Hu Et Al 2018 Dna Nanotechnology Enabled Drug Delivery Systems

Review
Cite This: Chem. Rev. 2019, 119, 6459−6506 pubs.acs.org/CR
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
◊
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
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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 nano-
medicine. 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 nanostructure-
based drug delivery.
CONTENTS 3.3.3. Proteins 6491

3.3.4. Inorganic Nanoparticles 6492
1. Introduction 6459 4. Cellular Fate of DNA Nanostructures 6493
2. Structural DNA Nanotechnology 6462 5. Challenges and Outlook 6493
2.1. Origin of Structural DNA Nanotechnology 6462 Author Information 6494
2.2. Tile-Based Bottom-Up Assembly 6462 Corresponding Authors 6494
2.3. Origami Assembly 6464 ORCID 6494
2.4. Single-Stranded Tile Assembly 6465 Author Contributions 6495
2.5. Hybrid of Oligonucleotide Tile, Origami, and Notes 6495
Single-Stranded Tile 6467 Biographies 6495
2.6. Nanoparticle-Templated DNA Nanostruc- Acknowledgments 6495
tures 6469 Abbreviations 6495
2.7. Other Strategies for the Construction of DNA References 6496
Nanostructures 6469
3. DNA Nanostructure-Based Drug Delivery 6471
3.1. Primary Targeting―Getting to the Organ 6471
3.2. Secondary Targeting―Getting to the Cell 1. INTRODUCTION
and Cellular Subunits 6471 Both chemical and biomolecular drugs (e.g., iRNA, antibody)
3.2.1. Gateways into the Cell 6471 have intrinsic issues in maximizing their drug functions in
3.2.2. Cellular Targeting 6475 healthcare. The former often has poor solubility and tends to
3.2.3. Creating Artificial Channels with DNA cause unwanted side effects and toxicity problems, whereas the
Nanotechnology 6479 latter is prone to enzymatic degradation and difficult to penetrate
3.2.4. Subcellular Targeting 6480
3.3. Delivery of Drug Molecules by DNA Nano-
Special Issue: Nucleic Acid Nanotechnology
technology 6482
3.3.1. Small Molecules 6482 Received: November 2, 2017
3.3.2. Oligonucleotides 6483 Published: February 21, 2018
© 2018 American Chemical Society 6459 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
Chemical Reviews Review
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.
the cell membrane. To overcome these barricades it is necessary toxic elements or residuals13 and are usually difficult to be
to develop active and targeted systems for delivery of drug degraded,14 resulting in safety concerns. As an emerging delivery
molecules. An ideal drug delivery system would be capable of system, DNA nanostructures exhibit great potential to fulfill the
preserving the activity of drug molecules by protecting them ultimate goal of drug delivery, reaching the maximal efficacy with
against degradation, improving the solubility, and mitigating the minimal toxicity. DNA is a genetic material in nature. Thus, it
toxicity and other biological side effects of drug molecules, is inherently biocompatible and biodegradable. In biology,
penetrating in vivo barriers like epithelium and cell membrane, through A-T, G-C Watson−Crick base pairing (bp), DNA
specifically targeting cells and controlling drug release. Many mainly adopts the B-form structure, which is a double-stranded
different drug delivery systems have been developed so far,1,2 helix with about 2 nm in diameter and 3.4 nm (or 10.4 bp) per
including natural systems such as viruses,3 biomimetic systems helical turn. The persistence length of B-DNA is about 50 nm or
such as red blood cell mimics,4 synthetic organic systems such as 150 bp, which makes DNA a relatively stiff polymer in the
liposomes5,6 and cationic dendric polymers,7 and synthetic nanoscale.
inorganic systems such as gold nanoparticles8 and carbon Invented by Seeman in the early 1980s,15,16 DNA nano-
nanomaterials.9 These natural, bioinspired, or synthetic plat- technology has grown exponentially in the past two decades.
forms bypass the physiochemical limitation and barriers of drug Owing to the intrinsic properties of DNA including predictable
molecules to increase the drug loading efficacy, the body intermolecular interactions (Watson−Crick base pairing),
circulation time, and the cellular uptake. Many of them have been convenient automated chemistry synthesis, convenient modify-
in clinical trials and several approved for clinical therapeutics by ing enzymes, locally stiff polymer, externally readable code, high
the Food and Drug Administration (FDA). functional group density, and prototype for many derivatives,
Despite the advances in the development of drug delivery numerous DNA or DNA-based nanostructures,17−62 from
systems, each of the current drug carriers has some limitation. simple to complex and from small to large, have been self-
For examples, viruses can only deliver short DNAs into the cells. assembled with precisely controlled size and shape at one, two, or
There are risks of random insertion sites, cytophathic effects, and three dimensions, as well as with controllable surface chemistry
mutagenesis.10 The cationic surface charges of dendric polymers and dynamic function. These characteristics make DNA
are inherently cytotoxic,11 and the heterogeneous distribution in nanostructures unique platforms with great delivery capacities
size not only affects the drug loading capacity but also increases and offer new prospects for the bottom-up construction of
the immune toxicity.12 Many inorganic nanomaterials contain nanoscale drug carriers.63−74
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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 Following this will be a brief description of primary and
delivery systems. We will start with the origin of DNA secondary targeting for drug delivery. Then we will discuss drug
nanotechnology, introducing the immobile Holliday junction
in DNA nanotechnology, the key element for the construction of molecules that have been loaded and delivered by DNA
DNA nanostructures. We will then move to the design nanostructures, including CpG oligos, aptamers, siRNA,
milestones in the development of DNA nanotechnology,
CRISPR-Cas9, nanoparticles, and so on. In addition, we will
including Seeman’s oligonucleotide tile-based bottom-up
approach, Rothemund’s DNA origami approach, as well as discuss the cellular fate of certain DNA nanostructures. Finally,
Yin’s single-stranded tile approach. We will also discuss the we will touch on RNA nanotechnology, a derivative of DNA
hybrid of the above three techniques and some others, including
design of the nanoparticle, the metal−organic, and the rolling- nanotechnology, and finish with challenges and our thoughts on
circle amplification (RCA) templated DNA nanostructures. the future direction of DNA nanostructure-based drug delivery.
6461 DOI: 10.1021/acs.chemrev.7b00663
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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
2.1. Origin of Structural DNA Nanotechnology
DNA nanotechnology in 1982.
DNA is a linear polymer that carries genetic information in Besides the assembly of 1D, 2D, and even 3D lattices from
biology. Occasionally nonlinear DNA occurs in cells. For individual DNA tiles, numerous DNA nano-objects have also
example, during genetic recombination, homologous DNAs been built with Seeman’s tile-based bottom-up strategy.
exchange genetic information through a Holliday junction, which Following the initial immobile 4-arm junction, junctions with
is a branched nucleic acid structure that contains four double- less or more arms including 3-arm,86 5-arm,87 6-arm,87 8-arm,88
stranded arms joined together (Figure 1A). Because of the and even 12-arm88 were created. On the basis of these building
sequence symmetry, Holliday junctions are mobile intermedi- blocks, polyhedra of different sizes and shapes such as cubes,20
ates. The four individual arms can slide through the junction back tetrahedron,89 and octahedron,90 were assembled (Figure 2C).
and forth. In 1982, Seeman proposed that by breaking the Through multistep ligations, Seeman’s group constructed a
sequence symmetry of the four strands that make up the Holliday covalently closed cube-like molecular complex containing 12
junction, one should be able to lock the junction in a stable state equal-length double-helical edges arranged about eight vertices.
(Figure 1B).15 Using four synthetic DNA strands with Each of the six faces of this object is a single-stranded cyclic DNA,
programmed sequences to avoid symmetry, Seeman demon- doubly catenated to four neighboring strands, and each vertex is
strated that such an immobile Holliday junction indeed connected by an edge to three others. However, to synthesize
formed.16 By adding cohesive ends or single-stranded overhangs such a cube, laborious work was required and the yield was not
to the four-arm branched junction, Seeman proposed this high. Later, Turberfield’s group reported a facile way to build a
junction could act as tiles to self-assemble into infinite two- DNA tetrahedron with four synthetic DNA strands. By a quick
dimensional lattices (Figure 1C). The number of branched arms annealing step (95 to 4 °C in 30 s), tetrahedrons assembled with
in a junction might not stop at four. With six synthetic strands, a a yield of around 95%. The shape of the tetrahedron can be
six-arm junction might be created and act as a tile to further visualized under an atomic force microscope (AFM).
assemble in the three dimensions (3D) to generate a 3D DNA Bundling more DNA duplexes together can increase the tile’s
lattice (Figure 1D). Seeman pointed out that such a 3D DNA rigidity, which has been demonstrated during the assembly of
lattice could serve as a 3D template to organize and orient periodic arrays with DNA tiles. This principle also applies for the
macromolecules, such as proteins through DNA−protein construction of robust DNA nano-objects self-assembled from
interactions, in a 3D crystalline fashion to enable their structure DNA tiles. Yan and LaBean reported an improved second
determination. The immobile Holliday junction together with generation of the four-arm junction tile, which contains four four-
the proposal of using 3D DNA scaffolds for structural biology is arm DNA branched junctions pointing in four directions (Figure
considered as the origin of “structural DNA nanotechnology”. 2E).91 This point star-like tile is composed of nine strands, with
Along the development trajectory of the field are several one strand participating in all four junctions. Bulged T loops were
breakthroughs in the design technique of DNA nanostructures. placed at each of the four corners inside the tile core to decrease
Also, branched from its initial goal, DNA nanotechnology has the probability of stacking interactions between adjacent four-
shown promising applications in many different aspects arm junctions and to allow the arms to point to four different
including biosensing, bioimaging, diagnosis, and drug delivery. directions. Comparing to Seeman’s original four-arm junction,
2.2. Tile-Based Bottom-Up Assembly the new generation contains two DNA duplexes bundled in each
The immobile Holliday junction was the first tile built in DNA arm instead of one. Hence, such a tile is more rigid and robust for
nanotechnology. It can be considered as one simple crossover various bottom-up assemblies.
between two DNA duplexes. Later, more robust DNA tiles, such Mao’s group further extended the design of point star
as double crossover (DX),26 triple crossover (TX),75 paranemic tiles.92−95 They exploited sequence symmetry to design a series
crossover (PX),29,56 paranemic crossover with two juxtaposed of DNA tiles, including three-point, four-point, and five-point
sites (JX2),29,56 six-helix bundles,76 etc., were constructed by star tiles (Figure 2D and 2E). Each arm of the point star tile is
Seeman’s group using multiple junctions to bundle two or more more rigid due to the bundling of two duplexes. The adjacent
DNA duplexes together (Figure 2A). By doing so, the rigidity of arms are linked by the bulged loops to provide appropriate
those tiles is greatly enhanced. For example, the DX molecules bending angles. With longer bulged loops, the point star tiles
were characterized twice as stiff as linear DNA duplex,77,78 and bend more from their original geometric plane. For the three-
the estimated persistence length for six-helix bundle was 1−5 μm, point star tiles, Mao’s group reported that tetrahedrons self-
about 20−100-fold stiffer than the DNA duplex.79 All of the tiles assembled from the tiles with a loop length of five bases and a low
mentioned above have shown the ability to self-assemble into concentration of 75 nM, while the tiles with a three-base loop and
large 1D and 2D arrays,80−84 including one-dimensional tubes a 50 nM concentration yielded dodecahedrons and a three-base
and ribbons and two-dimensional arrays with stripes, triangle, loop at a 500 nM concentration generated buckyball structures.
quadrilateral, and hexagon patterns, within micrometer scales Each of the above DNA nano-objects is around 14 nm in
through tile−tile interactions via cohesive ends. diameter. Similarly, by tuning the flexibility of four- and five-
Besides 1D and 2D DNA arrays, efforts to fabricate 3D DNA point star tiles, octahedrons and icosahedrons were successfully
lattices have never been stopped. In 2004, Mao’s group reported assembled, respectively (Figure 2E). The robustness of the
a triangular motif called a tensegrity triangle (Figure 2B).85 It is a polyhedrons was further confirmed by cryo-EM 3D reconstruc-
rigid DNA motif with 3-fold rotational symmetry, consisting of tion.
three helices that are directed along linearly independent vectors. In the first two decades after the origin of DNA nano-
Their helix axis directions do not share the same plane. Hence, technology, Seeman’s oligonucleotide-assembled tiles and such
the tensegrity triangle has the potential to extend in the three tile-based nanoarrays and nanostructures were the major
dimensions to self-assemble into 3D lattices. In 2009, Seeman research scope and topic in this nascent field. By exploiting
and Mao’s group reported the crystal structure at 4 Å resolution sequence symmetry, a couple of synthetic DNA strands in a one-
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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 control of the individual oligonucleotide strands, which are time
even 3D crystals. The elegance and powerfulness of this design consuming. The complexity of nanostructures is limited due to
approach are obvious. On the other hand, there are also some relatively simple geometric shapes or repetition of basic tiles.
constraints for this approach. To design a tile, the sequences Furthermore, the addressable surface area and diameter of the
must be optimized via computer-aided programs. Also, the self-assembled DNA nanoarrays and nano-objects are also
formation of a tile often requires exact stoichiometric and purity limited by the small size of the individual tiles. In the next
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section we will talk about another design approach that resolves closely stacked 3D object. Due to the multiple packing, 3D DNA
most if not all of the above issues. objects made by this strategy are often very rigid. A series of 3D
2.3. Origami Assembly nanostructures such as mololith, square nut, railed bridge, slotted
cross, and stacked cross with precisely controlled dimensions
In 2004, Shih and co-workers designed and synthesized a 1669- ranging from 10 to 100 nm were constructed on the honeycomb
nucleotide, single-stranded DNA molecule through clonal lattice and demonstrated under transmission electron micro-
production. With the help of five 40-mer synthetic oligo scope (TEM) (Figure 3B). Later, Shih’s group reported that
DNAs, the long DNA strand folded into an octahedron structure multilayer 3D DNA origami can be packed not only on a
by a simple denaturation−renaturation procedure.50 This work honeycomb lattice but also on a square lattice.98 The square
laid the foundation of the following DNA origami technique. In lattice provides a more natural framework for designing
2006, Rothemund described a simple method for folding long, rectangular structures and an option for a more densely packed
single-stranded DNA molecules into arbitrary two-dimensional architecture, as well as the ability to create surfaces which are
shapes.45 To design a desired shape, a 7-kilobase M13mp18 more flat than that given by the honeycomb lattice.
