with intravenously administered nanoparticles.151 Generally, metabolic products, hormones, and gases are exchanged across the blood−brain barrier.152 Under certain circumstances, for example, when a brain tumor develops, it may disrupt the integrity of the blood− brain barrier,153 giving opportunities for drug molecules to access the brain.154−157 Several groups reported that only small nanoparticles with sizes of less than 15 nm can efficiently go across the blood−brain barrier.155,158 For particles in the range of 15−100 nm, the ability to penetrate the brain decreases exponentially with size (Figure 8).158,159 Surface modifications with lipophilic moieties and neutral charge are known to enhance brain uptake of nanoparticles.159−162 In addition, by targeting leukocytes and macrophages in the circulation, nanoparticles might be trafficked into the brain and ultimately be released.163,164 The bone is actively involved in cancer metastases. However, little is known for targeting nanoparticles to the bone. Previous research focused on small molecules and proteins targeted to hydroxyapatite, the calcium-containing mineral component that constitutes up to 50% of bone. Compounds such as alendronate and aspartic acid adhere to the bone and have been harnessed for the accumulation of nanoparticles in the bone (Figure 8).165 Lymph nodes have mainly been targeted using cell-based nanotechnologies in an indirect process. Nanoparticles are usually designed to bind to the receptors on the surface of leukocytes, followed by being transferred to lymph nodes as part of a normal immune response.166 Generally, nanoparticles with negative charges are taken up faster by leukocytes (Figure 8).166,167 Interestingly, polyethylene glycol modification on the surface of nanoparticles can inhibit uptake by leukocytes.168−170 Moreover, other routes of administration can also achieve lymph node targeting. For example, through intrapulmonary administration, noncationic particles with a size range of 6−34 nm were reported to be trafficked rapidly to local lymph nodes.171 Also,

3.2. Secondary Targeting―Getting to the Cell and Cellular Subunits

3.2.1. Gateways into the Cell. The cell membrane, also known as the plasma membrane or cytoplasmic membrane, is the natural barrier to protect sophisticated cellular organelles from the extracellular environment and maintain the differences between the cytosol and the extracellular environment. It is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. Large molecules, including nanosized particles, can pass through the membrane via highly regulated processes called endocytosis.73,184−186 In general, endocytosis can be classified into two broad categories: phagocytosis, which is the uptake of particles usually larger than 250 nm, and pinocytosis, which is the uptake of fluids and small particles that are not taken by phagocytosis. Phagocytosis in M


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Figure 9. Gateways and intracellular fate of nanocarriers.

the cell membrane, resulting in the formation of large (>1 μm) and heterogeneous vesicles termed as macropinosomes. Later, these macropinosomes further fuse with lysosomes for degradation. Note that this internalization process is nonspecific.73 Clathrin is a main cytosolic protein that makes coated vesicles. Its triskelion shape allows the protein unit interacting with each other to form a polyhedral lattice that surrounds the vesicle. Clathrin-mediated endocytosis (CME), especially receptordependent CME, is the major pathway for the internalization of extracellular nanoscale materials. In this case, nanomaterials modified with ligands on their surface are first recognized by the receptors on the plasma membrane, which triggers the bending of the membrane and the assembly of clathrin-coated pits with the assistance of several accessory proteins. This results in the formation of a cargo-containing clathrin-coated vesicle in the cytosol (Figure 9). Then the coated proteins start to fall off from the vesicle, generating an early endosome of about 120 nm in diameter. Later, a prelysosomal vesicle involving various enzymes secreted by Golgi fuses with the early endosome to form a late endosome and eventually mature as a lysosome for the degradation of captured cargos.185,189,190 The other type of CME is receptor independent, in which cells recognize the cargos through the unselective hydrophobic and electrostatic interaction between the cargos and cell membrane. The receptorindependent CME has a much slower internalization rate comparing with the receptor-dependent one.191 Therefore, it is important to design drug carriers modified with ligands that can

mammals is restricted to specific cells including macrophages, neutrophils, dendritic cells, and monocytes, while pinocytosis is adopted by all types of cells through four different mechanisms: macropinocytosis, clathrin-mediated endocytosis, caveolaemediated endocytosis, as well as clathrin- and caveolaeindependent endocytosis.73,184−186 The gateways and intracellular pathways of engulfed molecules by different endocytic mechanisms are depicted in Figure 9. In some cells and single-celled organisms, phagocytosis is involved in the acquisition of nutrients. While in multicellular mammalian, phagocytosis has been harnessed to remove pathogens and cell debris. Phagocytosis generally begins with the recognition of the invaders such as viruses, microorganisms, and even nanovehicles used for drug delivery, followed by the engulfment of those particles by the actin-driven macrophages through specific ligand−receptor interactions, which in turn results in the formation of cellular phagosome. As the phagosome matures, it then fuses with the lysosome to form the phagolysosome, in which the acidic condition and various enzymes help to degrade the ingested particles.187,188 Generally, particles with a size of more than 250 nm can be internalized effectively through this pathway. Similar to phagocytosis, macropinocytosis is also an actindriven endocytic process (Figure 9). However, unlike phagocytosis in which specific substances are transported through the membrane, micropinocytosis takes up small particles nonselectively by forming membrane protrusions in the first step. Then these protrusions collapse onto the particles and fuse with N


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Figure 10. Examples for the endocytic mechanism of DNA nanostructures. (A) Schematics of cellular uptake, transport, and fate of DNA tetrahedron. Adapted with permission from ref 199. Copyright 2014 Wiley-VCH. (B) TEM images of cells incubated with SNAs, verifying the caveolae-mediated endocytosis. cav, caveolae; Nu, nucleus. Adapted with permission from ref 200. Copyright 2013 National Academy of Sciences. (C) Internalization of Zn/DNA clusters. Zn/DNA cluster was added to the cells that were preincubated in the presence of either MβCD or CPZ. (Bottom right panel) TEM image to illustrate the formation of macropinosomes, ruffling the cell surface into open cups. Adapted with permission from ref 203. Copyright 2015 Wiley-VCH.

present in many specific cells, such as smooth-muscle cells, fibroblasts, endothelial cells, and adipocytes.192 The most prevalent protein associated with caveolae structures is caveolin-1, which is pertinent not only to caveolae formation in the membrane but also the subsequent vesicular production.193 Caveolae vesicles move through the cytoplasm with the assistance of an activated signaling transduction cascade and can transfer to either nonenzymatic caveosomes or other nonlysosomal compartments, like endoplasmic reticulum, and eventually reach nucleus. During the transportation, the caveolar

specifically target their receptors on the cell membrane to trigger the receptor-dependent CME pathway for faster and more accurate drug delivery. Moreover, the drug carriers need to be able to escape from the early endosome as soon as possible to avoid lysosomal degradation and to reach the specific subcellular compartment if such destination is planned for targeting. Caveolae-mediated endocytosis (CvME) is another receptordependent pinocytosis (Figure 9). Caveolaes are defined as flaskshaped, cholesterol- and sphingolipid-rich smooth invaginations with a diameter of 50−80 nm in the plasma membrane. They are O


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demonstrating that sequence composition was another tunable property for the enhancement of delivery efficiency.202 It is worthy to note that the type of employed endocytic pathway greatly depends on the size of nanostructures. Caveolar vesicles can efficiently carry particles less than 80 nm in diameter. TDNs and SNAs are about 7 and 20 nm in diameter, respectively.199 Both in principle fall in the suitable size range for a caveolae-mediated pathway and were experimentally confirmed to go through CvME when incubated with certain types of cells. For particles generally larger than 100 nm, CME or macropinocytosis or multiple endocytic mechanisms may contribute to their cellular internalization. Lim et al.203 constructed the self-assembled Zn/DNA and Zn/siRNA nanoclusters with average sizes of ∼200 nm (Figure 10C upper) and studied their cellular uptake mechanism. The Zn/DNA cluster was prepared with yoyo-labeled DNA to monitor the cellular uptake by tracking the fluorescence signal. During the cellular uptake process, Lim et al. treated HEK293 cells with different endocytosis inhibitors, including chlorpromazine (CPZ), an inhibitor for a clathrin-mediated pathway, and methyl-βcyclodextrin (MβCD), an inhibitor for a clathrin-independent pathway. The results showed that the cellular uptake of the Zn/ DNA cluster decreased to 85% in the presence of CPZ and to 30% in the presence of MβCD (as shown in Figure 10C, bottom left), indicating that both clathrin-dependent and -independent pathways contributed to the cellular uptake of Zn/DNA clusters. In addition, the TEM image (Figure 10C, bottom right) illustrated that macropinocytosis was also one uptake pathway for Zn/DNA clusters. In parallel research, Chen et al.144 constructed periodic DNA nanoribbons with a size of ∼500 nm for intracellular pH sensing and gene silencing. They concluded that the cellular internalization of their DNA nanoribbons was via clathrin- and lipid raft-mediated endocytosis. Besides the size, the shape and 3D arrangement of DNA nanostructures also play critical roles for efficient cellular uptake. It has been reported that certain inorganic and biologically inspired nanomaterials with high aspect ratio exhibit unique properties including improved membrane penetration and increased cellular internalization, as well as decreased uptake and sequestration by phagocytic cells.204,205 Sleiman and coworkers constructed DNA nanotubes by RCA and observed an enhanced cellular penetration with these nanostructures. They suspected it was due to the dense display of DNA and the high aspect ratio in the RCA-nanotubes.146 In a separate study, our team also found that DNA nanostructures with high length-towidth ratios were more readily taken up by cells.143 The majority of in vivo-administered nanoparticles are likely sequestered by macrophages.206 Also, macrophage uptake correlates with nanomaterial size.207 Chan and co-workers used DNA as linkers and spacers to organize inorganic nanoparticles into the “core− satellite” colloidal superstructures.208 On a model cell system of J774A.1 murine macrophages, they found that although the core−satellite superstructure was 2.5 times bigger than its core component, the uptake by macrophages was lower by 2-fold. The data suggested that the spatial arrangement of satellites with DNA changed the surface chemistry of the superstructure, which inhibited its uptake by macrophages. With this DNA−nanoparticle superstructure, Chan and co-workers were able to reduce nanoparticle retention by macrophages and mitigate the socaused immune toxicity, as well as improve their in vivo tumor accumulation.

vesicle can avoid the involvement of any detrimental enzymes. In fact, many viruses are found to hijack this pathway to bypass lysosomal degradation for efficient transfection to host cells. Therefore, presumably it is valuable to design drug carriers with ligands, e.g., folic acid, albumin, and cholesterol, to be recognized by receptors of CvME for drug-complex evasion of the degradative CME pathway.194,195 Besides the pinocytosis pathways described above, there are other small “lipid rafts” with a diameter of about 40−50 nm that can form on the cytoplasmic side of any cell surface and fuse with any endocytic vesicles. Currently, the mechanism of such clathrin- and caveolae-independent endocytosis is not clear and requires further studies.196 The polyanionic nature of DNA can lead to strong electrostatic repulsion between DNA and the negatively charged cell membrane. This matches the observation that unmodified DNA oligonucleotides alone cannot be efficiently internalized by cells in the absence of transfection agents. However, in 2011 we and others found that certain DNA nanostructures, like the DNA tetrahedron, can enter several different types of cells readily without the aid of transfection agents.146,197,198 Due to the negatively charged hydrophilic property, passive uptake of DNA nanostructures by cells is unlikely. Enlightened by the entry pathway of nanoscale viruses,190 we and others suspected that the DNA nanostructures might be internalized into cells by an endocytosis pathway. We used a single-particle tracking approach to monitor the entry pathway of the tetrahedral DNA nanostructures (TDNs) in live Hela cells.199 We confirmed that the cellular entry of TDNs was an energy-dependent endocytosis process by a set of fluorescence imaging and biochemical experiments. TDNs were found to interact with the plasma membrane nonspecifically and then be rapidly internalized by the caveolae-mediated pathway. In our experiments we observed that the caveolar vesicles eventually directed the transportation of TDNs to lysosomes in a highly ordered, microtubule-dependent manner for degradation (Figure 10A). These unique cellular properties of TDNs, or more broadly framework nucleic acids (FNAs), shed new light on novel nucleic acid scaffold-based nanomedicine. The cellular entry mechanism of spherical nucleic acids (SNAs), the nanoparticle-templated DNA nanostructures, was also studied by Mirkin’s group in 2013.200 SNAs are 3D DNA nanostructures which typically consist of densely functionalized and highly oriented nucleic acids covalently attached to the surfaces of spherical nanoparticle cores.201 Mirkin and coworkers found that SNAs could be rapidly internalized into more than 50 different cell types in a manner irrespective of the presence of the nanoparticle core as well as the surface oligonucleotide sequences. They concluded that the phenomenon was due to the 3D arrangement of the oligonucleotides on SNA, which made them compete for and engage with the cell surface receptors more substantially than linear oligonucleotides. For C166 (mouse endothelial) cells, SNAs bound strongly to the class A scavenger receptor (SR-A) proteins on the cell surface, which, in turn, significantly promoted the cellular uptake of SNAs via the caveolae-mediated pathway, as verified by TEM images (Figure 10B). On the basis of the observation that the degree of SNAs’ correlated positively with the expression level of SR-A and caveolin-1 proteins, Mirkin and co-workers speculated that other cell types, such as epithelial cells and fibroblasts, might also adopt a similar mechanism to internalize SNAs. Further research showed that SNAs with higher G content had a higher degree of cellular uptake than those primarily composed of A, T, and Cs, P