genomic DNA was raster filled into the shape as a scaffold, and Using the rigid 3D DNA origami elements as building blocks,
over 200 short oligonucleotide staple strands were carefully Shih’s group hierarchically assembled a rigid heterotrimeric
chosen to hold the scaffold in place (Figure 3A). After being wireframe icosahedron.24 By connecting rigid DNA origami
mixed in a pot, the scaffold and staple strands self-assembled in a helix-bundle units into 3D shapes with multiple segments of
single step. Various shapes such as squares, triangles, smiley faces, unpaired scaffold, Shih’s group created tensegrity 3D DNA
and five-pointed stars were created and visualized under AFM origami structures with very high strength-to-weight ratios and
with a spatial resolution of 6 nm. Structures made by this so- great resilience.99 Furthermore, in the same year (2009) of the
called “DNA origami” approach are roughly 100 nm in diameter invention of 3D DNA origami, Shih also expanded the design
in Rothemund’s work. space of accessible 3D DNA origami shapes to include a rich
The invention of DNA origami is a milestone in the diversity of nanostructures with designed twist and curvature.21
development of DNA nanotechnology. There are several Shih and co-workers found that targeted insertions and deletions
advantages for this new design technique. First, the shape of an of base pairs in a 3D DNA origami caused the DNA bundles to
origami structure is usually well defined, with a size about an develop a twist of either handedness or to curve (Figure 3C). The
order of magnitude larger than the previously reported DNA degree of curvature could be quantitatively controlled, and a
nano-objects self-assembled in the tile-based bottom-up way. radius of curvature as tight as 6 nm was achieved. Shih and co-
Second, to design a desired DNA origami, sequence optimization workers used this strategy to build several multilayer 3D
is generally not required. In fact, since most of the origami shapes nanostructures with precisely controlled curvatures, such as
are built on M13mp18 genomic DNA, sequences of the staple square-toothed gears.
strands are actually predetermined once the folding pathway is Yan’s lab further developed the twist-and-curvature design
defined. Currently, a few modeling software programs have been approach. By precisely tuning the bending and twisting of a 2D
developed to assist users for the design of origami structures.96,97 DNA origami, they were able to make a Mobius strip.100 Later,
By setting up the scaffold pathway and its starting point, they trained the double-helical DNA to bend following the
sequences of hundreds of staple strands can be generated rounded contours of the target object. Concentric rings of DNA
automatically and exported in a single file, ready for the following were used to generate in-plane curvature, which is maintained
chemical synthesis. The DNA origami technique enables and stabilized by rationally designed geometries and crossover
convenient design and assembly, especially for beginners and networks (Figure 3D).101 Combining in-plane and out-of-plane
those that are interested in the field of DNA nanotechnology. curvatures, they assembled a series of single-layered DNA
Third, unlike the tile-based bottom-up approach which often nanostructures with high curvature, such as 2D concentric rings
requires a strict stoichiometric ratio and purity control of the and 3D spherical shells, ellipsoidal shells, and a nanoflask.
involved oligonucleotide strands, DNA origami can be assembled Besides the 3D structures with high curvature, single-layered
with raw, unpurified staple strands. The molar ratio of the staple container-like 3D origami objects have also been constructed by
strands to the M13mp18 long strand is usually 10 to 1 or even folding and joining multiple single-layered 2D origami
lower. Also, the yield of a desire shape usually reaches above 90%. sheets.102,103 For example, Andersen et al.102 reported that by
All of these properties make DNA origami a simple yet powerful folding six 2D DNA origami sheets along the M13mp18 genomic
design technique, which stimulates and accelerates the develop- DNA and by adding the staple strands to bridge the edges of the
ment of DNA nanotechnology. However, it should be noted that sheets that make the six faces of the box, a cuboid structure of
the basic concepts or the principles behind DNA origami remain external size 42 × 36 × 36 nm3 was generated. Similarly, Yan’s
unchanged, that is, using crossovers to connect or bundle DNA group constructed a tetrahedron origami container with the
duplexes together to form nanostructures. In the origami length of the edge around 54 nm by joining four 2D triangular
strategy, multiple crossovers are generated at every possible origami sheets into an enclosed configuration.103 These hollow
position within an area defined by M13mp18 scaffold. Perhaps it 3D DNA objects could be potential drug carriers with large
is the high density of crossovers and the packing of multiple capacity to encapsulate substantial molecules inside them, which
duplex domains at once that makes the origami shapes well will be discussed later.
defined. New progress on the origami design technique keeps coming
Rothemund’s original origami strategy is suitable for the out. Recently, Högberg’s team developed a general method to
creation of 2D flat shapes. In 2009, Shih’s group extended and fold arbitrary DNA polygonal digital meshes including bottle,
improved the origami strategy to building custom 3D shapes helix rod, and waving stickman.104 Using a routing algorithm
formed as pleated layers of helices constrained to a honeycomb based on graph theory and a relaxation simulation that traces
lattice (Figure 3B).24 Shih’s 3D origami strategy can be simply scaffold strands through the target structures, the team made the
considered as packing multiple single-layer 2D origamis into a design process highly automated. Similarly, Bathe’s group
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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.
reported a general, top-down strategy to design nearly arbitrary 2.4. Single-Stranded Tile Assembly
DNA architectures autonomously based only on target shape.105 In 2012, Yin’s group pushed Seeman’s tile-based assembly to the
Unlike conventional origami designs built from close-packed extreme of simplicity.31,55 Unlike Seeman’s tile-based assembly,
helices, structures assembled with the above two methods where a few oligonucleotide strands form tiles and through tile−
generally have a more open conformation with one or two helices tile interactions generates large assembly, Yin and co-workers
per edge and are therefore more stable under the low-salt designed single-stranded tiles (SST) consisting of 42-base DNA
biological condition. strands (Figure 4A).55 These strands can be divided into four
From Seeman’s oligonucleotide tile to Rothemund’s origami, domains, each acting as sticky ends to bind with four local
neighbors during self-assembly. To simplify the design process
DNA nanotechnology underwent a bottom-up to top-down
and minimize the cost for assembling many different shapes, Yin
transition in design techniques. The origami approach makes the and co-workers treated a desired shape as a molecular canvas.
design versatile, and computer-aided automation makes the Each of the constituent SST strands acts as a pixel and is folded
design handy. However, there are also certain drawbacks for into a 3 nm by 7 nm tile and attached to four neighboring tiles.
origami. Generally, hundreds of synthetic staple strands are The shape is produced by one-pot annealing of all of those
required for an origami construction, setting the total price strands that correspond to pixels covered by the target shape.
relatively high. Purifying and accumulating the intact origami Over 100 distinct 2D shapes, including the 26 capital letters of
structures to certain concentrations for biotechnological the Latin alphabet, 10 arabic numerals, 23 punctuation marks and
applications require tedious work and perhaps large amounts other standard keyboard symbols, 10 emoticons, 9 astrological
symbols, 6 Chinese characters, and various miscellaneous
of sample. The size of an origami object is restricted by the long
symbols, were constructed using this simple strategy.
single-stranded scaffold, which is mainly defined by the 7- Later, Yin’s group extended this strategy to 3D design.31 They
kilobase M13mp18 genomic DNA. Overcoming these draw- used 32-base short synthetic DNA strands as DNA bricks (Figure
backs would make DNA origami a more powerful design 4B). Similar to the 2D design, each brick is a modular component
technique for various applications. and binds to four local neighbors. A master brick collection
6465 DOI: 10.1021/acs.chemrev.7b00663
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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 length of the scaffold DNA. Unlike Seeman’s oligonucleotide
voxels. Each 8-base pair interaction between neighboring bricks tile-based assembly, purification and exact stoichiometry of DNA
defines a voxel with dimensions of 2.5 × 2.5 × 2.7 nm3. By components are not required in SST-based assembly. Prescribed
choosing subsets of bricks from the canvas, over 100 distinct 3D 2D and 3D shapes can form by choosing the right pixel- or voxel-
objects were created, including shapes with sophisticated surface corresponding SSTs for one-pot annealing. Despite these
features and intricate interior cavities and tunnels. advantages, there are still some drawbacks for the SST approach.
Single-stranded tile-based assembly represents another mile- As the size of the desired shape gets bigger, more synthetic SSTs
stone of design technique in DNA nanotechnology. Comparing are needed, meaning the increase in cost and complication in
to the DNA origami approach, the lack of a long single-stranded sequence design and optimization. Also, the assembly yield of
scaffold DNA in SST-based assembly removes the size restriction desired shapes is inversely proportional to the total number of
of a desired shape, which is previously defined by the limited SSTs in the reaction mixture.
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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 stoichiometric quantities and treated as oligonucleotide tiles for
Single-Stranded Tile bottom-up assembly, yielding a 2D origami array with 1 μm
In Seeman and Yin’s tile strategies, DNA nanostructures are diameter. Ke and co-workers assembled DNA brick crystals with
designed and self-assembled in a bottom-up way. For prescribed depths (Figure 5C).109 Single-stranded brick tiles and
Rothemund’s origami strategy, DNA nanostructures are strands that connect each crystal unit (also considered as single-
designed in a top-down fashion. Nevertheless, the tile and stranded bricks) were mixed together for one-step, nonhierarchi-
origami approaches are not against each other. In fact, they cally annealing. DNA crystals were obtained and grew to
complement each other very well in many aspects. For example, micrometer size in the lateral dimensions with depths up to 80
Winfree and co-workers demonstrated the use of origami as nm (Figure 5C). Sophisticated 3D features such as cavities and
seeds to guide the algorithmic self-assembly of DNA tiles (Figure tubular crystals were displayed in the report.
5A).106,107 A complex binary counting pattern was generated It is not surprising to see that these approaches can be mixed
using the origami seed to specify the initial value for the counter. together very well. Regardless of tile- or origami-based designs,
Seeman’s team explored the 2D crystallization of origami tiles the central concepts of the above design approaches are the same,
(Figure 5B).108 Two cross-shaped origamis were first assembled which are bundling or packing DNA helices via the immobile
with the origami strategy. Then they were mixed together in Holliday junction crossovers (half crossovers in SST approach)
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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 that they could harness the geometric arrangement of blunt-end
to the DNA helical axis as well as interacting through the stacking interactions to create diverse bonds to connect different
complementarity of Watson−Crick binding to extend the DNA origami shapes,110 which may guide strategies for molecular
nanostructure along the direction of the DNA helical axis. There recognition in systems beyond DNA nanostructures. In addition,
are alternative ways to Watson−Crick base-pairing-based besides tile, origami, and hybrid approaches, there are some other
elongation. For example, Rothermund and co-workers showed mature methods to create DNA-based nanosystems, such as
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nanoparticle-templated DNA nanostructures, which will be analogs to common molecules, including linear CO2, triangular
discussed next. BF3, and tetrahedral CH4, were also assembled using a similar
2.6. Nanoparticle-Templated DNA Nanostructures strategy (Figure 6B, bottom).131,132
In the 1990s, at the early stage of DNA nanotechnology, the 2.7. Other Strategies for the Construction of DNA
Nanostructures
rigidity of DNA nanostructures was a problem due to the not-so-
robust mechanical property of a single-DNA duplex. Seeman Other than the tile-based, origami-based, and nanoparticle-
figured out that the rigidity could be enhanced by packing templated construction strategies, transition metal ions are also
multiple DNA helices via a series of immobile Holliday junctions, frequently used to build metal−DNA junctions and assemblies.
leading to the creation of conformationally restricted and The plan for this strategy is to obtain DNA−metal hybridized
structurally robust DX, TX, PX, JX2, and 6-helix bundle tiles. nanostructures in which DNA duplex serves as a nanoscale rigid
Alternatively, Mirkin chose to blend in a rigid inorganic or molecular arm to spatially position addressable transition metals
organic nanoparticle such as gold nanoparticle (AuNP) and that are usually coordinated by certain organic molecular pocket
made it act as a core to template and organize DNA in a surface- and form vertices. In 2004, Sleiman and co-workers synthesized a
normal orientation to add rigidity to the assembly.111 The branched ruthenium(II)−DNA complex,133 in which two
success of Mirkin’s approach leads to the establishment of the parallel DNA strands are linked to a relatively rigid RuII
nanoparticle-templated DNA nanotechnology.70 To date, nano- tris(bipyridine) center. By designing the sequence of DNA to
particle-templated DNA nanostructures have been used in a allow complementary hybridization, they were able to self-
number of biological applications such as gene regulation112 and assemble a discrete metal−DNA cyclic nanostructure. Later,
molecular probing.113,114 Sleiman’s team explored more transition metal ions for DNA−
In 1996, Mirkin and co-workers reported a method for metal nanostructures.134−140 For example, Sleiman and co-
assembling colloidal gold nanoparticles rationally and reversibly workers used a DNA-templated method to form a chiral (right-
into macroscopic aggregates.111 With terminal thiol modifica- handed) metal−DNA junction, which contains a single copper-
tions, single-stranded DNA oligonucleotides bound to the (I)−bisphenanthroline unit at its central point and four single-
surfaces of two batches of 13 nm colloidal AuNPs. By adding stranded DNA arms of different sequences (Figure 7A).139 The
an oligonucleotide duplex (linker) with “sticky ends” that were copper-binding ligand diphenylphenanthroline (dpp) was
complementary to the two grafted sequences, the AuNPs self- chemically synthesized in the middle of two DNA strands. The
assembled into aggregates. Mirkin’s initial work focused on two strands were then brought together by a linker DNA to
exploring the linker DNA to hybridize the ssDNA−AuNP achieve close proximity of the two dpp ligands. Also, the addition
conjugates into macroscopic networks.115 Mirkin and co-workers of copper ions resulted in the formation of a highly stable
found that the interparticle distances in the assemblies were Cu(dpp)2 complex that even resisted PAGE denaturation. Using
proportional to the length of the DNA linkers,116,117 and these DNA−metal junctions as building blocks, Sleiman’s team
significant correlation between the particle positions was only generated DNA−metal nanotubular assemblies.139 Besides
clear when the DNA linkers were double stranded.117 These metals, the same team also demonstrated that hydrophobic−
results indicate that the rigid core and multiple duplexed linkers hydrophilic copolymers can be covalently linked with DNA and
(DNA bonds) together maintain the configuration of the introduced into DNA nanostructures for dynamic functions.141
nanoparticle-templated DNA assemblies. Ultrathin 2D nanosheets of layered transition metal
Using this strategy, well-ordered super lattices with consid- dichalcogenides (TMDs) such as MoS2 are emerging as a class
erable long-range periodicity were created with AuNPs (Figure of key materials in chemistry and electronics due to their
6A).118−120 Since the only requirement for building up the DNA unconventional properties. Very recently, in 2017, Li et al.142
bonds is that they are attached to a solid core, a variety of harnessed the S-atom defect sites on the exfoliated surface of
inorganic nanoparticles, including catalytic noble metals, MoS2 to incorporate S atom-terminated (thiolated) DNA anchor
magnetic oxides, and semiconductors, were subsequently molecules into the otherwise inert TMDs. The DNA anchors
conjugated with DNA and assembled by the same strat- were further programmed to form interspacers to assemble
egy.121−123 Moreover, with a molecule that provides an initiation multiple layered MoS2 TMDs into superstructures of stacked
site for silica growth on a glass surface, AuNP−DNA lattices have nanosheets.
been grown in a preferred crystallographic direction on the silica Rolling-circle amplification (RCA) has also been demon-
support (Figure 6A, bottom).124,125 The simplicity of the strated to be an efficient way for producing DNA nanostructures.
protocol allowed DNA−nanoparticle super lattices amenable During RCA, usually the Phi29 DNA polymerase goes around
to become solid-state materials, thus expanding their potential circular DNA templates many times to produce long single-
use in many aspects. stranded concatemeric DNAs with each repeated unit having
Later, Mirkin’s group and others put a lot of efforts on making sequences complementary to the template. Using the origami
asymmetric DNA functionality conjugated on the surface of strategy, the long DNA produced from RCA can be directly
nanoparticles.126−130 One example is to use anisotropic nano- folded into the scaffold for origami construction.143,144 Also,
particles that present regions of greater chemical reactivity that RCA has been used for the production of staple strands required
can be selectively functionalized with DNA (Figure 6B). This has by origami.145 Thus far a series of nanoshapes, including
been done at regions of high curvature, like tips and edges, where nanotubes,146 nanoribbons,143 nanoclews,147 and nanoballs,148
the native surfactant coating required for the synthesis of such has been created in the RCA fashion.
nanoparticles is less dense and thought to be replaced by thiol- In 2013, Ouyang et al. used only three 32-base staple strands to
modified DNA strands more readily. Anisotropic core−satellite fold each 96-base periodic unit of a RCA product into a rectangle
clusters were created by selective incorporation of DNA at the and simultaneously connect the adjacent rectangle units to form
tips and edges of plate-like triangular nanoprisms128 or at the tips nanoribbons.143 These RCA-based nanoribbons are easily
of gold nanorods (Figure 6B, top).130 In addition, colloidal internalized by cells. Later, Yan et al. developed a novel 3D
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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.
superstructure based on the growth and origami folding of DNA integrated into the nanoclew to enhance the loading of the
on AuNPs (Figure 7B).149 The 3D superstructure contains a anticancer drug doxorubicin.
nanoparticle core and dozens of 2D DNA nanoribbons (belts) Unlike the tile-, the origami-, or even the metal-based assembly
folded from long single-stranded DNAs grown in situ on the strategies, RCA usually produces nanostructures with undefined
nanoparticle by RCA. Multiple functional groups such as drugs size. However, RCA-based nanostructures generally have large
and targeting agents can be loaded onto the 3D superstructures loading capacity due to the repeated units. In addition, RCA not
only allows for the creation of nanostructures with unique shapes
by either a merging or an intercalating mechanism for theranostic
but also can integrate multiple functional groups, such as cancer-
purposes (Figure 7B). specific targeting molecules, into the nanostructure assemblies.
In 2014, Gu’s group developed a bioinspired cocoon-like Thus, it is also frequently used as a drug carrier for targeted
anticancer drug delivery system consisting of a DNase- delivery.
degradable DNA nanoclew, which was embedded with an acid- We herein have reviewed the history of structural DNA
responsive DNase nanocapsule (Figure 7C).147 The DNA nanotechnology and summarized most if not all of the current
nanoclew was assembled from a long-chain single-stranded strategies to construct DNA nanostructures, including tile-based,
DNA synthesized by RCA. Multiple GC-pair sequences were origami-based, hybridized, nanoparticle-templated, metal-assis-
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ted, and RCA-directed assembly approaches. In the next section, nanoparticles with a size less than 80 nm were trafficked to lymph
we will discuss the principles for targeted drug delivery and how nodes via subcutaneous administration.172,173
DNA nanostructures can enter cells as well as what molecules It is known that lipid and lipid-like materials tend to
have been delivered into cells by which DNA nanostructures. accumulate in the liver. In addition, particle size also plays an
important role for liver targeting (Figure 8). Nanoparticles with a
3. DNA NANOSTRUCTURE-BASED DRUG DELIVERY size less than 100 nm can cross liver fenestrae and target
hepatocytes, while intravenously administered nanoparticles
Currently, the major work on DNA nanostructure-based drug
with a size more than 100 nm are more likely taken up by
delivery targets cancer cells. In fact, before reaching cancer cells,
activated Kupffer cells that reside within and near the liver
nanoparticles must be able to first accumulate or localize in the
vasculature and so do not reach the hepatocytes.174,175
specific organ or organs where the cancel cells reside. This step is
Through inhalation or intravenous administration, nano-
called primary targeting. Once in the organ or organs, drug
particles can be delivered to the lungs. Inhaled particles with low
molecules carried on the nanoparticles need to be directed to the
density (<0.4 g per cm3) and of large size (>5 mm) are more
cancer cells and potentially to a specific subcellular location
likely retained in the lungs for prolonged periods of time (Figure
within a cancer cell, a step called secondary targeting.150 In
sections 3.1 and 3.2, we will review our current knowledge on 8).176 The route of intravenous administration was reported to
primary and secondary targeting as well as discuss the pros and be more effective using nanoparticles with sizes larger than 200
cons of using DNA nanostructures for targeting. In section 3.3, nm, because they are generally trapped in lung capillaries.177
we will review the drug molecules that have been delivered via the Moreover, nanoparticles with positive surface charge are more
aid of DNA nanotechnology. likely to accumulate and reside in the lungs.178
DNA nanotechnology enables the precise assembly of well-
3.1. Primary Targeting―Getting to the Organ defined nanostructures with sizes ranging from a few nanometers
Most life-threatening cancers occur in the brain, lung, liver, to over 100 nm,179 a range that can meet the size requirements
lymph, and bone. Particle size and shape, surface charge and for targeted delivery to most organs, including the brain, lung,
properties, material composition, and the route of admin- liver, and lymph nodes (Figure 8). Negative charges on the
istration, all affect the localization of nanoparticles to specific surface may favor targeted delivery of DNA nanoparticles to
organs (Figure 8). Due to the existence of the blood−brain organs like lymph nodes but disfavor organs like the lung and
barrier, only certain materials can cross from the circulation into brain (Figure 8). The latter situation could be resolved by taking
the cerebrospinal fluid. Thus, the brain is always considered as advantage of the maturation of DNA chemical synthesis, which
the most challenging organ to target with intravenously allows for hundreds of modifications on DNA. By modifying only
administered nanoparticles.151 Generally, metabolic products, a few component strands, the surface property of a DNA
hormones, and gases are exchanged across the blood−brain nanostructure can be completely changed. For example, both
barrier.152 Under certain circumstances, for example, when a Shih’ group and Lin’s group reported that thiol modification on
brain tumor develops, it may disrupt the integrity of the blood− DNA enabled covalent linking of DNA and lipid molecules,
brain barrier,153 giving opportunities for drug molecules to access which turned the hydrophilic surface of DNA nanostructures to
the brain.154−157 be hydrophobic.180,181 Alternative to chemical modifications,
Several groups reported that only small nanoparticles with synthesized cationic molecules with strong affinity to DNA could
sizes of less than 15 nm can efficiently go across the blood−brain potentially turn the surface charge of a DNA nanostructure from
barrier.155,158 For particles in the range of 15−100 nm, the ability negative to positive.182,183 In principle, DNA nanotechnology-
to penetrate the brain decreases exponentially with size (Figure enabled nanostructures can potentially meet almost all necessary
8).158,159 Surface modifications with lipophilic moieties and aspects including particle size and shape, surface charge and
neutral charge are known to enhance brain uptake of properties, material composition, and so on, for specific organ
nanoparticles.159−162 In addition, by targeting leukocytes and targeting.
macrophages in the circulation, nanoparticles might be trafficked Once in the organ, nanoparticles need to be further directed to
into the brain and ultimately be released.163,164 the diseased cells or even subcellular compartments. Before
The bone is actively involved in cancer metastases. However, discussing DNA nanotechnology-associated cellular targeting,
little is known for targeting nanoparticles to the bone. Previous we will first review the known gateways and mechanisms for cell
research focused on small molecules and proteins targeted to entry.
hydroxyapatite, the calcium-containing mineral component that
3.2. Secondary Targeting―Getting to the Cell and
constitutes up to 50% of bone. Compounds such as alendronate
Cellular Subunits
and aspartic acid adhere to the bone and have been harnessed for
the accumulation of nanoparticles in the bone (Figure 8).165 3.2.1. Gateways into the Cell. The cell membrane, also
Lymph nodes have mainly been targeted using cell-based known as the plasma membrane or cytoplasmic membrane, is the
nanotechnologies in an indirect process. Nanoparticles are natural barrier to protect sophisticated cellular organelles from
usually designed to bind to the receptors on the surface of the extracellular environment and maintain the differences
leukocytes, followed by being transferred to lymph nodes as part between the cytosol and the extracellular environment. It is
of a normal immune response.166 Generally, nanoparticles with selectively permeable to ions and organic molecules and controls
negative charges are taken up faster by leukocytes (Figure the movement of substances in and out of cells. Large molecules,
8).166,167 Interestingly, polyethylene glycol modification on the including nanosized particles, can pass through the membrane
surface of nanoparticles can inhibit uptake by leukocytes.168−170 via highly regulated processes called endocytosis.73,184−186 In
Moreover, other routes of administration can also achieve lymph general, endocytosis can be classified into two broad categories:
node targeting. For example, through intrapulmonary admin- phagocytosis, which is the uptake of particles usually larger than
istration, noncationic particles with a size range of 6−34 nm were 250 nm, and pinocytosis, which is the uptake of fluids and small
reported to be trafficked rapidly to local lymph nodes.171 Also, particles that are not taken by phagocytosis. Phagocytosis in
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Figure 9. Gateways and intracellular fate of nanocarriers.
mammals is restricted to specific cells including macrophages, the cell membrane, resulting in the formation of large (>1 μm)
neutrophils, dendritic cells, and monocytes, while pinocytosis is and heterogeneous vesicles termed as macropinosomes. Later,
adopted by all types of cells through four different mechanisms: these macropinosomes further fuse with lysosomes for
macropinocytosis, clathrin-mediated endocytosis, caveolae- degradation. Note that this internalization process is non-
mediated endocytosis, as well as clathrin- and caveolae- specific.73
independent endocytosis.73,184−186 Clathrin is a main cytosolic protein that makes coated vesicles.