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Figure 11. Strategies to enhance the uptake efficiency of DNA nanostructures. (A) Virus capsid protein (CP)-coated DNA origami. Adapted with permission from ref 212. Copyright 2014 American Chemical Society. (B) Lipid-coated DNA octahedron. Adapted with permission from ref 180. Copyright 2014 American Chemical Society. (C) Self-assembly of TDN modified with the tumor-penetrating peptide (TPP) via the click reaction (pTDN). Dox can be intercalated into the helix of double-stranded DNA. Adapted with permission from ref 226. Copyright 2016 American Chemical Society. (D) Schematics of the self-assembly of aptamer-tethered DNA nanotrains for transport of molecular drugs in theranostic applications. Adapted with permission from ref 234. Copyright 2013 National Academy of Sciences.

3.2.2. Cellular Targeting. Although certain DNA nanostructures can enter cells much better than oligonucleotides, in order to lift the uptake efficiency, enhance the uptake specificity, as well as reduce the immune toxicity, it is necessary to endorse DNA nanostructures excellent abilities to avoid immune recognition, locate target cells, penetrate cell membrane, and perhaps propel to subcellular organelles of interest.

Bioexisting nanocarriers such as the protein cage of viral particles have been explored as vehicles for drug delivery to achieve better biocompatibility, reduced toxicity, and higher uptake efficiency.97,209,210 It has also been reported that DNA can be adopted as a template for virus capsid proteins to gain long tubular structures.211 Combining those together, Mikkilä et al.212 coated DNA origami surface with cowpea chlorotic mottle virus Q


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Figure 12. (A and B) Guided fusion of liposomes with DNA tethers. Adapted with permissions from refs 239 and 240. Copyright 2015 Wiley-VCH and 2016 American Chemical Society.

which were demonstrated to be effectively taken by human cancer cells (nasopharyngeal epidermal carcinoma KB cells). They found that the amount of folate conjugated on DNA nanotubes correlated well with the efficiency of uptake in KB cells. In a parallel study, Lee et al.216 assembled DNA tetrahedral nanoparticles to deliver siRNAs into cells and silence target genes in tumors. They showed that at least three folate molecules per DNA tetrahedron are required to transfect DNA tetrahedron efficiently into KB cells. The follow-up research also demonstrates that the folate ligand-based strategy is a highly selective way for targeting many tumor cells.147,217 Besides folate, several groups reported that certain short peptides can specifically target transmembrane protein neuropilin-1 (NRP), which is overexpressed in glioblastoma cells and endothelial cells of angiogenic blood vessels.218−220 These peptides bind NRP tightly and can penetrate tumor cells.221−225 By appending the peptides to DNA tetrahedrons (p-TDN), Xia et al.226 found that the uptake rate of p-TDN by glioblastoma cell U87MG was effectively accelerated compared with bare TDN and double-stranded DNA (Figure 11C). 3.2.2.2. Aptamers. Aptamers are single-stranded DNA or RNA molecules that can form certain secondary and tertiary structures to specifically bind target molecules, including metal ions, metabolites, proteins, and so on.227,228 They can be evolved from a random oligonucleotide library by a selection procedure termed as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Like antibodies, aptamers can bind targets with high affinity (kD = picomolar to micromolar) and specificity. Due to their intrinsic properties, aptamers can be integrated into DNA nanostructures either through cohesive-

(CCMV) capsid proteins (CP) and further packed the DNA origami nanostructures inside the viral capsid in order to facilitate efficient cell transfection (Figure 11A). They found that the DNA origami−CP complex exhibited a 13-fold increase in the efficiency of delivery into human HEK293 cells compared to the bare DNA origamis. Similarly, Shih and co-workers180 reported that by covering DNA octahedron with PEGylated lipid bilayers (Figure 11B), immune activation was decreased by 2 orders of magnitude below controls and the pharmacokinetic bioavailability improved by about 17-fold. In another work, Sleiman and collaborators demonstrated that DNA nanostructures modified with hydrophobic dendritic chains exhibited rapid cellular uptake behavior, whereas the hydrophilic chain-modified version showed a slow and continuous uptake profile.213 Targeting specific cells is a paramount prerequisite of drug delivery. DNA nanotechnology enables the creation of nanostructures which can not only have a capacity for drug loading but also carry functional groups, such as small molecules, aptamers, and proteins, for sensing and targeting. Through chemical modifications or cohesive interactions, DNA nanostructures are coupled with these functional groups to target cancer cells via the ligand−receptor recognition strategy. Some of the functional groups are discussed in the following part. 3.2.2.1. Small Molecules. The folate receptor (FR), a membrane protein that binds folic acid with high affinity, is overexpressed in various human cancer cells.214 It is known that folic acid retains its receptor binding properties when derivatized via its γ-carboxyl. Therefore, folate conjugation can be explored for targeted drug delivery. With folate−DNA conjugation, Mao and co-workers215 assembled micrometer-long DNA nanotubes R


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Figure 13. Dual targeting with DNA tetrahedron carrying two modifications: SL2B aptamer and folate molecule. Adapted with permission from ref 245. Copyright 2017 Dove Medical Press.

ends triggered hybridization229,230 or as components in the assembly procedure231−233 in order to assist DNA nanostructures to enter cells that express the ligands on the membrane. Tan and co-workers234 designed an aptamer-tethered DNA nanotrain (aptNTr), which is a long linear DNA nanostructure self assembled simply from two relatively short DNA building blocks through a hybridization chain reaction (Figure 11D). The conditional formation of aptNTrs upon initiation with engineered aptamer trigger probes ensured that each resultant nanotrain was tethered with an aptamer moiety on one end of the nanoconstruct. These aptamer moieties, capable of selectively recognizing cognate cancer cells, operated like locomotives guiding a series of tandem dsDNA “boxcars” toward target cells. Due to the high aspect ratio, the aptNTrs were endocytosed easily by target cells once the aptamer moieties were recognized by the receptors on the surface of cells. Besides classic DNA nanostructures, aptamers have also been assembled into DNA nanohydrogels for targeted and stimuliresponsive gene therapy. Tan and co-workers used two Y-shaped monomer units and one DNA linker unit to prepare DNA nanohydrogels.235 Meanwhile, they incorporated aptamers, disulfide linkages, and therapeutic genes into each building unit during the preparation of hydrogels. Tan and co-workers chose S6 aptamer,235 which specifically targets A549 cancer cells (human lung adenocarcinoma epithelial cell line). They

confirmed that such aptamer hydrogels strongly inhibited proliferation and migration of A549 cells but had negligible effects on normal cells. 3.2.2.3. Proteins. Proteins are the most important biological molecules, which participate in almost all activities of life. It is reported that the curvature and deformation of lipid membranes are promoted by special membrane-sculpting proteins, such as clathrin, dynamin, and SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor).236−238 DNA nanostructures mimicking these proteins presumably have the potential to self-manipulate the endocytosis process. SNARE proteins are the key machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins which bridge the membranes and thus prepare SNAREs for docking and fusion, Rothman and co-workers developed a lipidconjugated ssDNA mimic of tethers that is capable of regulating SNARE function in situ (Figure 12A).239 The nonfusogenic DNA−lipid hybrid molecules which serve as artificial tethers were designed to contain three consecutive structural segments from the membrane-distal to the membrane-proximal ends: (1) a 21-bp hybridization region, responsible for physically bridging two membranes via parallel hybridization, (2) a linker region containing 5−63 consecutive thymidine (T) nucleotides, which was inserted to regulate liposome distance and minimize possible nonspecific fusion caused by the tethers alone, and (3) the lipid S


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Figure 14. DNA nanochannels that can penetrate the lipid membrane. (A) Syringe-like DNA nanostructure with cholesterol (orange) modifications. Adapted with permission from ref 247. Copyright 2012 American Association for the Advancement of Science. (B) DNA nanopore with porphyrin tags (magenta). Adapted with permissions from refs 248 and 249. Copyright 2013 American Chemical Society and 2013 Wiley-VCH. (C) DNA nanopore with cholesterol anchors (orange). Adapted with permission from ref 250. Copyright 2016 Nature Publishing Group.

anchor residing in the lipid bilayer, which was cross-linked to the DNA sequence via a thiol−maleimide linkage. Without direct interaction with SNAREs, the DNA−lipid tethers regulated SNARE function by capturing and keeping two opposed membranes in a controlled distance, within which SNAREs drove the membranes fusing with each other. Rothman and co-

workers found that in the presence of these artificial tethers, SNARE-mediated lipid mixing was significantly accelerated, and the maximum fusion rate was obtained with a linker shorter than 40 nucleotides. As a programmable tool, the DNA−lipid tethers can be further applied to regulate other biological processes. The T


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selectively taken up by cells via endocytosis, while small molecules such as ions can selectively pass through membrane via channels generated by pore-forming membrane proteins. Ion channels are located within the membrane of most cells and of many intracellular organelles. Their functions include regulating cell volume, shaping action potentials and other electrical signals by gating the flow of ions across the cell membrane, establishing a resting membrane potential, and so on. α-Hemolysin is a toxin that can cause cell death by binding to the outer membrane, with subsequent oligomerization of the toxin monomer forming the water-filled channels.246 Using αhemolysin as a model, Simmel, Dietz, and co-workers247 created an artificial channel with DNA origami that, when penetrating a lipid bilayer membrane, acted similar to a membrane channel (Figure 14A). The channel consists of two modules: a stem that penetrates and spans a lipid membrane and a barrel-shaped cap that is mediated by 26 cholesterol moieties and adheres to the cis side of the membrane. In a single-channel electrophysiological experiment, this artificial channel showed conductances on the order of 1 nanosiemens and channel gating which are similar to the response of natural ion channels. By manipulating the length of a DNA component strand in the stem domain to allow its protrusion into the artificial channel, Simmel and Dietz’s team observed greater gating responses.247 Single-molecule translocation experiments show that the synthetic channels can be applied for the discrimination of single-DNA molecules. In the work by Simmel and Dietz’s team,247 the aromatic membrane anchors drove the unmodified hydrophilic DNA stem penetrating the membrane. Another strategy to penetrate the membrane is to mimic membrane proteins that have an outer hydrophobic surface. Following this route, Howorka and coworkers248 designed a 6-helix-bundle-based synthetic DNA nanochannel, which carries an outer hydrophobic belt comprised of small chemical alkyl groups (Figure 14B, left). This modification masked the negatively charged oligonucleotide backbone and hence overcame the otherwise inherent energetic mismatch to the hydrophobic environment of the membrane. Such artificial DNA pores were structurally stable and demonstrated to support the transmembrane flow of water molecules by a range of analytical techniques.248 In the above two studies, hydrophobic chemical tags, either cholesterol-based lipid anchors covalently attached to DNA strands247 or ethyl-modified phosphorothioate groups that replace the negatively charged backbone phosphate to form a hydrophobic belt to mimic natural protein pores,248 were precisely positioned to anchor the strongly hydrophilic DNA nanostructures into lipid bilayers. However, to make such artificial pores, complex designs were required: The former tag was placed at up to 26 positions of the pore, while the latter was introduced 72 times into a DNA origami. With the intent to simplify nanochannel design and move toward minimal chemical intervention, Howorka’s team continued to explore whether other chemical tags of greater hydrophobicity can achieve the same level of membrane penetration by only a few tag copies. They found that with tetraphenylporphyrin (TPP) to couple with DNA, solely two tags achieved the membrane-anchoring task (Figure 14B, right).249 Porphyrin not only has a van der Waals surface area 12 times larger than ethane but also is a chromophore with an emission wavelength at 656 nm. Also, moreover, the insertion of porphyrins into lipid bilayers leads to a characteristic shift in their fluorescence spectrum. Howorka’s team reported that indeed they were able to visualize the TPPtagged DNA pore anchoring on the lipid bilayer through