The gateways and intracellular pathways of engulfed molecules Its triskelion shape allows the protein unit interacting with each
by different endocytic mechanisms are depicted in Figure 9. In other to form a polyhedral lattice that surrounds the vesicle.
some cells and single-celled organisms, phagocytosis is involved Clathrin-mediated endocytosis (CME), especially receptor-
in the acquisition of nutrients. While in multicellular mammalian, dependent CME, is the major pathway for the internalization
phagocytosis has been harnessed to remove pathogens and cell of extracellular nanoscale materials. In this case, nanomaterials
debris. Phagocytosis generally begins with the recognition of the modified with ligands on their surface are first recognized by the
invaders such as viruses, microorganisms, and even nanovehicles receptors on the plasma membrane, which triggers the bending
used for drug delivery, followed by the engulfment of those of the membrane and the assembly of clathrin-coated pits with
particles by the actin-driven macrophages through specific the assistance of several accessory proteins. This results in the
ligand−receptor interactions, which in turn results in the formation of a cargo-containing clathrin-coated vesicle in the
formation of cellular phagosome. As the phagosome matures, it cytosol (Figure 9). Then the coated proteins start to fall off from
then fuses with the lysosome to form the phagolysosome, in the vesicle, generating an early endosome of about 120 nm in
which the acidic condition and various enzymes help to degrade diameter. Later, a prelysosomal vesicle involving various enzymes
the ingested particles.187,188 Generally, particles with a size of secreted by Golgi fuses with the early endosome to form a late
more than 250 nm can be internalized effectively through this endosome and eventually mature as a lysosome for the
pathway. degradation of captured cargos.185,189,190 The other type of
Similar to phagocytosis, macropinocytosis is also an actin- CME is receptor independent, in which cells recognize the cargos
driven endocytic process (Figure 9). However, unlike through the unselective hydrophobic and electrostatic inter-
phagocytosis in which specific substances are transported action between the cargos and cell membrane. The receptor-
through the membrane, micropinocytosis takes up small particles independent CME has a much slower internalization rate
nonselectively by forming membrane protrusions in the first step. comparing with the receptor-dependent one.191 Therefore, it is
Then these protrusions collapse onto the particles and fuse with important to design drug carriers modified with ligands that can
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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.
specifically target their receptors on the cell membrane to trigger present in many specific cells, such as smooth-muscle cells,
the receptor-dependent CME pathway for faster and more fibroblasts, endothelial cells, and adipocytes.192 The most
accurate drug delivery. Moreover, the drug carriers need to be prevalent protein associated with caveolae structures is
able to escape from the early endosome as soon as possible to caveolin-1, which is pertinent not only to caveolae formation
avoid lysosomal degradation and to reach the specific subcellular in the membrane but also the subsequent vesicular produc-
compartment if such destination is planned for targeting. tion.193 Caveolae vesicles move through the cytoplasm with the
Caveolae-mediated endocytosis (CvME) is another receptor- assistance of an activated signaling transduction cascade and can
dependent pinocytosis (Figure 9). Caveolaes are defined as flask- transfer to either nonenzymatic caveosomes or other non-
shaped, cholesterol- and sphingolipid-rich smooth invaginations lysosomal compartments, like endoplasmic reticulum, and
with a diameter of 50−80 nm in the plasma membrane. They are eventually reach nucleus. During the transportation, the caveolar
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vesicle can avoid the involvement of any detrimental enzymes. In demonstrating that sequence composition was another tunable
fact, many viruses are found to hijack this pathway to bypass property for the enhancement of delivery efficiency.202
lysosomal degradation for efficient transfection to host cells. It is worthy to note that the type of employed endocytic
Therefore, presumably it is valuable to design drug carriers with pathway greatly depends on the size of nanostructures. Caveolar
ligands, e.g., folic acid, albumin, and cholesterol, to be recognized vesicles can efficiently carry particles less than 80 nm in diameter.
by receptors of CvME for drug-complex evasion of the TDNs and SNAs are about 7 and 20 nm in diameter,
degradative CME pathway.194,195 respectively.199 Both in principle fall in the suitable size range
Besides the pinocytosis pathways described above, there are for a caveolae-mediated pathway and were experimentally
other small “lipid rafts” with a diameter of about 40−50 nm that confirmed to go through CvME when incubated with certain
can form on the cytoplasmic side of any cell surface and fuse with types of cells. For particles generally larger than 100 nm, CME or
any endocytic vesicles. Currently, the mechanism of such macropinocytosis or multiple endocytic mechanisms may
clathrin- and caveolae-independent endocytosis is not clear and contribute to their cellular internalization. Lim et al.203
requires further studies.196 constructed the self-assembled Zn/DNA and Zn/siRNA nano-
The polyanionic nature of DNA can lead to strong clusters with average sizes of ∼200 nm (Figure 10C upper) and
electrostatic repulsion between DNA and the negatively charged studied their cellular uptake mechanism. The Zn/DNA cluster
cell membrane. This matches the observation that unmodified was prepared with yoyo-labeled DNA to monitor the cellular
DNA oligonucleotides alone cannot be efficiently internalized by uptake by tracking the fluorescence signal. During the cellular
cells in the absence of transfection agents. However, in 2011 we uptake process, Lim et al. treated HEK293 cells with different
and others found that certain DNA nanostructures, like the DNA endocytosis inhibitors, including chlorpromazine (CPZ), an
tetrahedron, can enter several different types of cells readily inhibitor for a clathrin-mediated pathway, and methyl-β-
without the aid of transfection agents.146,197,198 Due to the cyclodextrin (MβCD), an inhibitor for a clathrin-independent
negatively charged hydrophilic property, passive uptake of DNA pathway. The results showed that the cellular uptake of the Zn/
nanostructures by cells is unlikely. Enlightened by the entry DNA cluster decreased to 85% in the presence of CPZ and to
pathway of nanoscale viruses,190 we and others suspected that the 30% in the presence of MβCD (as shown in Figure 10C, bottom
DNA nanostructures might be internalized into cells by an left), indicating that both clathrin-dependent and -independent
endocytosis pathway. We used a single-particle tracking pathways contributed to the cellular uptake of Zn/DNA clusters.
approach to monitor the entry pathway of the tetrahedral In addition, the TEM image (Figure 10C, bottom right)
DNA nanostructures (TDNs) in live Hela cells.199 We confirmed illustrated that macropinocytosis was also one uptake pathway
that the cellular entry of TDNs was an energy-dependent
for Zn/DNA clusters. In parallel research, Chen et al.144
endocytosis process by a set of fluorescence imaging and
constructed periodic DNA nanoribbons with a size of ∼500
biochemical experiments. TDNs were found to interact with the
nm for intracellular pH sensing and gene silencing. They
plasma membrane nonspecifically and then be rapidly internal-
concluded that the cellular internalization of their DNA
ized by the caveolae-mediated pathway. In our experiments we
nanoribbons was via clathrin- and lipid raft-mediated endocy-
observed that the caveolar vesicles eventually directed the
tosis.
transportation of TDNs to lysosomes in a highly ordered,
microtubule-dependent manner for degradation (Figure 10A). Besides the size, the shape and 3D arrangement of DNA
These unique cellular properties of TDNs, or more broadly nanostructures also play critical roles for efficient cellular uptake.
framework nucleic acids (FNAs), shed new light on novel nucleic It has been reported that certain inorganic and biologically
acid scaffold-based nanomedicine. inspired nanomaterials with high aspect ratio exhibit unique
The cellular entry mechanism of spherical nucleic acids properties including improved membrane penetration and
(SNAs), the nanoparticle-templated DNA nanostructures, was increased cellular internalization, as well as decreased uptake
also studied by Mirkin’s group in 2013.200 SNAs are 3D DNA and sequestration by phagocytic cells.204,205 Sleiman and co-
nanostructures which typically consist of densely functionalized workers constructed DNA nanotubes by RCA and observed an
and highly oriented nucleic acids covalently attached to the enhanced cellular penetration with these nanostructures. They
surfaces of spherical nanoparticle cores.201 Mirkin and co- suspected it was due to the dense display of DNA and the high
workers found that SNAs could be rapidly internalized into more aspect ratio in the RCA-nanotubes.146 In a separate study, our
than 50 different cell types in a manner irrespective of the team also found that DNA nanostructures with high length-to-
presence of the nanoparticle core as well as the surface width ratios were more readily taken up by cells.143 The majority
oligonucleotide sequences. They concluded that the phenom- of in vivo-administered nanoparticles are likely sequestered by
enon was due to the 3D arrangement of the oligonucleotides on macrophages.206 Also, macrophage uptake correlates with
SNA, which made them compete for and engage with the cell nanomaterial size.207 Chan and co-workers used DNA as linkers
surface receptors more substantially than linear oligonucleotides. and spacers to organize inorganic nanoparticles into the “core−
For C166 (mouse endothelial) cells, SNAs bound strongly to the satellite” colloidal superstructures.208 On a model cell system of
class A scavenger receptor (SR-A) proteins on the cell surface, J774A.1 murine macrophages, they found that although the
which, in turn, significantly promoted the cellular uptake of SNAs core−satellite superstructure was 2.5 times bigger than its core
via the caveolae-mediated pathway, as verified by TEM images component, the uptake by macrophages was lower by 2-fold. The
(Figure 10B). On the basis of the observation that the degree of data suggested that the spatial arrangement of satellites with
SNAs’ correlated positively with the expression level of SR-A and DNA changed the surface chemistry of the superstructure, which
caveolin-1 proteins, Mirkin and co-workers speculated that other inhibited its uptake by macrophages. With this DNA−nano-
cell types, such as epithelial cells and fibroblasts, might also adopt particle superstructure, Chan and co-workers were able to reduce
a similar mechanism to internalize SNAs. Further research nanoparticle retention by macrophages and mitigate the so-
showed that SNAs with higher G content had a higher degree of caused immune toxicity, as well as improve their in vivo tumor
cellular uptake than those primarily composed of A, T, and Cs, accumulation.
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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 (p-
TDN). 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 nano- Bioexisting nanocarriers such as the protein cage of viral
structures can enter cells much better than oligonucleotides, in particles have been explored as vehicles for drug delivery to
order to lift the uptake efficiency, enhance the uptake specificity, achieve better biocompatibility, reduced toxicity, and higher
as well as reduce the immune toxicity, it is necessary to endorse uptake efficiency.97,209,210 It has also been reported that DNA can
DNA nanostructures excellent abilities to avoid immune be adopted as a template for virus capsid proteins to gain long
recognition, locate target cells, penetrate cell membrane, and tubular structures.211 Combining those together, Mikkilä et al.212
perhaps propel to subcellular organelles of interest. coated DNA origami surface with cowpea chlorotic mottle virus
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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.
(CCMV) capsid proteins (CP) and further packed the DNA which were demonstrated to be effectively taken by human
origami nanostructures inside the viral capsid in order to facilitate cancer cells (nasopharyngeal epidermal carcinoma KB cells).
efficient cell transfection (Figure 11A). They found that the They found that the amount of folate conjugated on DNA
DNA origami−CP complex exhibited a 13-fold increase in the nanotubes correlated well with the efficiency of uptake in KB
efficiency of delivery into human HEK293 cells compared to the cells. In a parallel study, Lee et al.216 assembled DNA tetrahedral
bare DNA origamis. Similarly, Shih and co-workers180 reported nanoparticles to deliver siRNAs into cells and silence target genes
that by covering DNA octahedron with PEGylated lipid bilayers in tumors. They showed that at least three folate molecules per
(Figure 11B), immune activation was decreased by 2 orders of DNA tetrahedron are required to transfect DNA tetrahedron
magnitude below controls and the pharmacokinetic bioavail- efficiently into KB cells. The follow-up research also demon-
ability improved by about 17-fold. In another work, Sleiman and strates that the folate ligand-based strategy is a highly selective
collaborators demonstrated that DNA nanostructures modified way for targeting many tumor cells.147,217
with hydrophobic dendritic chains exhibited rapid cellular uptake Besides folate, several groups reported that certain short
behavior, whereas the hydrophilic chain-modified version peptides can specifically target transmembrane protein neuro-
showed a slow and continuous uptake profile.213 pilin-1 (NRP), which is overexpressed in glioblastoma cells and
Targeting specific cells is a paramount prerequisite of drug endothelial cells of angiogenic blood vessels.218−220 These
delivery. DNA nanotechnology enables the creation of peptides bind NRP tightly and can penetrate tumor
nanostructures which can not only have a capacity for drug cells.221−225 By appending the peptides to DNA tetrahedrons
loading but also carry functional groups, such as small molecules, (p-TDN), Xia et al.226 found that the uptake rate of p-TDN by
aptamers, and proteins, for sensing and targeting. Through glioblastoma cell U87MG was effectively accelerated compared
chemical modifications or cohesive interactions, DNA nano- with bare TDN and double-stranded DNA (Figure 11C).
structures are coupled with these functional groups to target 3.2.2.2. Aptamers. Aptamers are single-stranded DNA or
cancer cells via the ligand−receptor recognition strategy. Some RNA molecules that can form certain secondary and tertiary
of the functional groups are discussed in the following part. structures to specifically bind target molecules, including metal
3.2.2.1. Small Molecules. The folate receptor (FR), a ions, metabolites, proteins, and so on.227,228 They can be evolved
membrane protein that binds folic acid with high affinity, is from a random oligonucleotide library by a selection procedure
overexpressed in various human cancer cells.214 It is known that termed as SELEX (Systematic Evolution of Ligands by
folic acid retains its receptor binding properties when derivatized Exponential Enrichment). Like antibodies, aptamers can bind
via its γ-carboxyl. Therefore, folate conjugation can be explored targets with high affinity (kD = picomolar to micromolar) and
for targeted drug delivery. With folate−DNA conjugation, Mao specificity. Due to their intrinsic properties, aptamers can be
and co-workers215 assembled micrometer-long DNA nanotubes integrated into DNA nanostructures either through cohesive-
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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 confirmed that such aptamer hydrogels strongly inhibited
assembly procedure231−233 in order to assist DNA nanostruc- proliferation and migration of A549 cells but had negligible
tures to enter cells that express the ligands on the membrane. Tan effects on normal cells.
and co-workers234 designed an aptamer-tethered DNA nanotrain 3.2.2.3. Proteins. Proteins are the most important biological
(aptNTr), which is a long linear DNA nanostructure self molecules, which participate in almost all activities of life. It is
assembled simply from two relatively short DNA building blocks reported that the curvature and deformation of lipid membranes
through a hybridization chain reaction (Figure 11D). The are promoted by special membrane-sculpting proteins, such as
conditional formation of aptNTrs upon initiation with clathrin, dynamin, and SNARE (soluble N-ethylmaleimide-
engineered aptamer trigger probes ensured that each resultant sensitive factor attachment protein receptor).236−238 DNA
nanotrain was tethered with an aptamer moiety on one end of the nanostructures mimicking these proteins presumably have the
nanoconstruct. These aptamer moieties, capable of selectively potential to self-manipulate the endocytosis process. SNARE
recognizing cognate cancer cells, operated like locomotives proteins are the key machinery to drive fusion of a vesicle with its
guiding a series of tandem dsDNA “boxcars” toward target cells. target membrane. Inspired by the tethering proteins which
Due to the high aspect ratio, the aptNTrs were endocytosed bridge the membranes and thus prepare SNAREs for docking
easily by target cells once the aptamer moieties were recognized and fusion, Rothman and co-workers developed a lipid-
by the receptors on the surface of cells. conjugated ssDNA mimic of tethers that is capable of regulating
Besides classic DNA nanostructures, aptamers have also been SNARE function in situ (Figure 12A).239 The nonfusogenic
assembled into DNA nanohydrogels for targeted and stimuli- DNA−lipid hybrid molecules which serve as artificial tethers
responsive gene therapy. Tan and co-workers used two Y-shaped were designed to contain three consecutive structural segments
monomer units and one DNA linker unit to prepare DNA from the membrane-distal to the membrane-proximal ends: (1) a
nanohydrogels.235 Meanwhile, they incorporated aptamers, 21-bp hybridization region, responsible for physically bridging
disulfide linkages, and therapeutic genes into each building unit two membranes via parallel hybridization, (2) a linker region
during the preparation of hydrogels. Tan and co-workers chose containing 5−63 consecutive thymidine (T) nucleotides, which
S6 aptamer,235 which specifically targets A549 cancer cells was inserted to regulate liposome distance and minimize possible
(human lung adenocarcinoma epithelial cell line). They nonspecific fusion caused by the tethers alone, and (3) the lipid
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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 workers found that in the presence of these artificial tethers,
DNA sequence via a thiol−maleimide linkage. Without direct SNARE-mediated lipid mixing was significantly accelerated, and
interaction with SNAREs, the DNA−lipid tethers regulated
the maximum fusion rate was obtained with a linker shorter than
SNARE function by capturing and keeping two opposed
membranes in a controlled distance, within which SNAREs 40 nucleotides. As a programmable tool, the DNA−lipid tethers
drove the membranes fusing with each other. Rothman and co- can be further applied to regulate other biological processes. The
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work establishes an important step toward controllable DNA− selectively taken up by cells via endocytosis, while small
nanostructure/cell-membrane interaction. molecules such as ions can selectively pass through membrane
In a subsequent work, how SNAREs drive membrane fusion via channels generated by pore-forming membrane proteins. Ion
was studied by using self-assembled DNA nanorings to template channels are located within the membrane of most cells and of
the formation of uniform-sized small unilamellar vesicles (SUVs) many intracellular organelles. Their functions include regulating
which contain predetermined maximal number of SNAREs cell volume, shaping action potentials and other electrical signals
facing externally (Figure 12B).240 The lipid-conjugated comple- by gating the flow of ions across the cell membrane, establishing a
mentary ssDNA strands as tethers were incorporated into the resting membrane potential, and so on.
vesicles for membrane targeting. This strategy enabled the bypass α-Hemolysin is a toxin that can cause cell death by binding to
of the rate-limiting docking step of fusion reactions and allowed the outer membrane, with subsequent oligomerization of the
direct observation of individual membrane-fusion events at toxin monomer forming the water-filled channels.246 Using α-
SNARE densities as low as one pair per vesicle. The results hemolysin as a model, Simmel, Dietz, and co-workers247 created
showed that at the single-event level, after docking of the an artificial channel with DNA origami that, when penetrating a
templated SUVs to the supported lipid bilayers (SBL), 1−2 pairs lipid bilayer membrane, acted similar to a membrane channel
of SNAREs were sufficient to drive fast lipid mixing. (Figure 14A). The channel consists of two modules: a stem that
Besides designing DNA nanostructures to mimick the penetrates and spans a lipid membrane and a barrel-shaped cap
behavior of certain membrane-sculpting proteins, modifying that is mediated by 26 cholesterol moieties and adheres to the cis
DNA nanostructures with protein ligands whose receptors dwell side of the membrane. In a single-channel electrophysiological
on the cell surface can also assist them to target specific cells via experiment, this artificial channel showed conductances on the
the antibody−antigen interactions. With terminal modifications, order of 1 nanosiemens and channel gating which are similar to
the current synthetic chemistry can ensure oligonucleotide the response of natural ion channels. By manipulating the length
strands with customized sequences covalently link to the cysteine of a DNA component strand in the stem domain to allow its
or lysine residues of an antibody.241,242 In principle, any protrusion into the artificial channel, Simmel and Dietz’s team
component DNA strand of a DNA nanostructure can be observed greater gating responses.247 Single-molecule trans-
designed to conjugate with an antibody. Thus, comparing to location experiments show that the synthetic channels can be
other nanomaterials, DNA nanostructures have enormous applied for the discrimination of single-DNA molecules.
flexibility in antibody conjugation: Not only the position where In the work by Simmel and Dietz’s team,247 the aromatic
the antibody is conjugated to the DNA nanostructure but also membrane anchors drove the unmodified hydrophilic DNA stem
the number of conjugated antibodies per DNA nanostructure penetrating the membrane. Another strategy to penetrate the
can be well programmed and controlled. membrane is to mimic membrane proteins that have an outer
Transferrin (Tf) is a key protein player in the metabolism of hydrophobic surface. Following this route, Howorka and co-
iron cellular transportation via receptor-mediated endocytosis. In workers248 designed a 6-helix-bundle-based synthetic DNA
order to map the cellular pH gradients with DNA nanomachines, nanochannel, which carries an outer hydrophobic belt comprised
Krishnan and colleagues chemically conjugated Tf to a DNA of small chemical alkyl groups (Figure 14B, left). This
nanodevice.243 This Tf−DNA nanocomplex was able to travel modification masked the negatively charged oligonucleotide
through the transferrin pathway and report the pH values of backbone and hence overcame the otherwise inherent energetic
subcellular organelles within the pathway. In a separate study, mismatch to the hydrophobic environment of the membrane.
Kjems and co-workers244 demonstrated that by modifying a Such artificial DNA pores were structurally stable and
planar DNA origami structure with Tf, a significant strong demonstrated to support the transmembrane flow of water
increase in cellular uptake was achieved in an established cancer molecules by a range of analytical techniques.248
cell line. The decoration of the 2D DNA origami roughly 100 nm In the above two studies, hydrophobic chemical tags, either
in diameter with approximately 32 Tf protein molecules resulted cholesterol-based lipid anchors covalently attached to DNA
in an up to 22-fold increase of cytoplasmic uptake compared to strands247 or ethyl-modified phosphorothioate groups that
unmodified structures, which is comparable to the lipofectamine- replace the negatively charged backbone phosphate to form a
based transfection. hydrophobic belt to mimic natural protein pores,248 were
If the receptors that overexpressed in cancer cells were also precisely positioned to anchor the strongly hydrophilic DNA
expressed in normal cells, targeting a single receptor would nanostructures into lipid bilayers. However, to make such
inevitably cause nonspecific drug uptake, resulting in toxicity to artificial pores, complex designs were required: The former tag
normal cells as well as reduced anticancer efficacy. Dual or was placed at up to 26 positions of the pore, while the latter was
multiple targeting could increase the specificity of drug delivery. introduced 72 times into a DNA origami. With the intent to
DNA nanostructures usually consist of several to hundreds of simplify nanochannel design and move toward minimal chemical
component strands, each of which in principle could be modified intervention, Howorka’s team continued to explore whether
to link with a ligand. Sun et al. reported that they modified a other chemical tags of greater hydrophobicity can achieve the
TDN with SL2B aptamer and folic acid.245 SL2B is a 26-mer same level of membrane penetration by only a few tag copies.
DNA strand which can specifically target the heparin binding They found that with tetraphenylporphyrin (TPP) to couple
domain (HBD) of vascular endothelial growth factor (VEGF165). with DNA, solely two tags achieved the membrane-anchoring
Such dual-functionalized TDNs could be simultaneously task (Figure 14B, right).249 Porphyrin not only has a van der
recognized by both VEGF and folate receptors on the surface Waals surface area 12 times larger than ethane but also is a
of HT-29 cancer cells (Figure 13). Sun et al. confirmed that the chromophore with an emission wavelength at 656 nm. Also,
dual-functionalized TDNs could cause sufficient HT-29 cell moreover, the insertion of porphyrins into lipid bilayers leads to a
inhibition at a much lower Dox concentration. characteristic shift in their fluorescence spectrum. Howorka’s
3.2.3. Creating Artificial Channels with DNA Nano- team reported that indeed they were able to visualize the TPP-
technology. Large molecules including nanoparticles can be tagged DNA pore anchoring on the lipid bilayer through
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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 charged group can be selectively distinguished to transport
the DNA pores exhibited two voltage-dependent conductance through the channel. It is exciting and encouraging that the
states: Low transmembrane voltages favor a stable high- artificial DNA channels can control when and which cargo is
conductance level corresponding to an unobstructed DNA transported across a lipid bilayer, which potentially paves the way
pore, whereas at higher voltages, the channel shows a main low- for controlled drug release.
conductance state probably caused by electric-field-induced 3.2.4. Subcellular Targeting. The route of uptake can affect
changes of the DNA pore in its conformation or orientation.48 which cellular compartment drug molecules are released into. As
In their recent work, Howorka and colleagues further explored we reviewed in section 3.2.1, clathrin-dependent endocytosis
the artificial DNA pore’s ability to selectively transport charged leads to the lysosomal pathway in which lysosomes keep their
molecules across a biological membrane.250 With the design of contents away from the cytosol and eventually degrade the
seven concatenated DNA strands, they constructed an atomisti- contents. In fact, many endocytic pathways result in entry to
cally determined molecular valve (Figure 14C). The valve can lysosomes, where the function of drug molecules may be
bind a specific ligand, triggering a nanomechanical change to inhibited due to the lack of process to the cytoplasm and nucleus
open up the membrane-spanning channel. Small organic or due to the damage caused by degradation. In order to
molecules that differ by the presence of a positively or negatively maximize drug efficacy, targeting with decent precision at
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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 endosome acidification by the proton sponge effect.252,253
perhaps should be called “tertiary targeting”.251 Retrograde endocytic pathway, a nonlysosomal pathway, has
Bypassing the lysosome is critical for subcellular targeting. been explored to deliver a pH-sensing DNA nanodevice to a
Drug vehicles can be engineered to break out of endosomes or specific subcellular compartment other than lysosome.243 In a
enter the cell through nonlysosomal pathways. Nanoparticles retrograde route, proteins or lipids targeted to endosomes from
with positive surface charges can induce osmotic lysis upon the Golgi apparatus or the plasma membrane are transported to
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the trans-Golgi network (TGN), Golgi membranes, or the macromolecular biosynthesis.258 While effectively killing cancer
endoplasmic reticulum (ER).254 Furin is a protein enriched in the cells, Dox also harms normal cells and causes toxicity to most
Golgi apparatus via a retrograde route.255 Krishnan and co- noncancerous organs. The adverse side effects and poor
workers managed to fuse a sequence-specific dsDNA binding selectivity force the Dox-based treatment to be dose limiting.259
protein (single-chain variable fragment recombinant antibody, or Over the years, many drug delivery systems, e.g., liposome,260
scFv)256 as a chimera with furin to build up the DNA−protein nanoparticle,261 and polymer micelle,262 have been developed to
partnership (Figure 15).243 The scFv specifically binds an 8-bp mitigate the adverse effects and improve drug efficiency. Dox can
DNA sequence, which was incorporated in a pH-responsive dwell itself in DNA by intercalating the GC-rich regions of the
DNA nanomachine. When the scFv-furin chimera was expressed double helices, which opens a wide range of possibilities to tailor
inside cells, it bound with the DNA nanomachine and hijacked DNA nanostructures as new carriers for Dox delivery.
the nanomachine along the retrograde furin endocytic pathway Högberg and co-workers optimized the delivery of Dox to
into the TGN. With pH-sensing groups and fluorophores human breast cancer cells with different DNA origami
designed in the DNA nanomachine, Krishnan and co-workers nanostructures.263 These DNA nanostructures exhibited varying
were able to not only map pH but also reveal the morphology of degrees of global twist, leading to different amounts of relaxation
organelles along the retrograde furin pathway, including sorting in the DNA double-helix structure (Figure 16A). By tuning the
the endosome, late endosome, and trans-Golgi network.243 The design, the encapsulation efficiency and the release rate of Dox
work resembles an excellent example to apply DNA nano- can be adjusted to increase cytotoxicity and lower the
technology for subcellular targeting. In principle, during intracellular elimination rate. Flow cytometry also verified the
retrograde transport, through the DNA−protein or DNA− enhanced apoptosis induced by the delivery system in breast
lipid partnership, DNA nanoparticles could be targeted to any cancer cells. In addition, Högberg and co-workers demonstrated
compartment where its partner is transported to. From this work, that the drug release kinetics could be rationally controlled and
we can see the great potential and advantages for using DNA tailored by tuning the twist degrees.263 Such tunable DNA
nanotechnology to guide subcellular targeting. In the next nanostructures resemble efficient delivery vehicles for Dox,
section, during the discussion of drug molecules delivered by through which can generate high degrees of internalization and
DNA nanotechnology, we will also show more examples of increased induction of programmed cell death in breast cancer
precise subcellular targeting with DNA nanotechnology. cells.