work establishes an important step toward controllable DNA− nanostructure/cell-membrane interaction. In a subsequent work, how SNAREs drive membrane fusion was studied by using self-assembled DNA nanorings to template the formation of uniform-sized small unilamellar vesicles (SUVs) which contain predetermined maximal number of SNAREs facing externally (Figure 12B).240 The lipid-conjugated complementary ssDNA strands as tethers were incorporated into the vesicles for membrane targeting. This strategy enabled the bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. The results showed that at the single-event level, after docking of the templated SUVs to the supported lipid bilayers (SBL), 1−2 pairs of SNAREs were sufficient to drive fast lipid mixing. Besides designing DNA nanostructures to mimick the behavior of certain membrane-sculpting proteins, modifying DNA nanostructures with protein ligands whose receptors dwell on the cell surface can also assist them to target specific cells via the antibody−antigen interactions. With terminal modifications, the current synthetic chemistry can ensure oligonucleotide strands with customized sequences covalently link to the cysteine or lysine residues of an antibody.241,242 In principle, any component DNA strand of a DNA nanostructure can be designed to conjugate with an antibody. Thus, comparing to other nanomaterials, DNA nanostructures have enormous flexibility in antibody conjugation: Not only the position where the antibody is conjugated to the DNA nanostructure but also the number of conjugated antibodies per DNA nanostructure can be well programmed and controlled. Transferrin (Tf) is a key protein player in the metabolism of iron cellular transportation via receptor-mediated endocytosis. In order to map the cellular pH gradients with DNA nanomachines, Krishnan and colleagues chemically conjugated Tf to a DNA nanodevice.243 This Tf−DNA nanocomplex was able to travel through the transferrin pathway and report the pH values of subcellular organelles within the pathway. In a separate study, Kjems and co-workers244 demonstrated that by modifying a planar DNA origami structure with Tf, a significant strong increase in cellular uptake was achieved in an established cancer cell line. The decoration of the 2D DNA origami roughly 100 nm in diameter with approximately 32 Tf protein molecules resulted in an up to 22-fold increase of cytoplasmic uptake compared to unmodified structures, which is comparable to the lipofectaminebased transfection. If the receptors that overexpressed in cancer cells were also expressed in normal cells, targeting a single receptor would inevitably cause nonspecific drug uptake, resulting in toxicity to normal cells as well as reduced anticancer efficacy. Dual or multiple targeting could increase the specificity of drug delivery. DNA nanostructures usually consist of several to hundreds of component strands, each of which in principle could be modified to link with a ligand. Sun et al. reported that they modified a TDN with SL2B aptamer and folic acid.245 SL2B is a 26-mer DNA strand which can specifically target the heparin binding domain (HBD) of vascular endothelial growth factor (VEGF165). Such dual-functionalized TDNs could be simultaneously recognized by both VEGF and folate receptors on the surface of HT-29 cancer cells (Figure 13). Sun et al. confirmed that the dual-functionalized TDNs could cause sufficient HT-29 cell inhibition at a much lower Dox concentration. 3.2.3. Creating Artificial Channels with DNA Nanotechnology. Large molecules including nanoparticles can be U


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Figure 15. DNA nanodevices that can go through the furin (Fu) and transferrin (Tf) pathways. Adapted with permission from ref 243. Copyright 2013 Nature Publishing Group. (A) Schematic of IFu. IFu contains a nicked duplex and cytosine-rich sticky ends. It undergoes a pH-dependent conformational change due to i-motif formation (green). FRET pair of Alexa 546 (magenta) and Alexa 647 (blue) is labeled to monitor the conformational change. dsDNA domain (gray) acts as a recognition site for a recombinant antibody (scFv, gray cylinders) to guide IFu through the Fu pathway. (B) Schematic of ITf. ITf is a duplex carrying a pH-responsive element (purple). It forms an intramolecular i-motif at acidic condition. Similarly, a FRET pair of Alexa 488 (green) and Alexa 647 (blue) is labeled to monitor this structural change. Coupling transferrin (Tf) to ITf confines the complex to the Tf receptor pathway. (C) Different pathways by the two DNA nanodevices. In pathway A, An scFv-furin chimera (gray) retrogradely transports IFu into the TGN via the SE and LE. In pathway B, Tf-ITf marks the SE en route to the RE.

fluorescence microscopic imaging.249 Also, they established that the DNA pores exhibited two voltage-dependent conductance states: Low transmembrane voltages favor a stable highconductance level corresponding to an unobstructed DNA pore, whereas at higher voltages, the channel shows a main lowconductance state probably caused by electric-field-induced changes of the DNA pore in its conformation or orientation.48 In their recent work, Howorka and colleagues further explored the artificial DNA pore’s ability to selectively transport charged molecules across a biological membrane.250 With the design of seven concatenated DNA strands, they constructed an atomistically determined molecular valve (Figure 14C). The valve can bind a specific ligand, triggering a nanomechanical change to open up the membrane-spanning channel. Small organic molecules that differ by the presence of a positively or negatively

charged group can be selectively distinguished to transport through the channel. It is exciting and encouraging that the artificial DNA channels can control when and which cargo is transported across a lipid bilayer, which potentially paves the way for controlled drug release. 3.2.4. Subcellular Targeting. The route of uptake can affect which cellular compartment drug molecules are released into. As we reviewed in section 3.2.1, clathrin-dependent endocytosis leads to the lysosomal pathway in which lysosomes keep their contents away from the cytosol and eventually degrade the contents. In fact, many endocytic pathways result in entry to lysosomes, where the function of drug molecules may be inhibited due to the lack of process to the cytoplasm and nucleus or due to the damage caused by degradation. In order to maximize drug efficacy, targeting with decent precision at V


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Figure 16. Delivery of Dox by different DNA nanostructures. (A) Straight and twisted DNA nanotubes as vehicles. Adapted with permission from ref 263. Copyright 2012 American Chemical Society. (B) Tubular and triangular DNA origami as carriers. Adapted with permission from ref 264. Copyright 2012 American Chemical Society. (C) AptNAs as carriers. Adapted with permission from ref 266. Copyright 2013 American Chemical Society. (D) Delivery of Dox by DOX/FA-NCl/NCa assemblies. Adapted with permission from ref 147. Copyright 2014 American Chemical Society.

subcellular resolution is needed. This is an emerging field and perhaps should be called “tertiary targeting”.251 Bypassing the lysosome is critical for subcellular targeting. Drug vehicles can be engineered to break out of endosomes or enter the cell through nonlysosomal pathways. Nanoparticles with positive surface charges can induce osmotic lysis upon

endosome acidification by the proton sponge effect.252,253 Retrograde endocytic pathway, a nonlysosomal pathway, has been explored to deliver a pH-sensing DNA nanodevice to a specific subcellular compartment other than lysosome.243 In a retrograde route, proteins or lipids targeted to endosomes from the Golgi apparatus or the plasma membrane are transported to W


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macromolecular biosynthesis.258 While effectively killing cancer cells, Dox also harms normal cells and causes toxicity to most noncancerous organs. The adverse side effects and poor selectivity force the Dox-based treatment to be dose limiting.259 Over the years, many drug delivery systems, e.g., liposome,260 nanoparticle,261 and polymer micelle,262 have been developed to mitigate the adverse effects and improve drug efficiency. Dox can dwell itself in DNA by intercalating the GC-rich regions of the double helices, which opens a wide range of possibilities to tailor DNA nanostructures as new carriers for Dox delivery. Högberg and co-workers optimized the delivery of Dox to human breast cancer cells with different DNA origami nanostructures.263 These DNA nanostructures exhibited varying degrees of global twist, leading to different amounts of relaxation in the DNA double-helix structure (Figure 16A). By tuning the design, the encapsulation efficiency and the release rate of Dox can be adjusted to increase cytotoxicity and lower the intracellular elimination rate. Flow cytometry also verified the enhanced apoptosis induced by the delivery system in breast cancer cells. In addition, Högberg and co-workers demonstrated that the drug release kinetics could be rationally controlled and tailored by tuning the twist degrees.263 Such tunable DNA nanostructures resemble efficient delivery vehicles for Dox, through which can generate high degrees of internalization and increased induction of programmed cell death in breast cancer cells. Similar research was carried out by Ding and co-workers, who designed and assembled 2D and 3D Dox-loaded DNA origami structures that exhibited a high level of Dox loading efficiency, as well as greater cellular uptake of Dox (Figure 16B).264 Ding and co-workers found that the DNA origami/Dox complex exhibited prominent cytotoxicity not only to regular human breast adenocarcinoma cancer cells (MCF 7) but more importantly to Dox-resistant MCF 7 cells due to the increase in the cellular uptake of Dox and redistribution of Dox to action sites by the origami vehicle. In the follow-up project, Ding and co-workers studied the therapeutic efficacy of the origami/Dox system in vivo.265 After conducting fluorescence imaging and safety evaluation experiments, they found that Dox-loaded DNA origami exhibited remarkable antitumor efficacy without observable systemic toxicity in nude mice bearing orthotopic breast tumors. These findings hint that DNA origami nanostructures could be innovative platforms for the efficient and safe drug delivery of cancer therapeutics in vivo. To avoid complicated structure design and lower the cost of regular DNA origami, Yan et al. invented a novel strategy to construct 3D AuNP−DNA superstructure by simultaneously growing DNA and folding origami on AuNPs.149 The strategy features the combination of the rigidity of nanoparticles with the flexibility of DNA nanostructures (Figure 7B). The as-fabricated 3D superstructures have a high molecule-loading capacity, which enables the simultaneous transport of signal reporters and drug molecules with high efficiency for cellular imaging and drug delivery.149 Besides DNA origami, other self-assembled DNA structures were also explored as nanocarriers for Dox delivery. Tan and coworkers constructed a multifunctional aptamer-based DNA assembly (AptNA) for targeted cancer therapy.266 Multiple DNA subunits with different functions, including targeting aptamers, intercalated anticancer drugs, and therapeutic antisense oligonucleotides, were first self-assembled to form Y-shaped functional domains (Figure 16C). They were then linked to Xshaped connectors through hybridization to generate building

the trans-Golgi network (TGN), Golgi membranes, or the endoplasmic reticulum (ER).254 Furin is a protein enriched in the Golgi apparatus via a retrograde route.255 Krishnan and coworkers managed to fuse a sequence-specific dsDNA binding protein (single-chain variable fragment recombinant antibody, or scFv)256 as a chimera with furin to build up the DNA−protein partnership (Figure 15).243 The scFv specifically binds an 8-bp DNA sequence, which was incorporated in a pH-responsive DNA nanomachine. When the scFv-furin chimera was expressed inside cells, it bound with the DNA nanomachine and hijacked the nanomachine along the retrograde furin endocytic pathway into the TGN. With pH-sensing groups and fluorophores designed in the DNA nanomachine, Krishnan and co-workers were able to not only map pH but also reveal the morphology of organelles along the retrograde furin pathway, including sorting the endosome, late endosome, and trans-Golgi network.243 The work resembles an excellent example to apply DNA nanotechnology for subcellular targeting. In principle, during retrograde transport, through the DNA−protein or DNA− lipid partnership, DNA nanoparticles could be targeted to any compartment where its partner is transported to. From this work, we can see the great potential and advantages for using DNA nanotechnology to guide subcellular targeting. In the next section, during the discussion of drug molecules delivered by DNA nanotechnology, we will also show more examples of precise subcellular targeting with DNA nanotechnology. 3.3. Delivery of Drug Molecules by DNA Nanotechnology