3.3. Delivery of Drug Molecules by DNA Nanotechnology
Similar research was carried out by Ding and co-workers, who
designed and assembled 2D and 3D Dox-loaded DNA origami
We have discussed the principles of cellular endocytosis and the structures that exhibited a high level of Dox loading efficiency, as
way DNA nanostructures enter cells. Through sophisticated well as greater cellular uptake of Dox (Figure 16B).264 Ding and
design and modification, DNA nanostructures can specifically co-workers found that the DNA origami/Dox complex exhibited
locate at the plasma membrane of target cells, followed by prominent cytotoxicity not only to regular human breast
endocytosis mostly via a clathrin- or caveoleo-mediated pathway. adenocarcinoma cancer cells (MCF 7) but more importantly
In cells, DNA nanostructures can also be engineered to hijack the to Dox-resistant MCF 7 cells due to the increase in the cellular
retrograde route to bypass lysosomal degradation and target uptake of Dox and redistribution of Dox to action sites by the
specific subcellular compartment. In addition, DNA nano- origami vehicle. In the follow-up project, Ding and co-workers
channels can be constructed to mimic the natural trans- studied the therapeutic efficacy of the origami/Dox system in
membrane pores for controllable transportation of cargos across vivo.265 After conducting fluorescence imaging and safety
the plasma membrane. In this section, we will give an in-depth evaluation experiments, they found that Dox-loaded DNA
and detailed review on what drug molecules have been delivered origami exhibited remarkable antitumor efficacy without
into cells with DNA nanotechnology observable systemic toxicity in nude mice bearing orthotopic
3.3.1. Small Molecules. 3.3.1.1. Fluorescent Dyes. breast tumors. These findings hint that DNA origami
Fluorescent dyes are the most commonly used molecules for nanostructures could be innovative platforms for the efficient
cellular analysis. Due to the maturation of DNA synthesis and safe drug delivery of cancer therapeutics in vivo.
techniques, various fluorescent dyes can be easily modified on To avoid complicated structure design and lower the cost of
DNA strands to track the cycling, distribution, and lifetime of regular DNA origami, Yan et al. invented a novel strategy to
DNA nanostructures in living cells by fluorescent microscopy.257 construct 3D AuNP−DNA superstructure by simultaneously
Usually fluorescent dyes along with other functional molecules growing DNA and folding origami on AuNPs.149 The strategy
are integrated in one DNA nanostructure for simultaneous features the combination of the rigidity of nanoparticles with the
imaging and delivery. For example, Tan and co-workers designed flexibility of DNA nanostructures (Figure 7B). The as-fabricated
aptamer-conjugated FRET (fluorescent resonance energy trans- 3D superstructures have a high molecule-loading capacity, which
fer) nanoflowers (NFs) for multiplexed cellular imaging and enables the simultaneous transport of signal reporters and drug
traceable targeted drug delivery.231 The NFs are modified to molecules with high efficiency for cellular imaging and drug
carry three fluorescent dyes, fluorescein (FAM), cyanine 3 delivery.149
(Cy3), and 6-carboxyl-X-rhodamine (ROX). By optimizing the Besides DNA origami, other self-assembled DNA structures
amount of dye molecules in FRET NFs, the emission spectra can were also explored as nanocarriers for Dox delivery. Tan and co-
be tuned so that only the dye with the longest emission workers constructed a multifunctional aptamer-based DNA
wavelength exhibits significant fluorescent signals. When tested assembly (AptNA) for targeted cancer therapy.266 Multiple DNA
in cancer cells, the NFs system exhibited high fluorescence subunits with different functions, including targeting aptamers,
intensity and excellent photostability.231 intercalated anticancer drugs, and therapeutic antisense
3.3.1.2. Doxorubicin. Doxorubicin (Dox), as one of the most oligonucleotides, were first self-assembled to form Y-shaped
potent chemotherapeutic drugs approved by the FDA, has been functional domains (Figure 16C). They were then linked to X-
used to treat a wide range of cancers through the inhibition of shaped connectors through hybridization to generate building
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Figure 17. Delivery of Dox by MoS2−DNA superstructures. Adapted with permission from ref 142. Copyright 2017 American Chemical Society.
units. Hundreds of these units were further photo-cross-linked to and Dox-binding DNA for drug delivery. The densely packed
form the multifunctional and programmable aptamer-based drug-binding motifs and porous intrastructures endow NFs with
nanoassembly structure. Through calculation, Tan and co- a high loading capacity (71.4%, wt/wt). The Dox-loaded NFs
workers reported that each AptNA contained about 100−200 were stable at physiological pH, and drug release was facilitated
building units, and each unit was able to provide a high-loading under acidic or basic conditions.
capacity of more than 220 Dox loading sites. In addition, the Recently, layer-by-layer self-assembled stacked MoS2−DNA
incorporation of therapeutic antisense oligonucleotides resulted superstructures were demonstrated for protective and autono-
in the inhibition of P-gp expression (a drug efflux pump to mous delivery of Dox in cancer cells. Li et al.142 reported that
increase excretion of anticancer drugs), which enhanced the drug they could functionalize MoS2 nanosheets with terminal-
efficacy. Moreover, the AptNAs showed excellent stability and thiolated DNAs via strong binding to S-atom defect vacancies
integrity in the physiological environment, avoiding unnecessary on MoS2 surfaces (Figure 17). By a linker ATP-aptamer that
leaking of intercalated drugs during the delivery process. induced interlayer assembly, they successfully guided the
Similarly, Zhang et al. studied controllable and targeted delivery formation of stacked MoS 2−DNA superstructures. Dox
of Dox to cancer cells with an aptamer-based dendritic DNA molecules were loaded on the DNA interspacers between
nanostructure.267 neighboring MoS2 nanosheets. In the presence of a high level of
More complex and sophisticated DNA nanosystems have been ATP molecules in many cancer cells, the multilayer MoS2−DNA
developed to control and target Dox to cancers. Gu and co- superstructures disassembled due to the stronger binding of ATP
workers147 engineered a bioinspired cocoon-like anticancer drug with the linking aptamers, which in turn released the Dox cargos.
delivery system consisting of a deoxyribonuclease (DNase)- These superstructures offer a protective armor-like shell of MoS2
degradable DNA nanoclew (NCl) embedded with an acid- nanosheets, which keeps the DNA away from nuclease digestion
responsive DNase I nanocapsule (NCa). The NCl was while it remains responsive to small and infiltrating ATP
assembled from a long-chain single-stranded DNA synthesized molecules. Thus, they could be an enhanced stimuli-responsive
by RCA (Figure 7C). Repeated GC-pair sequences were drug release system for targeted Dox-based chemotherapy.
integrated into the NCl for enhanced loading capacity of Dox. 3.3.2. Oligonucleotides. 3.3.2.1. CpGs. CpGs (unmethy-
After cellular uptake of the complex via FR-mediated pathway, lated cytosine-phosphate-guanine dinucleotides) are frequently
DNase I in the nanocapsule was activated through the acid- seen in microbial genomes but rare in vertebrate genomes.
triggered shedding of the polymeric shell of the NCa, which in Bacterial DNA and synthetic oligonucleotides containing CpG
turn led to the self-degradation of NCl and the release of Dox motifs are recognized as dangerous signals by mammalian innate
(Figure 16D). Tan and co-workers268 used a similar strategy to immune systems and could trigger strong immune responses.269
engineer multifunctional DNA nanoflowers (NFs) for targeted For synthetic CpGs, the stimulatory effects are mediated by Toll-
drug delivery to both chemosensitive and multidrug resistance like-receptor 9 (TLR9) proteins located in the endosome of host
(MDR) cancer cells that circumvented MDR in both leukemia cells, leading to secretion of the pro-inflammatory cytokines,
and breast cancer cell models. NFs were also self-assembled by such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and
RCA. They contain a few functional domains, including aptamers so on.270 Because of this, CpGs have been actively explored in
for specific cancer cell recognition, fluorophores for bioimaging, both basic research and clinical trials as a type of potent and safe
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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 Wiley-
VCH.
vaccine adjuvant for immunotherapy of infectious diseases and (Figure 18A).197 We demonstrated that the DNA nanostructure
cancer.271 Naked CpG dinucleotides are poor in cellular uptake complex was resistant to nuclease degradation and remained
and prone to nuclease degradation. To address the issue, various substantially intact in fetal bovine serum and in cells for at least
approaches have been developed.272,273 For instance, phosphor- several hours. The complex was also able to efficiently and
othioate (PT) modification of the backbone of CpG nucleotides noninvasively enter macrophage-like RAW264.7 cells without
is frequently used to enhance the stability of CpG against the the aid of transfection agents. After cellular uptake, CpG motifs
DNase degradation.274 However, several side effects, including were recognized by TLR9, which in turn activated the
reduced immune response and lymphoid follicle destruction, downstream pathways to induce immunostimulatory effects,
were found to be related to the thiol modification.275 resulting in high-level secretion of various pro-inflammatory
In recent years, DNA nanotechnology-enabled nanostructures cytokines such as TNF-R, IL-6, and IL-12.197
have been explored as carriers for CpG delivery. Due to their Liedl and co-workers tested the immune responses induced by
inherent compatibility, CpG-rich sequences can be easily a 30-helix-bundled DNA origami tube, which was decorated with
incorporated into DNA nanostructures to increase their stability up to 62 CpG sequences, in spleen cells.276 By monitoring
and targeting specificity. In 2011, our group designed a cytokine production and immune cell activation, they confirmed
multivalent DNA tetrahedron to load and deliver CpG motifs that such decorated origami tubes triggered much higher
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immunostimulation than equal amounts of naked CpG urgent to develop strategies to prevent cancer recurrence after
oligonucleotides. Later, we synthesized RCA-based DNA surgery. On the basis of recent successes of cancer
nanoribbons to increase the payload of CpG sequences.143 immunotherapy,285 we think that it can be utilized to prevent
Theoretically, each periodic unit of DNA nanoribbons could cancer recurrence. Programmed death 1 (PD-1) is a key
couple one CpG sequence. We found that the length and immune-checkpoint receptor expressed by activated T cells,
concentration of DNA nanoribbon were highly correlated with and it mediates immunosuppression. The interaction between
the yield of TNF-α. For DNA nanoribbons ∼1 μm in length at a PD-1 and its ligand 1 and 2 (PD-L1/2) is a key pathway hijacked
10 nM concentration, the released amount of CpG was high by tumors to suppress immune response. Therefore, disrupting
enough to stimulate the production of ∼5100 pg/mL TNF-α, the interaction between PD-1 and PD-L1 by anti-PD antibodies
much higher than that triggered by the previously reported can in principle boost the immune response against cancer cells.
carrier systems. Tan and co-workers further simplified the Also, how to improve the therapeutic efficacy with insignificant
synthesis of CpG-decorated DNA nanostructures.277 They side effects, such as autoimmune disorders, is a central theme for
constructed novel immuno-nanoflowers (NFs) self-assembled the anti-PD-based cancer immunotherapy. Recently, Gu and co-
from long DNA strands, which were integrated with tandem workers developed an innovative DNA nanococoon (DNC)
CpGs through RCA, for protection of CpGs from nuclease carrier for controlled release of loaded anti-PD-1 antibody
degradation. In a model of macrophage-like cells, the CpG NFs (aPD1) and CpGs (CpG ODNs) in response to inflammation
were proved to be potent immunostimulators triggering the conditions.286 The inflammatory environment is conducive to
secretion of TNF-R, IL-6, and IL-12 to induce cancer cell immunotherapy by converting quiescent precursor lymphocytes
apoptosis and necrosis.277 into activated lymphocytes, which are required for tumor
We also explored gold nanoparticle-aided nanostructures for eradication. The DNC carrier was built on a long single-stranded
CpG delivery.278 With thiolated modification on the end, DNA DNA amplified by RCA, which contains repeated units with
sequences carrying CpGs were successfully conjugated to interval CpG sequences and cutting sites for restriction enzyme
AuNPs. We used the AuNPs as nanocarriers to noninvasively HhaI (Figure 18C). Multiple CpG fragments would be generated
deliver the synthetic CpGs into cells. Compared to unconjugated once the carrier was digested by HhaI. To make the release event
CpG-containing ssDNA, the self-assembled polyvalent CpG- bioresponsive, Gu and co-workers loaded DNCs with aPD1,
AuNP conjugates were demonstrated to enhance the efficiency trapped HhaI in cages of triglycerol monostearate (TGMS)
of cellular uptake and stimulate secretion of cytokines. The nanoparticles, and attached them with each other to form DNC-
immunostimulatory activity of the conjugates increased with the TGMS complexes.286 TGMS is an amphiphile whose ester
density of the CpG motifs at the surface of AuNPs. Further linkage enables cleavage by esterases and matrix metal-
studies showed that nonthiolated, diblock oligonuleotides loproteinases (MMPs) that are highly expressed at the wound
containing a CpG motif and a polyadenine (polyA) tail could sites for developmental tissue remodeling.287 Therefore,
readily self-assemble on the surface of AuNPs with controllable triggered by the inflammatory condition in the wound site of
and tunable density.279 Under optimal conditions, the polyA- the tumor resection incision, TGMS can be cleaved, leading to
CpG-AuNPs exhibited significantly higher immunostimulatory the release of HhaI, which can further sequentially convert DNCs
activity than their thiolated counterpart. to CpG fragments and release aPD1. The released CpGs can
In addition, Mohri et al.280 constructed ligation-free DNA activate dendritic cells to drive T cell immune response against
dendrimers for CpG delivery. Using CpG-incorporated DNA cancer cells, while the release aPD1 further boosts the immune
units with elongated adhesive ends, they were able to assemble response with PD-1 blockade (Figure 18C). The controlled
DNA dendrimers without the common use of DNA ligases. bioresponsive release of CpG and aPD1 is way more effective
Mohri et al.280 found that the cellular uptake of DNA dendrimers than the treatment with free CpG nucleotides and aPD1. In
by mouse macrophage-like RAW264.7 cells and subsequent addition, the synergistic action of aPD1 and CpG can also
release of TNF-α were dependent on the structural complexity of prevent the potential risk of toxic peak dosage of aPD1 in the
the dendrimers. The results indicate that DNA dendrimers are a body.