We have discussed the principles of cellular endocytosis and the way DNA nanostructures enter cells. Through sophisticated design and modification, DNA nanostructures can specifically locate at the plasma membrane of target cells, followed by endocytosis mostly via a clathrin- or caveoleo-mediated pathway. In cells, DNA nanostructures can also be engineered to hijack the retrograde route to bypass lysosomal degradation and target specific subcellular compartment. In addition, DNA nanochannels can be constructed to mimic the natural transmembrane pores for controllable transportation of cargos across the plasma membrane. In this section, we will give an in-depth and detailed review on what drug molecules have been delivered into cells with DNA nanotechnology 3.3.1. Small Molecules. 3.3.1.1. Fluorescent Dyes. Fluorescent dyes are the most commonly used molecules for cellular analysis. Due to the maturation of DNA synthesis techniques, various fluorescent dyes can be easily modified on DNA strands to track the cycling, distribution, and lifetime of DNA nanostructures in living cells by fluorescent microscopy.257 Usually fluorescent dyes along with other functional molecules are integrated in one DNA nanostructure for simultaneous imaging and delivery. For example, Tan and co-workers designed aptamer-conjugated FRET (fluorescent resonance energy transfer) nanoflowers (NFs) for multiplexed cellular imaging and traceable targeted drug delivery.231 The NFs are modified to carry three fluorescent dyes, fluorescein (FAM), cyanine 3 (Cy3), and 6-carboxyl-X-rhodamine (ROX). By optimizing the amount of dye molecules in FRET NFs, the emission spectra can be tuned so that only the dye with the longest emission wavelength exhibits significant fluorescent signals. When tested in cancer cells, the NFs system exhibited high fluorescence intensity and excellent photostability.231 3.3.1.2. Doxorubicin. Doxorubicin (Dox), as one of the most potent chemotherapeutic drugs approved by the FDA, has been used to treat a wide range of cancers through the inhibition of X


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Figure 17. Delivery of Dox by MoS2−DNA superstructures. Adapted with permission from ref 142. Copyright 2017 American Chemical Society.

and Dox-binding DNA for drug delivery. The densely packed drug-binding motifs and porous intrastructures endow NFs with a high loading capacity (71.4%, wt/wt). The Dox-loaded NFs were stable at physiological pH, and drug release was facilitated under acidic or basic conditions. Recently, layer-by-layer self-assembled stacked MoS2−DNA superstructures were demonstrated for protective and autonomous delivery of Dox in cancer cells. Li et al.142 reported that they could functionalize MoS2 nanosheets with terminalthiolated DNAs via strong binding to S-atom defect vacancies on MoS2 surfaces (Figure 17). By a linker ATP-aptamer that induced interlayer assembly, they successfully guided the formation of stacked MoS 2−DNA superstructures. Dox molecules were loaded on the DNA interspacers between neighboring MoS2 nanosheets. In the presence of a high level of ATP molecules in many cancer cells, the multilayer MoS2−DNA superstructures disassembled due to the stronger binding of ATP with the linking aptamers, which in turn released the Dox cargos. These superstructures offer a protective armor-like shell of MoS2 nanosheets, which keeps the DNA away from nuclease digestion while it remains responsive to small and infiltrating ATP molecules. Thus, they could be an enhanced stimuli-responsive drug release system for targeted Dox-based chemotherapy. 3.3.2. Oligonucleotides. 3.3.2.1. CpGs. CpGs (unmethylated cytosine-phosphate-guanine dinucleotides) are frequently seen in microbial genomes but rare in vertebrate genomes. Bacterial DNA and synthetic oligonucleotides containing CpG motifs are recognized as dangerous signals by mammalian innate immune systems and could trigger strong immune responses.269 For synthetic CpGs, the stimulatory effects are mediated by Tolllike-receptor 9 (TLR9) proteins located in the endosome of host cells, leading to secretion of the pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and so on.270 Because of this, CpGs have been actively explored in both basic research and clinical trials as a type of potent and safe

units. Hundreds of these units were further photo-cross-linked to form the multifunctional and programmable aptamer-based nanoassembly structure. Through calculation, Tan and coworkers reported that each AptNA contained about 100−200 building units, and each unit was able to provide a high-loading capacity of more than 220 Dox loading sites. In addition, the incorporation of therapeutic antisense oligonucleotides resulted in the inhibition of P-gp expression (a drug efflux pump to increase excretion of anticancer drugs), which enhanced the drug efficacy. Moreover, the AptNAs showed excellent stability and integrity in the physiological environment, avoiding unnecessary leaking of intercalated drugs during the delivery process. Similarly, Zhang et al. studied controllable and targeted delivery of Dox to cancer cells with an aptamer-based dendritic DNA nanostructure.267 More complex and sophisticated DNA nanosystems have been developed to control and target Dox to cancers. Gu and coworkers147 engineered a bioinspired cocoon-like anticancer drug delivery system consisting of a deoxyribonuclease (DNase)degradable DNA nanoclew (NCl) embedded with an acidresponsive DNase I nanocapsule (NCa). The NCl was assembled from a long-chain single-stranded DNA synthesized by RCA (Figure 7C). Repeated GC-pair sequences were integrated into the NCl for enhanced loading capacity of Dox. After cellular uptake of the complex via FR-mediated pathway, DNase I in the nanocapsule was activated through the acidtriggered shedding of the polymeric shell of the NCa, which in turn led to the self-degradation of NCl and the release of Dox (Figure 16D). Tan and co-workers268 used a similar strategy to engineer multifunctional DNA nanoflowers (NFs) for targeted drug delivery to both chemosensitive and multidrug resistance (MDR) cancer cells that circumvented MDR in both leukemia and breast cancer cell models. NFs were also self-assembled by RCA. They contain a few functional domains, including aptamers for specific cancer cell recognition, fluorophores for bioimaging, Y


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Figure 18. Delivery of CpG with different DNA nanostructures. (A) With DNA tetrahedron. Adapted with permission from ref 197. Copyright 2011 American Chemical Society. (B) With DNA dendrimers. Adapted with permission from ref 281. Copyright 2017 American Chemical Society. (C) Codelivery of CpG and aPD1 by DNA nanococoon under inflammation conditions. Adapted with permission from ref 286. Copyright 2015 WileyVCH.

(Figure 18A).197 We demonstrated that the DNA nanostructure complex was resistant to nuclease degradation and remained substantially intact in fetal bovine serum and in cells for at least several hours. The complex was also able to efficiently and noninvasively enter macrophage-like RAW264.7 cells without the aid of transfection agents. After cellular uptake, CpG motifs were recognized by TLR9, which in turn activated the downstream pathways to induce immunostimulatory effects, resulting in high-level secretion of various pro-inflammatory cytokines such as TNF-R, IL-6, and IL-12.197 Liedl and co-workers tested the immune responses induced by a 30-helix-bundled DNA origami tube, which was decorated with up to 62 CpG sequences, in spleen cells.276 By monitoring cytokine production and immune cell activation, they confirmed that such decorated origami tubes triggered much higher

vaccine adjuvant for immunotherapy of infectious diseases and cancer.271 Naked CpG dinucleotides are poor in cellular uptake and prone to nuclease degradation. To address the issue, various approaches have been developed.272,273 For instance, phosphorothioate (PT) modification of the backbone of CpG nucleotides is frequently used to enhance the stability of CpG against the DNase degradation.274 However, several side effects, including reduced immune response and lymphoid follicle destruction, were found to be related to the thiol modification.275 In recent years, DNA nanotechnology-enabled nanostructures have been explored as carriers for CpG delivery. Due to their inherent compatibility, CpG-rich sequences can be easily incorporated into DNA nanostructures to increase their stability and targeting specificity. In 2011, our group designed a multivalent DNA tetrahedron to load and deliver CpG motifs Z


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urgent to develop strategies to prevent cancer recurrence after surgery. On the basis of recent successes of cancer immunotherapy,285 we think that it can be utilized to prevent cancer recurrence. Programmed death 1 (PD-1) is a key immune-checkpoint receptor expressed by activated T cells, and it mediates immunosuppression. The interaction between PD-1 and its ligand 1 and 2 (PD-L1/2) is a key pathway hijacked by tumors to suppress immune response. Therefore, disrupting the interaction between PD-1 and PD-L1 by anti-PD antibodies can in principle boost the immune response against cancer cells. Also, how to improve the therapeutic efficacy with insignificant side effects, such as autoimmune disorders, is a central theme for the anti-PD-based cancer immunotherapy. Recently, Gu and coworkers developed an innovative DNA nanococoon (DNC) carrier for controlled release of loaded anti-PD-1 antibody (aPD1) and CpGs (CpG ODNs) in response to inflammation conditions.286 The inflammatory environment is conducive to immunotherapy by converting quiescent precursor lymphocytes into activated lymphocytes, which are required for tumor eradication. The DNC carrier was built on a long single-stranded DNA amplified by RCA, which contains repeated units with interval CpG sequences and cutting sites for restriction enzyme HhaI (Figure 18C). Multiple CpG fragments would be generated once the carrier was digested by HhaI. To make the release event bioresponsive, Gu and co-workers loaded DNCs with aPD1, trapped HhaI in cages of triglycerol monostearate (TGMS) nanoparticles, and attached them with each other to form DNCTGMS complexes.286 TGMS is an amphiphile whose ester linkage enables cleavage by esterases and matrix metalloproteinases (MMPs) that are highly expressed at the wound sites for developmental tissue remodeling.287 Therefore, triggered by the inflammatory condition in the wound site of the tumor resection incision, TGMS can be cleaved, leading to the release of HhaI, which can further sequentially convert DNCs to CpG fragments and release aPD1. The released CpGs can activate dendritic cells to drive T cell immune response against cancer cells, while the release aPD1 further boosts the immune response with PD-1 blockade (Figure 18C). The controlled bioresponsive release of CpG and aPD1 is way more effective than the treatment with free CpG nucleotides and aPD1. In addition, the synergistic action of aPD1 and CpG can also prevent the potential risk of toxic peak dosage of aPD1 in the body. 3.3.2.2. siRNA. siRNA (small interfering RNA) operates within the fundamental RNA interference (RNAi) pathway in eukaryotic cells. It functions by targeting and inducing the cleavage of a certain complementary mRNA, which leads to the shutdown of the expression of mRNA-encoded proteins.288 SiRNAs are usually 21−23 bases in length and produced by the enzyme Dicer in cells.289 Alternatively, chemically synthesized siRNAs can be directly introduced into cells and bind with complementary mRNA to induce a cellular RNAi process.290,291 Thus, synthetic siRNAs represent a class of potent nucleic acidbased drug candidates for RNAi therapies. Many strategies have been developed for delivery of siRNAs, including the conventional complexation or encapsulation of siRNAs with polymers, lipids, or other nanoparticles.292−294 However, conventional vehicles such as liposomes and polymeric systems are usually heterogeneous in size, composition, and surface chemistry, which can lead to potential toxicity, lack of tissue specificity, and suboptimal performance.11,291,295 DNA nanostructures can be assembled with well-defined sizes, and the component strands are easily programmed to carry siRNAs with DNA−RNA