potent system for the delivery of immunostimulatory CpG DNA 3.3.2.2. siRNA. siRNA (small interfering RNA) operates
to immune cells. Similarly, Ding and co-workers constructed within the fundamental RNA interference (RNAi) pathway in
programmable DNA dendrimers decorated with CpG loops.281 eukaryotic cells. It functions by targeting and inducing the
They found that the CpG-loop decoration triggered stronger cleavage of a certain complementary mRNA, which leads to the
immune response characterized by pro-inflammatory cytokines shutdown of the expression of mRNA-encoded proteins.288
production, in contrast to linear CpG (Figure 18B). Further SiRNAs are usually 21−23 bases in length and produced by the
modification with TAT peptide (a typical cell-penetrating enzyme Dicer in cells.289 Alternatively, chemically synthesized
peptide) on the surface of DNA dendrimers enabled enhanced siRNAs can be directly introduced into cells and bind with
cellular internalization and cytokines production. Ding and co- complementary mRNA to induce a cellular RNAi process.290,291
workers reported that the TAT−DNA dendrimer-CpG loop Thus, synthetic siRNAs represent a class of potent nucleic acid-
constructs did not affect the viability of immune cells, and no based drug candidates for RNAi therapies. Many strategies have
detectable cytotoxicity was observed. Their results further been developed for delivery of siRNAs, including the conven-
demonstrated that the DNA dendrimers can serve as designable tional complexation or encapsulation of siRNAs with polymers,
and safe vehicles for delivery of CpGs.281 lipids, or other nanoparticles.292−294 However, conventional
Currently, surgical treatment is the most effective therapeutic vehicles such as liposomes and polymeric systems are usually
method for many solid tumors. However, many patients develop heterogeneous in size, composition, and surface chemistry, which
recurrent disease postsurgery, which could lead to significant can lead to potential toxicity, lack of tissue specificity, and
morbidity and mortality for cancer patients.282,283 Following suboptimal performance.11,291,295 DNA nanostructures can be
tumor resection, the inflammatory processes during wound assembled with well-defined sizes, and the component strands
healing may also promote cancer progression.284 Therefore, it is are easily programmed to carry siRNAs with DNA−RNA
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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.
hybridization, thus representing a novel type of vehicle for siRNA environment. In 2014, Sleiman and co-workers generated gene
delivery. silencing 3D DNA prisms by integrating antisense therapeutic
Lee et al.216 designed a self-assembled DNA tetrahedral oligonucleotides within them. They showed that antisense
nanostructure for delivery of siRNAs in vivo to silence target strands tethered on DNA cages could readily induce gene
genes in tumors (Figure 19A). They precisely controlled the silencing in mammalian cells and maintain gene knockdown
diameter of the DNA tetrahedron to be around 30 nm to avoid levels more effectively than single- and double-stranded oligo
renal filtration, which is a typical outcome for monomeric siRNA. controls, because the DNA cages increased the stability of the
In addition, the spatial orientation of cancer-targeting ligands on bound antisense oligo units.296 Through rational design and
the tetrahedron as well as the density of the ligands such as optimization, Sleiman and co-workers further assembled the
peptides and folate on the surface of the tetrahedron were also DNA “nanosuitcase” that could encapsulate a siRNA construct
optimized. Lee et al.216 found that at least three folate molecules and release it upon recognition of an oligonucleotide trigger
per tetrahedron were required for the optimal delivery of siRNAs (Figure 19B).297 The trigger could be an mRNA or a microRNA
into targeted cancer cells. In addition, gene silencing occurred which offers potential for dual or synergistic therapy. Sleiman and
only when the ligands were assembled in the appropriate spatial co-workers demonstrated that the DNA suitcase could sustain
orientation. When three folate molecules were decorated on the biological conditions and protect siRNA cargos, as well as release
tetrahedron so that the local density was maximized, targeted cargos on demand.297
gene silencing was observed. Otherwise, silencing disappeared. Nanoparticle-assisted DNA nanostructures have also been
Lee et al.216 suspect that this phenomenon might be due to the explored for siRNA delivery. Mirkin and co-workers constructed
higher local density of folates influencing the intracellular a RNAi-based nanomedicine platform by assembling spherical
trafficking pathway of the DNA tetrahedron through the cells. nucleic acid (SNA) nanostructures consisting of gold nano-
During the delivery of siRNA, DNA nanostructures can act as particles covalently functionalized with densely packed, highly
not only an umbrella to protect the cargo against nuclease oriented siRNA duplexes.298 They preclinically evaluated the
degradation but also a platform to read and respond to the platform to neutralize oncogene expression in Glioblastoma
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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 anti-
microRNA sticky ends for microRNA quenching. Adapted with permission from ref 313. Copyright 2017 Royal Society of Chemistry.
multiforme (GBM), a neurologically debilitating disease that of active siRNA. Hammond and co-workers reported that more
culminates in death 14−16 months after diagnosis.299−301 Mirkin than one-half a million copies of siRNA could be delivered to a
and co-workers found that the siRNA-loaded SNA penetrated cell with the uptake of a single RNAi-microsponge. Also, due to
the blood−brain barrier and blood−tumor barrier to disseminate the high density of siRNAs, roughly 3 orders of magnitude less
throughout xenogeneic glioma explants. SNAs targeting the carrier was required to achieve the same degree of gene silencing
oncoprotein Bcl2L12 were effective in knocking down as a conventional particle-based vehicle. Recently, Hammond
endogenous Bcl2L12 mRNA and protein levels and sensitized and co-workers took a step forward by making a multi-RNAi
glioma cells toward therapy-induced apoptosis. Mirkin and co- microsponge platform for simultaneous controlled delivery of
workers also observed that systemically delivered SNAs reduced multiple siRNAs.303 They showed that the microsponge-based
Bcl2L12 expression in intracerebral GBM, increased intratumoral siRNA delivery could be potentially applied for the treatment of
apoptosis, and reduced tumor burden and progression in cancer, genetic disorders, and viral infections.
xenografted mice without adverse side effects.298 SNAs represent 3.3.2.3. microRNA. microRNA (miRNA) is similar to siRNA
a new approach for RNAi therapy for GBM and possibly other in terms of size and function. The drawbacks of the conventional
lethal malignancies. vehicles for siRNA delivery also exist in miRNA delivery.
Similar to SNAs, which can load and deliver a large amount of Commercially available transfection reagents for miRNAs may
siRNAs, microsponges comprised of repeated siRNA sequences work well on cultured cells, but these reagents are typically
were developed by Hammond and co-workers for efficient liposomes with a wide size distribution which can lead to
siRNA delivery.302 Using rolling-circle transcription (RCT) to toxicity.304 Much effort has been put into developing better
generate repeated units of combined carrier and cargo (siRNA) miRNA delivery strategies,305−309 but none of them reaches the
sequences, Hammond and co-workers constructed RNAi expectation. In fact, in many cases delivery is only the first step of
polymers that can self-assemble into nanoscale-pleated sheets miRNAs’ in vivo journey. Once into cells, the exogeneous
of hairpin RNA, which in turn form sponge-like microspheres miRNAs would undergo lysosomal degradation, which greatly
(Figure 19C). The microspheres provided protection for siRNA diminishes the actual working concentration of the therapeutic
until they were internalized into the cell, where the cellular RNAi miRNAs. To avoid lysosomal degradation, plasmid or viral
machinery converted the stable hairpin RNAi to short fragments vectors have been used to overexpress miRNAs in cancer cells.
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Figure 21. DNA NC-based CRISPR-Cas9 delivery system. Adapted with permission from ref 325. Copyright 2015 Wiley-VCH.
However, this strategy has limited real efficacy in clinical settings excellent customizable platforms for miRNA-based cancer
due to collateral damage to noncancer cells and an overly therapies.
complicated multistage process. Comparing to conventional Besides acting as tumor suppressors, certain miRNAs
vehicles, DNA nanostructures are more readily synthesized with (oncomiRs) are also associated with cancer. Nahar et al.313
well-defined sizes. In addition, when miRNAs are loaded on assembled a programmable anti-miR branched DNA nanostruc-
DNA nanostructures via DNA−RNA hybridization, the ture carrying single-stranded anti-miRNA overhangs for
nanostructure entity can provide steric hindrance to protect simultaneous selective quenching oncomiRs miRNA-27a, 96,
miRNA from RNase’s degradation. and 182 (Figure 20B), which collectively downregulate
MiRNA-145 is recognized as an important therapeutic FOXO1a, a transcription factor that regulates multiple genes
molecule due to its powerful tumor suppressive effect on cancer. involved in cell cycle progression, apoptosis, and cellular
In most cancer cell lines and tissues miRNA-145 was reported to metabolism. Reduced levels of FOXO1a are known to contribute
be downregulated.310 Reinstating miRNA-145 could almost to malignant transformation and oncogenic state.314 Nahar et al.
immediately suppress tumor growth and block cell invasion and observed enhanced stability of the anti-miR-branched DNA
metastasis.311 In 2017 Leong and co-workers designed and nanostructures than naked anti-miRNAs in serum. The former
assembled a DNA star motif that each can carry three miRNA- were able to knockdown the three oncomiRs with up to a 50%
145 molecules and form a Shuriken-like shape upon miRNA-145 greater repression as compared to the latter. The synergestic
loading (Figure 20A).312 Shuriken’s multipronged configuration miRNA repression led to restoration of FOXO1a protein levels
increased the residence time for robust uptake by cells without which in turn inhibits G1-S transition in cancer cells. This study
any transfection agents. In several cancer cell lines, Leong and co- further demonstrates that the programmability of DNA
workers observed close to 2-fold improvement in the DNA nanostructures can be explored as a powerful tool for miRNA
Shuriken performance when compared to utilizing the common delivery and regulation.
transfection agent, Lipofectamine 2000. The DNA shuriken has 3.3.2.4. CRISPR-Cas9. CRISPR-Cas9 is a prokaryotic immune
an average hydrodynamic diameter of 24.0 ± 8.3 nm with the system that confers resistance to foreign genetic elements such as
miRNA cargos within less than 5 nm of the DNA crossover plasmid and phage DNAs. It has quickly turned from an immune
segment and the triad arms of the Shuriken (Figure 20A). Leong defense system in prokaryotes to a facile genome-editing method
and co-workers believed that the close proximity could provide in biotechnology.315 The engineered CRISPR-Cas9 system
steric shielding from degradative RNase enzymes. They did find requires only two factors to function: A single-guide RNA
that the intracellular miRNA-145 level is more than 5000 or 30 (sgRNA) and the Cas9 protein. The sgRNA recognizes
times higher than that of the untreated control or naked miR- complementary DNA sequences flanked by a 5′-NGG PAM
145-treated cells, respectively, even after 24 h. On the highly motif and directs Cas9 to cleave the recognized DNA.315−318 By
proliferative colorectal cancer cell line DLD-1, the DNA designing the guide sequence of sgRNA to hybridize with target
Shuriken also showed significant antiproliferative effects. In DNA, CRISPR-Cas9 can be engineered to edit almost any
addition, on a 3D spheroid tumor model the DNA Shuriken- genomic region of interest. As the CRISPR-Cas9 system
treated tumor was 31% smaller than the untreated one after 2 undergoes development toward human therapeutics, efficient
days. The work indicates that DNA nanostructures can be delivery poses the major challenge. Viral vectors have been
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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 antibodies. When incorporated into DNA nanostructures,
However, the random integration of viral DNA into the genome besides assisting them to target specific cells for drug delivery
of host cells can potentially lead to genetic diseases.321 Also, often as described in section 3.2.2, aptamers can also endow DNA
the overexpression of Cas9 and sgRNA in vivo increases the off- nanostructures with the ability to sense and respond to the
target effect.322,323 An alternative is to codeliver the Cas9/sgRNA cellular environment, making them “smart” carriers. Meanwhile,
complex in a controllable fashion.324 However, both proteins and DNA nanostructures can make therapeutic aptamers more
RNA suffer from poor cell-membrane permeability due to their resistant to nuclease degradation, resulting in enhanced drug
large size and low solubility as well as the negative charges, efficacy. Comparing to protein therapeutics, aptamers exhibit
respectively. An ideal carrier should be able to shield the protein significant advantages in terms of size, synthetic accessibility, and
and RNA from detrimental physiological environment and escort modification by medicinal chemistry. 227 Blending DNA
them to the cell nucleus where they function. DNA nanostructures with aptamers have been studied by many
nanostructures have been explored by Gu and co-workers as a laboratories to assist drug delivery.
safe and efficient way for codelivery of Cas9/sgRNA.325 They AS1411, one of the popular aptamers, has been used as a
synthesized a DNA nanoclew (NC) by RCA and loaded the NC cancer-targeting ligand.327−330 Its receptor is known as nucleolin,
with Cas9/sgRNA complex through the partial hybridization which is a glycoprotein upregulated on the plasma membrane of
between the RCA-generated ssDNA and the guide sequence of several cancer cells.331−333 In 2014 Bermudez and co-workers
sgRNA (Figure 21). Gu and co-workers fused the nuclear- investigated the uptake and efficacy of pyramidal DNA
localization-signal peptides to Cas9 to promote the trans- nanostructures bearing multiple copies of the AS1411 aptamer
portation of Cas9/sgRNA complex from cytoplasm to nuclei. In in a human cervical cancer cell line (HeLa).334 They found that
addition, they also coated the complex with cationic polymer the AS1411−DNA pyramids exhibited enhanced intracellular
polyethylenimine (PEI) to facilitate the induced endosomal uptake in the absence of any transfection reagents and selectively
escape.326 With cultured cells and tumor-bearing mice models, inhibited the growth of cancer cells. Furthermore, the complex
Gu and co-workers demonstrated that the DNA NCs efficiently was substantially more resistant to nuclease degradation than the
delivered Cas9/sgRNA to the nuclei and in there Cas9/sgRNA aptamers alone.334
effectively drove the formation of indels through targeted DNA Aptamers have been mostly codelivered with other drug
cleavage.325 Although the potential immunogenicity associated molecules to maximize the drug efficacy. One example is by
with DNA NCs is not clear at this stage, DNA nanotechnology Huang and co-workers, who created aptamer-conjugated and
has opened a door to deliver Cas9/sgRNA for human Dox-intercalated DNA icosahedron.335 The aptamer targets
therapeutics. Note that because the current DNA nano- MUC1, which is an important class of tumor surface marker that
technology-enabled delivery of the CRISPR-Cas9 system takes is uniquely and abundantly expressed on various epithelial cancer
advantage of the programmable interaction between the DNA cells.336,337 In addition, MUC1 is also rapidly recycled through
nanostructure and sgRNA, we place this RNA−protein intracellular compartments338,339 and thus can serve as an entry
hybridized drug system in the Oligonucleotides section for portal for its aptamer.340 Under confocal microscope, Huang and
discussion. co-workers observed that only the aptamer-conjugated icosahe-
3.3.2.5. Aptamers. Aptamers are single-stranded DNA or dron efficiently internalized into MUC1-positive cells and that
RNA sequences that can recognize and bind a wide range of the distribution of Dox surrounded the nucleus for killing the
target molecules with affinities and specificities comparable to epithelial cancer cells.335
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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 cells induced the conformational change of the ATP aptamer,
nucleolin and MUC1 but also small metabolic molecules. Cyclic- resulting in the release of Dox to produce strong cytotoxicity and
di-GMP (c-di-GMP) is an important bacterial second messenger induce apoptosis. Gu and co-workers demonstrated that this
and regulates many pathways. Krishnan and co-workers designed sophisticated ATP-responsive system exhibited ∼4-fold en-
a DNA icosahedron integrated with c-di-GMP aptamers and hanced cytotoxicity in the MDA-MB-231 cancer cells than the
certain internal cargosthe fluorescent dextran.341 In the in systems without ATP aptamers.342
vitro test, the binding of c-di-GMP leads to a shape shift of its In the following work, Gu and co-workers applied similar
corresponding aptamer, which in turn resulted in the disassembly design principles to build a fusogenic liposome with a protein−
of the DNA icosahedron, and the subsequent release of DNA complex core containing an ATP-responsive DNA aptamer
fluorescent cargos verified by fluorescent microscopy. Later, scaffold loaded with Dox343 and ATP-responsive aptamer-
Gu and co-workers built an ATP-responsive DNA nanostructure graphene hybridized nanoaggregates for anticancer drug
and demonstrated its drug delivery functionality in vivo.342 The delivery.344 These studies indicate that the stimuli-responsive
whole complex contains three functional domains: an ATP- nanovehicles including DNA nanostructures can be engineered
binding DNA aptamer loaded with Dox, a protamine, and a to promote physiological specificity and on-demand therapeutic
hyaluronic acid (HA)-cross-linked shell (Figure 22). Dox was efficacy of anticancer drugs.