immunostimulation than equal amounts of naked CpG oligonucleotides. Later, we synthesized RCA-based DNA nanoribbons to increase the payload of CpG sequences.143 Theoretically, each periodic unit of DNA nanoribbons could couple one CpG sequence. We found that the length and concentration of DNA nanoribbon were highly correlated with the yield of TNF-α. For DNA nanoribbons ∼1 μm in length at a 10 nM concentration, the released amount of CpG was high enough to stimulate the production of ∼5100 pg/mL TNF-α, much higher than that triggered by the previously reported carrier systems. Tan and co-workers further simplified the synthesis of CpG-decorated DNA nanostructures.277 They constructed novel immuno-nanoflowers (NFs) self-assembled from long DNA strands, which were integrated with tandem CpGs through RCA, for protection of CpGs from nuclease degradation. In a model of macrophage-like cells, the CpG NFs were proved to be potent immunostimulators triggering the secretion of TNF-R, IL-6, and IL-12 to induce cancer cell apoptosis and necrosis.277 We also explored gold nanoparticle-aided nanostructures for CpG delivery.278 With thiolated modification on the end, DNA sequences carrying CpGs were successfully conjugated to AuNPs. We used the AuNPs as nanocarriers to noninvasively deliver the synthetic CpGs into cells. Compared to unconjugated CpG-containing ssDNA, the self-assembled polyvalent CpGAuNP conjugates were demonstrated to enhance the efficiency of cellular uptake and stimulate secretion of cytokines. The immunostimulatory activity of the conjugates increased with the density of the CpG motifs at the surface of AuNPs. Further studies showed that nonthiolated, diblock oligonuleotides containing a CpG motif and a polyadenine (polyA) tail could readily self-assemble on the surface of AuNPs with controllable and tunable density.279 Under optimal conditions, the polyACpG-AuNPs exhibited significantly higher immunostimulatory activity than their thiolated counterpart. In addition, Mohri et al.280 constructed ligation-free DNA dendrimers for CpG delivery. Using CpG-incorporated DNA units with elongated adhesive ends, they were able to assemble DNA dendrimers without the common use of DNA ligases. Mohri et al.280 found that the cellular uptake of DNA dendrimers by mouse macrophage-like RAW264.7 cells and subsequent release of TNF-α were dependent on the structural complexity of the dendrimers. The results indicate that DNA dendrimers are a potent system for the delivery of immunostimulatory CpG DNA to immune cells. Similarly, Ding and co-workers constructed programmable DNA dendrimers decorated with CpG loops.281 They found that the CpG-loop decoration triggered stronger immune response characterized by pro-inflammatory cytokines production, in contrast to linear CpG (Figure 18B). Further modification with TAT peptide (a typical cell-penetrating peptide) on the surface of DNA dendrimers enabled enhanced cellular internalization and cytokines production. Ding and coworkers reported that the TAT−DNA dendrimer-CpG loop constructs did not affect the viability of immune cells, and no detectable cytotoxicity was observed. Their results further demonstrated that the DNA dendrimers can serve as designable and safe vehicles for delivery of CpGs.281 Currently, surgical treatment is the most effective therapeutic method for many solid tumors. However, many patients develop recurrent disease postsurgery, which could lead to significant morbidity and mortality for cancer patients.282,283 Following tumor resection, the inflammatory processes during wound healing may also promote cancer progression.284 Therefore, it is AA


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Figure 19. Delivery of siRNA with different DNA nanostructures. (A) With DNA tetrahedron. Adapted with permission from ref 216. Copyright 2012 Nature Publishing Group. (B) With DNA prism. Adapted with permission from ref 291. Copyright 2016 American Chemical Society. (C) Delivery of siRNA by RNAi-microsponge self-assembled from rolling circle transcription (RCT) products. Adapted with permission from ref 302. Copyright 2012 Nature Publishing Group.

environment. In 2014, Sleiman and co-workers generated gene silencing 3D DNA prisms by integrating antisense therapeutic oligonucleotides within them. They showed that antisense strands tethered on DNA cages could readily induce gene silencing in mammalian cells and maintain gene knockdown levels more effectively than single- and double-stranded oligo controls, because the DNA cages increased the stability of the bound antisense oligo units.296 Through rational design and optimization, Sleiman and co-workers further assembled the DNA “nanosuitcase” that could encapsulate a siRNA construct and release it upon recognition of an oligonucleotide trigger (Figure 19B).297 The trigger could be an mRNA or a microRNA which offers potential for dual or synergistic therapy. Sleiman and co-workers demonstrated that the DNA suitcase could sustain biological conditions and protect siRNA cargos, as well as release cargos on demand.297 Nanoparticle-assisted DNA nanostructures have also been explored for siRNA delivery. Mirkin and co-workers constructed a RNAi-based nanomedicine platform by assembling spherical nucleic acid (SNA) nanostructures consisting of gold nanoparticles covalently functionalized with densely packed, highly oriented siRNA duplexes.298 They preclinically evaluated the platform to neutralize oncogene expression in Glioblastoma

hybridization, thus representing a novel type of vehicle for siRNA delivery. Lee et al.216 designed a self-assembled DNA tetrahedral nanostructure for delivery of siRNAs in vivo to silence target genes in tumors (Figure 19A). They precisely controlled the diameter of the DNA tetrahedron to be around 30 nm to avoid renal filtration, which is a typical outcome for monomeric siRNA. In addition, the spatial orientation of cancer-targeting ligands on the tetrahedron as well as the density of the ligands such as peptides and folate on the surface of the tetrahedron were also optimized. Lee et al.216 found that at least three folate molecules per tetrahedron were required for the optimal delivery of siRNAs into targeted cancer cells. In addition, gene silencing occurred only when the ligands were assembled in the appropriate spatial orientation. When three folate molecules were decorated on the tetrahedron so that the local density was maximized, targeted gene silencing was observed. Otherwise, silencing disappeared. Lee et al.216 suspect that this phenomenon might be due to the higher local density of folates influencing the intracellular trafficking pathway of the DNA tetrahedron through the cells. During the delivery of siRNA, DNA nanostructures can act as not only an umbrella to protect the cargo against nuclease degradation but also a platform to read and respond to the AB


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Figure 20. Design of different DNA nanostructures for microRNA delivery and regulation. (A) DNA Shuriken carrying 3 miR-145 strands for microRNA delivery. Adapted with permission from ref 312. Copyright 2017 Royal Society of Chemistry. (B) Branched DNA scaffold containing antimicroRNA sticky ends for microRNA quenching. Adapted with permission from ref 313. Copyright 2017 Royal Society of Chemistry.

of active siRNA. Hammond and co-workers reported that more than one-half a million copies of siRNA could be delivered to a cell with the uptake of a single RNAi-microsponge. Also, due to the high density of siRNAs, roughly 3 orders of magnitude less carrier was required to achieve the same degree of gene silencing as a conventional particle-based vehicle. Recently, Hammond and co-workers took a step forward by making a multi-RNAi microsponge platform for simultaneous controlled delivery of multiple siRNAs.303 They showed that the microsponge-based siRNA delivery could be potentially applied for the treatment of cancer, genetic disorders, and viral infections. 3.3.2.3. microRNA. microRNA (miRNA) is similar to siRNA in terms of size and function. The drawbacks of the conventional vehicles for siRNA delivery also exist in miRNA delivery. Commercially available transfection reagents for miRNAs may work well on cultured cells, but these reagents are typically liposomes with a wide size distribution which can lead to toxicity.304 Much effort has been put into developing better miRNA delivery strategies,305−309 but none of them reaches the expectation. In fact, in many cases delivery is only the first step of miRNAs’ in vivo journey. Once into cells, the exogeneous miRNAs would undergo lysosomal degradation, which greatly diminishes the actual working concentration of the therapeutic miRNAs. To avoid lysosomal degradation, plasmid or viral vectors have been used to overexpress miRNAs in cancer cells.

multiforme (GBM), a neurologically debilitating disease that culminates in death 14−16 months after diagnosis.299−301 Mirkin and co-workers found that the siRNA-loaded SNA penetrated the blood−brain barrier and blood−tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2L12 were effective in knocking down endogenous Bcl2L12 mRNA and protein levels and sensitized glioma cells toward therapy-induced apoptosis. Mirkin and coworkers also observed that systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice without adverse side effects.298 SNAs represent a new approach for RNAi therapy for GBM and possibly other lethal malignancies. Similar to SNAs, which can load and deliver a large amount of siRNAs, microsponges comprised of repeated siRNA sequences were developed by Hammond and co-workers for efficient siRNA delivery.302 Using rolling-circle transcription (RCT) to generate repeated units of combined carrier and cargo (siRNA) sequences, Hammond and co-workers constructed RNAi polymers that can self-assemble into nanoscale-pleated sheets of hairpin RNA, which in turn form sponge-like microspheres (Figure 19C). The microspheres provided protection for siRNA until they were internalized into the cell, where the cellular RNAi machinery converted the stable hairpin RNAi to short fragments AC


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Figure 21. DNA NC-based CRISPR-Cas9 delivery system. Adapted with permission from ref 325. Copyright 2015 Wiley-VCH.

excellent customizable platforms for miRNA-based cancer therapies. Besides acting as tumor suppressors, certain miRNAs (oncomiRs) are also associated with cancer. Nahar et al.313 assembled a programmable anti-miR branched DNA nanostructure carrying single-stranded anti-miRNA overhangs for simultaneous selective quenching oncomiRs miRNA-27a, 96, and 182 (Figure 20B), which collectively downregulate FOXO1a, a transcription factor that regulates multiple genes involved in cell cycle progression, apoptosis, and cellular metabolism. Reduced levels of FOXO1a are known to contribute to malignant transformation and oncogenic state.314 Nahar et al. observed enhanced stability of the anti-miR-branched DNA nanostructures than naked anti-miRNAs in serum. The former were able to knockdown the three oncomiRs with up to a 50% greater repression as compared to the latter. The synergestic miRNA repression led to restoration of FOXO1a protein levels which in turn inhibits G1-S transition in cancer cells. This study further demonstrates that the programmability of DNA nanostructures can be explored as a powerful tool for miRNA delivery and regulation. 3.3.2.4. CRISPR-Cas9. CRISPR-Cas9 is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmid and phage DNAs. It has quickly turned from an immune defense system in prokaryotes to a facile genome-editing method in biotechnology.315 The engineered CRISPR-Cas9 system requires only two factors to function: A single-guide RNA (sgRNA) and the Cas9 protein. The sgRNA recognizes complementary DNA sequences flanked by a 5′-NGG PAM motif and directs Cas9 to cleave the recognized DNA.315−318 By designing the guide sequence of sgRNA to hybridize with target DNA, CRISPR-Cas9 can be engineered to edit almost any genomic region of interest. As the CRISPR-Cas9 system undergoes development toward human therapeutics, efficient delivery poses the major challenge. Viral vectors have been

However, this strategy has limited real efficacy in clinical settings due to collateral damage to noncancer cells and an overly complicated multistage process. Comparing to conventional vehicles, DNA nanostructures are more readily synthesized with well-defined sizes. In addition, when miRNAs are loaded on DNA nanostructures via DNA−RNA hybridization, the nanostructure entity can provide steric hindrance to protect miRNA from RNase’s degradation. MiRNA-145 is recognized as an important therapeutic molecule due to its powerful tumor suppressive effect on cancer. In most cancer cell lines and tissues miRNA-145 was reported to be downregulated.310 Reinstating miRNA-145 could almost immediately suppress tumor growth and block cell invasion and metastasis.311 In 2017 Leong and co-workers designed and assembled a DNA star motif that each can carry three miRNA145 molecules and form a Shuriken-like shape upon miRNA-145 loading (Figure 20A).312 Shuriken’s multipronged configuration increased the residence time for robust uptake by cells without any transfection agents. In several cancer cell lines, Leong and coworkers observed close to 2-fold improvement in the DNA Shuriken performance when compared to utilizing the common transfection agent, Lipofectamine 2000. The DNA shuriken has an average hydrodynamic diameter of 24.0 ± 8.3 nm with the miRNA cargos within less than 5 nm of the DNA crossover segment and the triad arms of the Shuriken (Figure 20A). Leong and co-workers believed that the close proximity could provide steric shielding from degradative RNase enzymes. They did find that the intracellular miRNA-145 level is more than 5000 or 30 times higher than that of the untreated control or naked miR145-treated cells, respectively, even after 24 h. On the highly proliferative colorectal cancer cell line DLD-1, the DNA Shuriken also showed significant antiproliferative effects. In addition, on a 3D spheroid tumor model the DNA Shurikentreated tumor was 31% smaller than the untreated one after 2 days. The work indicates that DNA nanostructures can be AD


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Figure 22. ATP-responsive DNA assemblies loaded with Dox, protamine, and a HA-cross-linked gel shell. Adapted with permission from ref 342. Copyright 2014 Nature Publishing Group.

frequently used to coexpress Cas9 and sgRNA in vivo.319,320 However, the random integration of viral DNA into the genome of host cells can potentially lead to genetic diseases.321 Also, often the overexpression of Cas9 and sgRNA in vivo increases the offtarget effect.322,323 An alternative is to codeliver the Cas9/sgRNA complex in a controllable fashion.324 However, both proteins and RNA suffer from poor cell-membrane permeability due to their large size and low solubility as well as the negative charges, respectively. An ideal carrier should be able to shield the protein and RNA from detrimental physiological environment and escort them to the cell nucleus where they function. DNA nanostructures have been explored by Gu and co-workers as a safe and efficient way for codelivery of Cas9/sgRNA.325 They synthesized a DNA nanoclew (NC) by RCA and loaded the NC with Cas9/sgRNA complex through the partial hybridization between the RCA-generated ssDNA and the guide sequence of sgRNA (Figure 21). Gu and co-workers fused the nuclearlocalization-signal peptides to Cas9 to promote the transportation of Cas9/sgRNA complex from cytoplasm to nuclei. In addition, they also coated the complex with cationic polymer polyethylenimine (PEI) to facilitate the induced endosomal escape.326 With cultured cells and tumor-bearing mice models, Gu and co-workers demonstrated that the DNA NCs efficiently delivered Cas9/sgRNA to the nuclei and in there Cas9/sgRNA effectively drove the formation of indels through targeted DNA cleavage.325 Although the potential immunogenicity associated with DNA NCs is not clear at this stage, DNA nanotechnology has opened a door to deliver Cas9/sgRNA for human therapeutics. Note that because the current DNA nanotechnology-enabled delivery of the CRISPR-Cas9 system takes advantage of the programmable interaction between the DNA nanostructure and sgRNA, we place this RNA−protein hybridized drug system in the Oligonucleotides section for discussion. 3.3.2.5. Aptamers. Aptamers are single-stranded DNA or RNA sequences that can recognize and bind a wide range of target molecules with affinities and specificities comparable to

antibodies. When incorporated into DNA nanostructures, besides assisting them to target specific cells for drug delivery as described in section 3.2.2, aptamers can also endow DNA nanostructures with the ability to sense and respond to the cellular environment, making them “smart” carriers. Meanwhile, DNA nanostructures can make therapeutic aptamers more resistant to nuclease degradation, resulting in enhanced drug efficacy. Comparing to protein therapeutics, aptamers exhibit significant advantages in terms of size, synthetic accessibility, and modification by medicinal chemistry. 227 Blending DNA nanostructures with aptamers have been studied by many laboratories to assist drug delivery. AS1411, one of the popular aptamers, has been used as a cancer-targeting ligand.327−330 Its receptor is known as nucleolin, which is a glycoprotein upregulated on the plasma membrane of several cancer cells.331−333 In 2014 Bermudez and co-workers investigated the uptake and efficacy of pyramidal DNA nanostructures bearing multiple copies of the AS1411 aptamer in a human cervical cancer cell line (HeLa).334 They found that the AS1411−DNA pyramids exhibited enhanced intracellular uptake in the absence of any transfection reagents and selectively inhibited the growth of cancer cells. Furthermore, the complex was substantially more resistant to nuclease degradation than the aptamers alone.334 Aptamers have been mostly codelivered with other drug molecules to maximize the drug efficacy. One example is by Huang and co-workers, who created aptamer-conjugated and Dox-intercalated DNA icosahedron.335 The aptamer targets MUC1, which is an important class of tumor surface marker that is uniquely and abundantly expressed on various epithelial cancer cells.336,337 In addition, MUC1 is also rapidly recycled through intracellular compartments338,339 and thus can serve as an entry portal for its aptamer.340 Under confocal microscope, Huang and co-workers observed that only the aptamer-conjugated icosahedron efficiently internalized into MUC1-positive cells and that the distribution of Dox surrounded the nucleus for killing the epithelial cancer cells.335 AE


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Figure 23. Various drug molecules delivered by DNA nanostructure-based systems. (A) DNA nanorobot loaded with a protein payload. Adapted with permission from ref 360. Copyright 2012 American Association for the Advancement of Science. (B) â-gal functionalized with DNA shell. Adapted with permission from ref 370. Copyright 2015 American Chemical Society. (C) Nanoparticle-templated core−satellite DNA nanostructures with payloads encapsulated either via intercalating (orange hexagon) or hybridizing (green circle) to the DNA strands within the superstructure. Adapted with permission from ref 208. Copyright 2014 Nature Publishing Group.

Aptamers can specifically bind not only macromolecules like nucleolin and MUC1 but also small metabolic molecules. Cyclicdi-GMP (c-di-GMP) is an important bacterial second messenger and regulates many pathways. Krishnan and co-workers designed a DNA icosahedron integrated with c-di-GMP aptamers and certain internal cargosthe fluorescent dextran.341 In the in vitro test, the binding of c-di-GMP leads to a shape shift of its corresponding aptamer, which in turn resulted in the disassembly of the DNA icosahedron, and the subsequent release of fluorescent cargos verified by fluorescent microscopy. Later, Gu and co-workers built an ATP-responsive DNA nanostructure and demonstrated its drug delivery functionality in vivo.342 The whole complex contains three functional domains: an ATPbinding DNA aptamer loaded with Dox, a protamine, and a hyaluronic acid (HA)-cross-linked shell (Figure 22). Dox was intercalated in the duplex region of the aptamer. The positively charged protamine was applied to compress the Dox-loaded aptamer into a cationic core complex to facilitate cell penetration and endosomal escape. It has been reported that HAase is rich in many malignant tumor matrices and the tumor cellular endocytic vesicles. Thus, the HA shell can be more rapidly degraded in tumor sites by HAase to release the cationic complex for intracellular transportation. With protamine, the Dox−aptamer complex escaped from the endosome and transported into the cytosol, where the high concentration of cytosolic ATP in tumor

cells induced the conformational change of the ATP aptamer, resulting in the release of Dox to produce strong cytotoxicity and induce apoptosis. Gu and co-workers demonstrated that this sophisticated ATP-responsive system exhibited ∼4-fold enhanced cytotoxicity in the MDA-MB-231 cancer cells than the systems without ATP aptamers.342 In the following work, Gu and co-workers applied similar design principles to build a fusogenic liposome with a protein− DNA complex core containing an ATP-responsive DNA aptamer scaffold loaded with Dox343 and ATP-responsive aptamergraphene hybridized nanoaggregates for anticancer drug delivery.344 These studies indicate that the stimuli-responsive nanovehicles including DNA nanostructures can be engineered to promote physiological specificity and on-demand therapeutic efficacy of anticancer drugs. 3.3.2.6. Deoxyribozymes. Deoxyribozymes (DNAzymes) are single-stranded DNA molecules that form structures capable of catalyzing chemical reactions. Numerous DNAzymes have been reported to sense various metal ions,345 including the physiological ones like Na+ 346,347 and Ca2+,348 to catalyze a series of chemical reactions such as the cleavage of RNA. Some DNAzymes are also known to recruit amino acids like Lhistidine349 as cofactors for RNA cleavage. RNA-cleaving DNAzymes have attracted significant attention for both therapeutic and diagnostic applications due to their excellent AF


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programmability, stability, and activity.350 They can be designed to cleave a specific mRNA to downregulate gene expression in vivo for tumor suppression.351 However, efficient delivery of DNAzymes into cells remains challenging. Because of their negative charges, DNAzymes have poor cell membrane permeability. Also, once inside cells, they can hardly escape lysosomal degradation. Several DNAzymes have been delivered into cells using AuNPs.352−354 However, the biosafety of AuNPs is another concern. DNA nanostructures like tetrahedrons are known to be able to enter several types of cells readily without the aid of transfection agents.146,197,198 They recently were explored to carry DNAzyme probes for multiplexed detection of intraellular metal ionsUO22+ and Pb2+in living cells.355 DNA dendrimer nanostructures were also proved as an efficient nanocarrier of the histidine-dependent DNAzyme for intracellular molecular sensing.356 The two examples show the feasibility of using DNA nanotechnology to facilitate the delivery of DNAzyme-based molecular probes. Meanwhile, they hint at the possibility of DNA nanotechnology-enabled delivery of therapeutic DNAzymes in the future. 3.3.3. Proteins. As the engine of life, proteins perform essential functions in cells, including catalyzing metabolic reactions, gene replication and regulation, signal transduction, and so on. Many life-threatening diseases such as cancers exhibit abnormal expression patterns and/or functions of certain proteins. Thus, most of the current cancer therapies target or are based on proteins. For example, the popular monoclonal antibody therapy uses monoclonal antibodies to bind monospecifically with certain cells or proteins to stimulate the patient’s immune system to attack those cells. However, the main barrier for protein-based therapeutics comes from their intrinsic properties such as the vulnerability to enzymatic degradation and difficulty to penetrate the membrane.357−359 A series of vehicles, including lipid-mediated colloidal systems, polymeric nanocarriers, and inorganic systems, have been developed to escort proteins to the targeted cellular locations. Recently, DNA nanovehicles have also been explored for protein delivery. In 2012, Church and co-workers reported a logic-gated DNA nanorobot for targeted transport of molecular payloads including antibodies.360 Using the origami technique, they engineered a hexagonal DNA barrel that could respond to a wide array of cues through an aptamer-encoded logic gate (Figure 23A). Payloads such as antibody fragments were loaded inside the barrel. By designing the cues to be cell surface signals that can be recognized by the aptamer, the barrel sensed the cell surface inputs and triggered the opening of itself to release the payloads. When testing the robustness of payload release by this system, Church and co-workers loaded a combination of antibodies to human CD33 and CDw328 and observed induced growth arrest in NKL (natural-killer leukemia) cells in a dose-dependent fashion.360 The results matched well with the report that the two antibodies can induce growth arrest in leukemic cells.361 Other antibody fragments that target human CD33 and flagellin362 were also successfully loaded and delivered by this DNA nanorobot to enhance T-cell activation.360 In the same year, Chang, Yan, and co-workers used DNA tetrahedron nanostructures as platforms to assemble a model antigen (streptavidin) and CpG adjuvants together into synthetic nanoscale vaccines.363 In the immunized mice, they found that compared to a mixture of CpG and antigen, the assembled antigen−adjuvant−DNA complexes induced strong and longlasting antibody responses without stimulating a reaction to the DNA nanostructure itself. The work demonstrates the potential

of DNA nanostructures to serve as general platforms for the rational design and construction of various vaccines. Similarly, through the avidin−biotin modification, in 2014 Zhao and co-workers designed a simple, versatile, multivalent ligand system that was made of a RCA-produced polymeric DNA scaffold decorated with antibodies to specifically and efficiently interrogate and modulate cell receptor signaling and function.364 Using CD20 clustering-mediated apoptosis in B-cell cancer cells as a model system, they demonstrated that the system with multivalent CD20 antibodies was significantly more effective at inducing apoptosis of target cancer cells than the monovalent antibody. Although binding cell-surface receptors to mediate downstream cellular signaling through multivalent clustering has been fulfilled with the aid of other materials such as synthetic polymers,365−368 this multivalent DNA nanosystem is easier to manipulate and represents a new chemical biology tool for the development of therapeutics. Besides using chemical or biological modifications to load proteins in a DNA nanostructure as described above, Crawford et al. also showed that proteins could be encapsulated in a DNA cage through the noncovalent DNA−protein interaction.369 They designed a DNA tetrahedron, on one edge of which contains a 22-bp DNA recognition site for a transcription factor protein, CAP (catabolite activator protein), and showed that CAP could stably bind inside the DNA cage at a 1:1 ratio by bending the edge to accommodate the protein. In principle, this approach can also be extended to encapsulate other DNAbinding proteins in DNA nanostructures.369 In order to increase the cellular uptake efficiency of proteins, Mirkin and co-workers reported a strategy to chemically modify a functional protein core, β-galactosidase, with a dense shell of oligonucleotides (Figure 23B).370 The protein/DNA complex was termed as ProSNAs, in which the protein enzyme retained its native structure and catalytic ability, despite the functionalization of its surface with ∼25 DNA strands. The cellular uptake of SNAs is known to be superior relative to that of their individual components.200,202 This enhanced cellular internalization of SNAs is likely derived from the 3D architecture of the conjugates and its ability to engage scavenger receptors on the surfaces of most cells.200,371 Mirkin and co-workers found that the cellular uptake efficiency of the ProSNAs was enhanced by up to ∼280fold compared with the bare enzyme.370 In addition, the in vivo working concentration of ProSNAs could be as low as 100 pM. They also confirmed that the β-galactosidase delivered by ProSNAs effectively catalyzed the hydrolysis of β-glycosidic linkages once endocytosed, whereas equal concentrations of proteins alone showed little to no intracellular catalytic activity. The RCA-based DNA nanoclews have been frequently explored by Gu and co-workers as nanocarriers for the efficient delivery of Dox147 and CRISPR-Cas9 systems.325 Recently, they reported that the transformable DNA nanoclews could be engineered for plasma membrane-targeted delivery of cytokine.372 Cytokines represent a class of anticancer therapeutics due to their specific activities in inducing apoptosis in cancer cells.373 Most of the current anticancer protein delivery systems were designed for intracellular delivery by harnessing the sizedependent endocytosis of nanoparticles.374−376 Gu and coworkers designed two DNA nanoclews carrying complementary sequences in the core which were covered by liposome shells.372 The model cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), a cytokine that interacts with death receptors on the plasma membrane and induces tumor specific apoptosis,377,378 was loaded within the Ni2+-modified core AG


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sequences via a Ni2+−polyhistidine affinity. Gu and co-workers demonstrated that when the DNA nanoclew mixtures entered the tumor microenvironment, the liposome shells were degraded by phospholipase A2, an enzyme overexpressed by various tumors.379 This in turn released the TRAIL-loaded DNA nanoclews into the extracellular environment. Subsequent hybridization of the complementary DNA sequences occurred extracellularly, transforming the compact DNA nanoparticles into DNA nanofibers with microscale lengths. The nanofibers served as multivalent scaffolds to efficiently present TRAIL to death receptors on the cancer cell membrane and amplified the apoptotic signaling with reduced TRAIL internalization.372 3.3.4. Inorganic Nanoparticles. Inorganic nanoparticles can be synthesized in the scale of 1−100 nm with precise shape as well as controllable surface chemistry and physical properties.380,381 In recent decades, inorganic nanoparticles have been studied for biosensing, bioimaging, and drug delivery by many laboratories.381−384 As a class of inorganic nanoparticles, plasmonic nanoparticles exhibit interesting scattering, absorbance, and coupling properties based on their geometries and relative positions. These unique properties have made them a focus of research in many applications including cancer treatment with photothermal therapy.385,386 A remaining issue for clinical application of inorganic nanoparticles is the concern about the safety issues, like the high toxicity and poor biodegradability.387 Many different inorganic nanoparticles can be conjugated with DNA oligonucleotides through either chemical bonding, e.g., the disulfide bond, or biological interaction, e.g., the biotin−avidin interaction. On the basis of the great programmability and addressability of DNA nanotechnology, researchers have fabricated highly ordered inorganic nanoparticle−DNA assemblies with controllable aspect ratios and spatial distributions in order to improve the targeting specificity and/or reroute the endocytosis pathway of inorganic nanoparticles to enhance their drug efficacy and mitigate their toxicity. Meanwhile, when conjugated to DNA nanostructures, inorganic nanoparticles can be excellent labels for tracking the intracellular location of DNA nanostructures via fluorescence or electron microscopy.388 Moreover, the complex of inorganic nanoparticle-conjugated DNA nanostructure can possess both the programmable property intrinsic to DNA and the physical properties associated with inorganic nanoparticles, such as plasmonic and magnetic features. In 2014, Mao and co-workers reported that self-assembled, well-defined soft DNA polyhedron can be used for the encapsulation of rigid AuNPs into their interior spaces through a swallow mechanism.389 These AuNPs were conjugated with short DNA oligo strands through thiol linkages. The subsequent hybridization of the conjugated DNA oligos with their complementary sequences inside the polyhedron sucked in the AuNPs and positioned them there. Mao and co-workers demonstrated that the AuNP guest can be released in a controllable way from the polyhedron host.389 This work hints at the potential applications in surface engineering and cargo delivery of inorganic nanoparticles with DNA nanotechnology. As described in section 2.6, DNA and inorganic nanoparticles can be blended together to produce core−satellite superstructures. Chan and co-workers built such superstructures with one or multiple layers of satellite AuNPs surrounding a central core AuNP through the DNA linkages (Figure 23C).208 The architecture of the assembled superstructure can be controlled by adjusting both nanoparticle geometry and DNA

grafting density. The outer surface of the superstructure was coated with PEG for interfacing with biological systems. Chan and co-workers chose J774A.1 murine macrophages as a model cell system to study the uptake of nanoparticle superstructures.208 Macrophages are known to sequester the majority of in vivo administrated nanoparticles,206 and macrophage uptake correlates with nanomaterial size and surface charge.207 Interestingly, Chan and co-workers observed that the core− satellite superstructure was 2.5-fold bigger than its core component but resulted in 2-fold lower uptake into macrophages. They also found that in macrophages the superstructures were disassembled into their respective building blocks by enzymatic degradation. Although many nanoparticle formulations have been reported to aggregate under such environments,390 Chan and co-workers found that these components remained dispersed following breakdown and eventually escaped from the vesicles and were distributed throughout the cellular cytoplasm.208 They also confirmed that a certain percent of the dispersed building blocks could escape from the macrophages following uptake, implicating the facilitated in vivo clearance by the core−satellite design. In addition, Chan and co-workers demonstrated that these superstructures could improve their accumulation in tumor sites as well as facilitate their elimination from the body.208 In a following study, Chan and co-workers constructed DNAassembled gold−nanorod “core−satellite” superstructures for controllable drug loading and release.391 They found that by reasonable design of the DNA linker sequences, the loading capacity of Dox could be rationally controlled. To thermally denature DNA, Chan and co-workers selected gold−nanorods (AuNRs) as the core nanoparticle because they have a high absorption cross section and photothermal conversion within the medical window (700−1000 nm).392 Rapid Dox release was achieved by photothermal-induced linker DNA denaturation, resulting in a 2.1-fold increase in therapeutic effect compared to no laser treatment. This study presents a new strategy to control drug release from DNA−nanoparticle-assembled superstructures.391 Using a similar strategy, Ding and co-workers designed a DNA origami as a platform to integrate Dox, AuNRs, and a tumorspecific aptamer MUC-1 to realize the effective circumvention of drug resistance.393 Dox was loaded through base-pair intercalation, and oligos-conjugated AuNRs were assembled onto the origami through DNA hybridization. The MUC-1 aptamer enabled targeted delivery of the origami complex to multidrug resistant MCF-7 cells. After the targeted internalization, Ding and co-workers shined cells with a near-infrared laser and observed that the multidrug resistance pump protein Pglycoprotein was significantly downregulated, indicating the achievement of the synergistically chemotherapeutic (Dox) and photothermal (AuNRs) effects. To further explore the functionality of DNA-assembled inorganic nanoparticles for navigating complex biological environments, Chan and co-workers constructed DNA-controlled dynamic colloidal nanoparticle systems.394 The systems consist of a core nanoparticle surrounded by small satellites, whose conformation can be transformed by a toe-hold DNA displacement mechanism. Chan and co-workers found that by changing the surface display of targeting ligands (e.g., uncovering folic acid conjugated on the core to allow its interaction with the receptor on the cell membrane), the cellular targeting efficiency was increased 2.5 times.394 The study indicates that the core− satellite DNA−nanoparticle assemblies have the potential for AH


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the tetrahedron with nucleus-targeting signaling peptides, we observed the transport of the DNA nanostructure to nuclei. Besides caveolae-mediated endocytosis, there are a few other ways that can be harnessed to avoid the lysosomal degradation of DNA nanostructures. One is to take the retrograde endocytic pathways to deliver DNA nanostructures to microtubules, endoplasmic reticulum, trans-Golgi network, and/or other organelles.243,388 An alternative is to use nanoscale needles to penetrate the cell membrane and subsequently release surfacebound DNA nanostructures directly into cell cytosol.401,402 Chan and Lo demonstrated that the silicon nanoneedle (SiNN) arrays enabled the delivery of 3D DNA nanocages into cells with high uptake efficiency, enhanced stability, low cytotoxicity, and little damage to the cellular membrane.402 By decorating the DNA nanocages with organelle-localization signal peptides, Chan and Lo found that the nanocages could automatically transport to a specific subcellular compartment such as the mitochondria or nucleus.402 After accomplishing their delivery tasks inside cells, unlike inorganic nanoparticles that can often resist nuclease degradation and result in in vivo persistence, DNA nanostructures are likely to be degraded due to their inherent biodegradability. Perhaps the degraded DNA products (nucleotides) are served as nutrients and absorbed by cells. Interestingly, according to Chan and co-worker’s report,208 inorganic nanoparticletemplated DNA nanostructures could be a solution to promote the exocytosis of inorganic nanoparticles and mitigate their cellular persistence and the resulting cytotoxicity.

mimicking the diverse functions of a protein to selectively control the biological functions of the engineered nanosystem.

4. CELLULAR FATE OF DNA NANOSTRUCTURES Within cells, the DNA components of DNA nanostructures are believed to be degraded eventually by the natural DNases. However, when and where the degradation is processed is related to the uptake pathway of DNA nanostructures, which depends on various factors, including their size, shape, surface chemistry, and physical properties, as well as the type of cell for uptake. Generally, the majority of in vivo-administered nanoparticles, including DNA nanostructures, are sequestered by macrophages or other phagocytes via phagocytosis, wherein various enzymes in phagolysosome eventually degrade the ingested particles. In 2013 Krishnan and co-workers exploited the coelomocytes of the multicellular model organism Caenorhabditis elegans to study the lifetime of various DNA nanostructures in vivo.395 Coelomocytes are phagocytic leukocytes that appear in the bodies of animals that have a coelom. They found that reducing single-stranded domains on DNA nanostructures enhanced the nanostructures’ in vivo stability: A DNA duplex carrying two single-stranded domains was estimated with an in vivo half-life of 8 h, while removing some of the single-stranded domains increased its halflife to 11 h. Moreover, Krishan and co-workers observed that a DNA icosahedron396 without free termini was able to remain intact inside lysosomes over 24 h of investigation.395 An explanation of the enhanced in vivo stability of DNA icosahedron was that it maintained the structural integrity even at the physiological Mg2+ concentration (1−2 mM), and such an icosahedron conformation protected the DNA nanostructure from DNase attack. If DNA nanostructures circumvented phagocytosis, they could be internalized by cells via an active energy-dependent transport process, such as the clathrin- or caveolae-mediated endocytosis. As discussed earlier in this review, in clathrin-mediated endocytosis, DNA nanostructures are usually encapsulated in vesicles and transported to the early endosomes as well as the late endosomes. They finally arrive at lysosomes for endolysosomal degradation.147,268,397 Similar to what happens in phagolysosomes, the acidic environment and various DNase in lysosomes facilitate the degradation of DNA nanostructures and the release of drug molecules. Before reaching lysosomes, according to Krishan and co-worker’s work,243,388,398−400 there could be up to hours for DNA nanostructures to maintain their structural integrity in vivo and travel through the early endosomes/sorting endosomes and the late endosomes. In the above case, lysosomes are the terminal stop of DNA nanostructures. However, in some circumstances, drug molecules need to be delivered to specific subcellular compartments for drug functionalization. Several strategies have been developed to either break DNA nanostructures out of endosomes or train DNA nanostructures to ride through other pinocytosis pathway, such as the caveolae-mediated endocytosis, to continuously travel for their destination. For examples, it is known that through the proton sponge effect, positively charged nanoparticles can induce osmotic lysis upon endosome acidification.252,253 Gu and co-workers took advantage of that and decorated a DNA nanoclew vehicle with cationic polyethylenimine. By doing so, they were able to make the DNA nanostructure escape from the endosomal transportation and deliver the carried Cas9/sgRNA from cytoplasm to nuclei.326 We reported that a DNA tetrahedron could enter live Hela cells through the caveolae-mediated pathway.199 Also, by decorating

5. CHALLENGES AND OUTLOOK In this review, we briefly summarized recent advances and developments of DNA nanotechnology. The high programmability of DNA enables the design and self-assembly of numerous well-defined 2D and 3D nanostructures, many of which have been demonstrated to be elegant drug carriers. The intrinsic biocompatibility and biodegradability of DNA makes DNA− nanostructures highly intriguing drug delivery vehicles. Static and dynamic DNA nanocarriers have been engineered to be able to either passively or actively release payloads at specific sites. The various practicable chemical modifications on DNA even allow the addition of more functional groups to DNA nanostructures, resulting in the generation of versatile hybrid nanostructures of DNA and other materials. For example, with thiolated end labeling, DNA can be conjugated with AuNPs111,115−117 and lipid molecules180,181 for the assembly of AuNP-templated DNA nanostructures and DNA−nanostructure-guided liposome, respectively. A number of design strategies, including tile-based bottom-up, origami-based top-down, nanoparticle-templated, metal-assisted, and RCA assisted, have been applied for the assembly of DNA nanostructures to intercalate, conjugate, encapsulate, or noncovalently bind drug molecules to enhance their biostability, elongate their circulation time, change their surface and mechanical properties, as well as add functions such as responding to environmental cues and targeting specific cells. The unique feature of DNA nanostructure-based drug delivery systems is the modularity, meaning that the size of a DNA nanoobject and the positions of modifications (ligands or drug molecules) on or within it can be precisely controlled at nanoscale, as well as the shape and flexibility of the DNA object can be fine tuned. It is the modularity that makes DNA nanotechnology a robust platform to bring in a lot of aspects in a controllable manner for targeted drug delivery. In the past AI


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recently enzymatic digestion of RCA products has also been explored to generate high-quality short single-stranded oligonucleotides. For example, Högberg and co-workers reported using a restriction enzyme to digest RCA products to generate desired DNAs with 14−378 bases in length.145 Our team succeeded in using hydrolytic deoxyribozymes to self-digest RCA products for desired DNAs with length ranging from 50 to over 100 bases.410,411 Combining these alternative DNA preparation methods, it is likely that we could reduce the cost for DNA− nanostructure drug vehicles down to that of antibodies ($300 per gram412 or less) or aptamers ($50 per gram227 or less). However, still there is a big gap that needs to be filled before being able to compete with polymer materials which can cost less than $1 per gram. Third, biosafety is another concern. DNA is biodegradable and biocompatible. However, things could be changed when DNA is engineered to become a nanostructure. Before using DNA nanostructures for clinical bioapplications, we must systematically investigate their potential immunostimulatory properties. In certain early studies, DNA nanostructures performed very well as drug vehicles. For example, Anderson and co-workers demonstrated a well-defined DNA tetrahedron can deliver siRNAs into cells and silence target genes in tumors.216 In their mouse model, they did not observe antibody response against the DNA nanostructure. However, several critical questions remain to be answered, including (a) How do the physiochemical properties of DNA nanostructures affect the renal system, (b) would the foreign DNA sequences in the DNA nanostructures cause any harmful genetic recombination, and (c) for those nanoparticletemplated or metal-assisted DNA nanostructures what is the final fate for those metal elements? Are those metal elements toxic to cells? We believe these DNA nanostructure-related biosafety questions are potentially attractive topics to be studied in future. Cells evolved to manipulate DNA in a complex yet exquisite way. It is believed that the total amount of DNA in a cell is strictly maintained at a certain level.413−415 When DNA nanostructures penetrate the cell membrane, it must cause a temporary increase of DNA level in the cell, which could result in unexpected problems. An alternative approach to carry and deliver drug molecules into cells could be using RNA nanostructure instead of DNA. As DNA’s biological counterpart, RNA is more versatile and the total amount of a cell’s RNA can fluctuate in a wide range.416 Nanotechnologists have already confirmed that the design principles of DNA nanotechnology can be perfectly applied for assembling RNA nanostructures, some of which were used for drug delivery.417−422 Certain RNA nanostructures have been demonstrated to self-assemble from the RNA transcripts in cells423 or in transcription buffer,424 and the former has been harnessed to organize intracellular reactions.423 As a derivative of DNA nanotechnology, RNA nanotechnology is definitely another field that is worthy to watch and follow, especially when it comes to drug delivery.

decade, DNA nanostructures have made remarkable transition from the in vitro to the in vivo environment. Structures such as tetrahedron, octahedron, and origami-based objects have been demonstrated to be able to carry siRNA, AuNPs, CpG, aptamers, antibodies, or small-molecule drugs for targeted delivery. With increasing efforts on applying DNA nanotechnology for modern drug delivery, we strongly believe that this nascent and developing field will have a huge impact on advanced healthcare sciences in the near future. Despite the advantages, the potential, and expectations, there are undoubtedly certain obstacles that need to be addressed before making DNA nanostructures competitive with existing drug vehicles such as polymers, liposomes, viruses, and so on. First, little is known for the pharmacokinetics (in vivo circulation, distribution, metabolism, excretion, etc.) of DNA nanostructures. Origami-based DNA nanostructures have been reported to hold the structural integrity for a long time in cell lysate.170 However, it is unclear how the physical and chemical properties, including geometry, surface charges, base combinations, and oligonucleotide modifications, of DNA nanostructures affect the pharmacokinetic bioavailability. From primary targeting to secondary targeting, there are a couple of barriersthe blood−brain barrier and the plasma membrane of cellthat prevent efficient passage of DNA nanostructures. Little has been studied for penetrating the blood−brain barrier by DNA nanostructures. Also, the current cellular uptake of most DNA nanostructures mainly relies on the insufficient endocytosis or pinocytosis pathway by themselves. Thus, it is very urgent and important to design approaches for selective uptake of DNA nanostructures by specific organ(s) and diseased cells while minimizing nonselective uptake by normal organ(s) and cells. This is a common challenge for any drug vehicle. However, the completely programmable, precisely controllable, and fully surface-addressable virtue makes DNA nanostructures top candidates to tackle this problem. Second, production cost is a concern. For practical biomedical applications, we need to be able to prepare functional DNA nanostructures with high purity at a perhaps gram scale. Several groups have reported convenient and cost-effective purification methods in the laboratory level, but demonstration of these methods at a larger scale has not been performed. For example, Shih and co-workers developed an agarose-gel-based separation method to purify intact DNA origami nanostructures with high yields.403 In a following work, Lin and Shih found that ultracentrifugation provides scalable and contamination-free means to recover DNA origami nanostructures.404 To reduce the cost for scale-up chemical synthesis of DNA, alternative methods including asymmetric PCR, RCA, fermentation, and incell production have been explored to produce a large amount of DNA at a relatively cheap cost. For example, Weuster-Botz and co-workers developed high-cell-density bioreactors via fermentation to efficiently generate large quantities of M13 phage genome,405,406 the major component for most DNA origami nanostructures. Yan and co-workers designed a single-stranded PX DNA nanostructure for efficient amplification in live cells.407 To efficiently prepare long single-stranded DNA other than M13mp18 genome as the scaffold in DNA origami, Pound et al. modified the PCR protocol and succeeded in obtaining singlestranded DNAs up to 10 000 bases in length.408 We further extended the limit of scaffold length up to 26 kilobases with asymmetric PCR,409 which resulted in the assembly of supersized DNA origami with more capacity to carry payloads. Besides the PCR method for the production of long single-stranded DNA,

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Lihua Wang: 0000-0002-6198-7561 Chunhai Fan: 0000-0002-7171-7338 AJ


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Author Contributions

ABBREVIATIONS

⊥

Q.H. and H.L.: These authors contributed equally to this work. AFM AptNA aptNTr AuNPs bp CCMV c-di-GMP CME CP CpG

Notes

The authors declare no competing financial interest. Biographies Qinqin Hu obtained her Ph.D. degree in Biosystem Engineering from Zhejiang University in 2016. She now works as a postdoctoral fellow under the supervision of Professor Hongzhou Gu at the Institute of Biomedical Sciences of Fudan University. Her research focuses on the selection of functional oligonucleotides for biosensing and targeted drug delivery.

CPZ CvME Cy3 DNase DNC Dox dpp DX ER FAM FDA FR FRET GBM HA HBD IL JX2 MDR MMPs MβCD NCa NCl NFs NKL NRP PD-1 PEI polyA PT PX RCA RNAi ROX SELEX

Hua Li did his doctoral training under the supervision of Professor Junbo Ge at Fudan University. He was awarded his Ph.D. degree in Cardiology in 2013 and then moved to the University of California, Los Angeles (UCLA) for his postdoctoral training in Professor Peipei Ping’s group. He is now Principle Investigator in Zhongshan Hospital of Fudan University. His research focuses on novel nanomaterial-based drug delivery and cell biology in Cardiology. Lihua Wang is a Professor at Shanghai Institute of Applied Physics, CAS. She obtained her Ph.D. degree in Inorganic Chemistry from the Shanghai Institute of Applied Physics, CAS, in 2003. Her research focuses on the development of novel biosensors using aptamers and nanomaterials, high-resolution cell imaging, and multimodality in vivo imaging. Hongzhou Gu is a Professor in the Institutes of Biomedical Sciences at Fudan University and holds a joint faculty appointment at Fudan University Shanghai Cancer Center. He obtained his Ph.D. degree in Chemistry from New York University in 2010 and then worked as a postdoctoral researcher in Molecular Biology at Yale University. His research focuses on the selection of aptamer-based sensors for molecular probing and imaging, as well as the development of smart DNA or RNA nanodevices for targeted drug delivery. Chunhai Fan is Professor and Chief of the Division of Physical Biology at SINAP and the Center of Bioimaging at the Shanghai Synchrotron Radiation Facility (SSRF). He obtained his B.S. and Ph.D. degrees from Nanjing University in 1996 and 2000, respectively. After postdoctoral research at the University of California, Santa Barbara, he joined the faculty at Shanghai Institute of Applied Physics (SINAP), Chinese Academy of Sciences (CAS), in 2004. He is an elected fellow of the American Association for the Advancement of Science (AAAS), the Royal Society of Chemistry (RSC), and the International Society of Electrochemistry (ISE). He is also an Associate Editor of ACS Applied Materials & Interfaces. His research interests are biosensors, bioimaging, and DNA nanotechnology.

sgRNA SiNN siRNA SNARE

ACKNOWLEDGMENTS The involved research in this work was partially supported by the China Postdoctoral Science Foundation (2017M611453), the Project of Thousand Youth Talents (KHH1340004), the National Natural Science Foundation of China (21673050, 81500229, 21675167, 21390414), the National Key Research and Development Program of China (2016YFC1306400, 2016YFA0201200, 2016YFA0400900), and the Key Research Program of Frontier Sciences, CAS, Grant No. QYZDJ-SSWSLH031. We thank Prof. Yimin Wang and Dr. Jiantao Wu from Shandong Buchang Pharmaceutical Co., Ltd. for their helpful comments.

SNAs SR-A SST TDNs TEM Tf TGMS TGN TLR9 AK

atomic force microscope aptamer-based DNA assembly aptamer-tethered DNA nanotrain gold nanoparticles base pairing cowpea chlorotic mottle virus cyclic-di-GMP clathrin-mediated endocytosis capsid proteins unmethylated cytosine-phosphate-guanine dinucleotides chlorpromazine caveolae-mediated endocytosis cyanine 3 deoxyribonuclease DNA nanococoon doxorubicin diphenylphenanthroline double crossover endoplasmic reticulum fluorescein Food and Drug Administration folate receptor fluorescent resonance energy transfer glioblastoma multiforme hyaluronic acid heparin binding domain interleukin paranemic crossover with two juxtaposed sites multidrug resistance matrix metalloproteinases methyl-β-cyclodextrin DNase I nanocapsule DNA nanoclew nanoflowers natural-killer leukemia neuropilin-1 programmed death 1 polyethylenimine poly adenine phosphorothioate paranemic crossover rolling-circle amplification RNA interference 6-carboxyl-X-rhodamine systematic evolution of ligands by exponential enrichment single guide RNA silicon nanoneedle small interfering RNA soluble N-ethylmaleimide-sensitive factor attachment protein receptor spherical nucleic acids class A scavenger receptor single-stranded tiles tetrahedral DNA nanostructures transmission electron microscope transferrin triglycerol monostearate trans-Golgi network toll-like-receptor 9 DOI: 10.1021/acs.chemrev.7b00663 Chem. Rev. XXXX, XXX, XXX−XXX

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transition metal dichalcogenides tumor necrosis factor tetraphenylporphyrin tumor necrosis factor-related apoptosis-inducing ligand triple crossover vascular endothelial growth factor

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