intercalated in the duplex region of the aptamer. The positively 3.3.2.6. Deoxyribozymes. Deoxyribozymes (DNAzymes) are
charged protamine was applied to compress the Dox-loaded single-stranded DNA molecules that form structures capable of
aptamer into a cationic core complex to facilitate cell penetration catalyzing chemical reactions. Numerous DNAzymes have been
and endosomal escape. It has been reported that HAase is rich in reported to sense various metal ions,345 including the
many malignant tumor matrices and the tumor cellular endocytic physiological ones like Na+ 346,347 and Ca2+,348 to catalyze a
vesicles. Thus, the HA shell can be more rapidly degraded in series of chemical reactions such as the cleavage of RNA. Some
tumor sites by HAase to release the cationic complex for DNAzymes are also known to recruit amino acids like L-
intracellular transportation. With protamine, the Dox−aptamer histidine349 as cofactors for RNA cleavage. RNA-cleaving
complex escaped from the endosome and transported into the DNAzymes have attracted significant attention for both
cytosol, where the high concentration of cytosolic ATP in tumor therapeutic and diagnostic applications due to their excellent
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programmability, stability, and activity.350 They can be designed of DNA nanostructures to serve as general platforms for the
to cleave a specific mRNA to downregulate gene expression in rational design and construction of various vaccines.
vivo for tumor suppression.351 However, efficient delivery of Similarly, through the avidin−biotin modification, in 2014
DNAzymes into cells remains challenging. Because of their Zhao and co-workers designed a simple, versatile, multivalent
negative charges, DNAzymes have poor cell membrane ligand system that was made of a RCA-produced polymeric DNA
permeability. Also, once inside cells, they can hardly escape scaffold decorated with antibodies to specifically and efficiently
lysosomal degradation. Several DNAzymes have been delivered interrogate and modulate cell receptor signaling and function.364
into cells using AuNPs.352−354 However, the biosafety of AuNPs Using CD20 clustering-mediated apoptosis in B-cell cancer cells
is another concern. DNA nanostructures like tetrahedrons are as a model system, they demonstrated that the system with
known to be able to enter several types of cells readily without the multivalent CD20 antibodies was significantly more effective at
aid of transfection agents.146,197,198 They recently were explored inducing apoptosis of target cancer cells than the monovalent
to carry DNAzyme probes for multiplexed detection of antibody. Although binding cell-surface receptors to mediate
intraellular metal ionsUO22+ and Pb2+in living cells.355 downstream cellular signaling through multivalent clustering has
DNA dendrimer nanostructures were also proved as an efficient been fulfilled with the aid of other materials such as synthetic
nanocarrier of the histidine-dependent DNAzyme for intra- polymers,365−368 this multivalent DNA nanosystem is easier to
cellular molecular sensing.356 The two examples show the manipulate and represents a new chemical biology tool for the
feasibility of using DNA nanotechnology to facilitate the delivery development of therapeutics.
of DNAzyme-based molecular probes. Meanwhile, they hint at Besides using chemical or biological modifications to load
the possibility of DNA nanotechnology-enabled delivery of proteins in a DNA nanostructure as described above, Crawford et
therapeutic DNAzymes in the future. al. also showed that proteins could be encapsulated in a DNA
3.3.3. Proteins. As the engine of life, proteins perform cage through the noncovalent DNA−protein interaction.369
essential functions in cells, including catalyzing metabolic They designed a DNA tetrahedron, on one edge of which
reactions, gene replication and regulation, signal transduction, contains a 22-bp DNA recognition site for a transcription factor
and so on. Many life-threatening diseases such as cancers exhibit protein, CAP (catabolite activator protein), and showed that
abnormal expression patterns and/or functions of certain CAP could stably bind inside the DNA cage at a 1:1 ratio by
proteins. Thus, most of the current cancer therapies target or bending the edge to accommodate the protein. In principle, this
are based on proteins. For example, the popular monoclonal approach can also be extended to encapsulate other DNA-
antibody therapy uses monoclonal antibodies to bind mono- binding proteins in DNA nanostructures.369
specifically with certain cells or proteins to stimulate the patient’s In order to increase the cellular uptake efficiency of proteins,
immune system to attack those cells. However, the main barrier Mirkin and co-workers reported a strategy to chemically modify a
for protein-based therapeutics comes from their intrinsic functional protein core, β-galactosidase, with a dense shell of
properties such as the vulnerability to enzymatic degradation oligonucleotides (Figure 23B).370 The protein/DNA complex
and difficulty to penetrate the membrane.357−359 A series of was termed as ProSNAs, in which the protein enzyme retained its
vehicles, including lipid-mediated colloidal systems, polymeric native structure and catalytic ability, despite the functionalization
nanocarriers, and inorganic systems, have been developed to of its surface with ∼25 DNA strands. The cellular uptake of SNAs
escort proteins to the targeted cellular locations. Recently, DNA is known to be superior relative to that of their individual
nanovehicles have also been explored for protein delivery. components.200,202 This enhanced cellular internalization of
In 2012, Church and co-workers reported a logic-gated DNA SNAs is likely derived from the 3D architecture of the conjugates
nanorobot for targeted transport of molecular payloads including and its ability to engage scavenger receptors on the surfaces of
antibodies.360 Using the origami technique, they engineered a most cells.200,371 Mirkin and co-workers found that the cellular
hexagonal DNA barrel that could respond to a wide array of cues uptake efficiency of the ProSNAs was enhanced by up to ∼280-
through an aptamer-encoded logic gate (Figure 23A). Payloads fold compared with the bare enzyme.370 In addition, the in vivo
such as antibody fragments were loaded inside the barrel. By working concentration of ProSNAs could be as low as 100 pM.
designing the cues to be cell surface signals that can be They also confirmed that the β-galactosidase delivered by
recognized by the aptamer, the barrel sensed the cell surface ProSNAs effectively catalyzed the hydrolysis of β-glycosidic
inputs and triggered the opening of itself to release the payloads. linkages once endocytosed, whereas equal concentrations of
When testing the robustness of payload release by this system, proteins alone showed little to no intracellular catalytic activity.
Church and co-workers loaded a combination of antibodies to The RCA-based DNA nanoclews have been frequently
human CD33 and CDw328 and observed induced growth arrest explored by Gu and co-workers as nanocarriers for the efficient
in NKL (natural-killer leukemia) cells in a dose-dependent delivery of Dox147 and CRISPR-Cas9 systems.325 Recently, they
fashion.360 The results matched well with the report that the two reported that the transformable DNA nanoclews could be
antibodies can induce growth arrest in leukemic cells.361 Other engineered for plasma membrane-targeted delivery of cyto-
antibody fragments that target human CD33 and flagellin362 kine.372 Cytokines represent a class of anticancer therapeutics
were also successfully loaded and delivered by this DNA due to their specific activities in inducing apoptosis in cancer
nanorobot to enhance T-cell activation.360 cells.373 Most of the current anticancer protein delivery systems
In the same year, Chang, Yan, and co-workers used DNA were designed for intracellular delivery by harnessing the size-
tetrahedron nanostructures as platforms to assemble a model dependent endocytosis of nanoparticles.374−376 Gu and co-
antigen (streptavidin) and CpG adjuvants together into synthetic workers designed two DNA nanoclews carrying complementary
nanoscale vaccines.363 In the immunized mice, they found that sequences in the core which were covered by liposome shells.372
compared to a mixture of CpG and antigen, the assembled The model cytokine TRAIL (tumor necrosis factor-related
antigen−adjuvant−DNA complexes induced strong and long- apoptosis-inducing ligand), a cytokine that interacts with death
lasting antibody responses without stimulating a reaction to the receptors on the plasma membrane and induces tumor specific
DNA nanostructure itself. The work demonstrates the potential apoptosis,377,378 was loaded within the Ni2+-modified core
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sequences via a Ni2+−polyhistidine affinity. Gu and co-workers grafting density. The outer surface of the superstructure was
demonstrated that when the DNA nanoclew mixtures entered coated with PEG for interfacing with biological systems. Chan
the tumor microenvironment, the liposome shells were degraded and co-workers chose J774A.1 murine macrophages as a model
by phospholipase A2, an enzyme overexpressed by various cell system to study the uptake of nanoparticle super-
tumors.379 This in turn released the TRAIL-loaded DNA structures.208 Macrophages are known to sequester the majority
nanoclews into the extracellular environment. Subsequent of in vivo administrated nanoparticles,206 and macrophage
hybridization of the complementary DNA sequences occurred uptake correlates with nanomaterial size and surface charge.207
extracellularly, transforming the compact DNA nanoparticles Interestingly, Chan and co-workers observed that the core−
into DNA nanofibers with microscale lengths. The nanofibers satellite superstructure was 2.5-fold bigger than its core
served as multivalent scaffolds to efficiently present TRAIL to component but resulted in 2-fold lower uptake into macro-
death receptors on the cancer cell membrane and amplified the phages. They also found that in macrophages the superstructures
apoptotic signaling with reduced TRAIL internalization.372 were disassembled into their respective building blocks by
3.3.4. Inorganic Nanoparticles. Inorganic nanoparticles enzymatic degradation. Although many nanoparticle formula-
can be synthesized in the scale of 1−100 nm with precise shape as tions have been reported to aggregate under such environ-
well as controllable surface chemistry and physical proper- ments,390 Chan and co-workers found that these components
ties.380,381 In recent decades, inorganic nanoparticles have been remained dispersed following breakdown and eventually escaped
studied for biosensing, bioimaging, and drug delivery by many from the vesicles and were distributed throughout the cellular
laboratories.381−384 As a class of inorganic nanoparticles, cytoplasm.208 They also confirmed that a certain percent of the
plasmonic nanoparticles exhibit interesting scattering, absorb- dispersed building blocks could escape from the macrophages
ance, and coupling properties based on their geometries and following uptake, implicating the facilitated in vivo clearance by
relative positions. These unique properties have made them a the core−satellite design. In addition, Chan and co-workers
focus of research in many applications including cancer demonstrated that these superstructures could improve their
treatment with photothermal therapy.385,386 A remaining issue accumulation in tumor sites as well as facilitate their elimination
for clinical application of inorganic nanoparticles is the concern from the body.208
about the safety issues, like the high toxicity and poor In a following study, Chan and co-workers constructed DNA-
biodegradability.387 assembled gold−nanorod “core−satellite” superstructures for
Many different inorganic nanoparticles can be conjugated with controllable drug loading and release.391 They found that by
DNA oligonucleotides through either chemical bonding, e.g., the reasonable design of the DNA linker sequences, the loading
disulfide bond, or biological interaction, e.g., the biotin−avidin capacity of Dox could be rationally controlled. To thermally
interaction. On the basis of the great programmability and denature DNA, Chan and co-workers selected gold−nanorods
addressability of DNA nanotechnology, researchers have (AuNRs) as the core nanoparticle because they have a high
fabricated highly ordered inorganic nanoparticle−DNA assem- absorption cross section and photothermal conversion within the
blies with controllable aspect ratios and spatial distributions in medical window (700−1000 nm).392 Rapid Dox release was
order to improve the targeting specificity and/or reroute the achieved by photothermal-induced linker DNA denaturation,
endocytosis pathway of inorganic nanoparticles to enhance their resulting in a 2.1-fold increase in therapeutic effect compared to
drug efficacy and mitigate their toxicity. Meanwhile, when no laser treatment. This study presents a new strategy to control
conjugated to DNA nanostructures, inorganic nanoparticles can drug release from DNA−nanoparticle-assembled superstruc-
be excellent labels for tracking the intracellular location of DNA tures.391
nanostructures via fluorescence or electron microscopy.388 Using a similar strategy, Ding and co-workers designed a DNA
Moreover, the complex of inorganic nanoparticle-conjugated origami as a platform to integrate Dox, AuNRs, and a tumor-
DNA nanostructure can possess both the programmable specific aptamer MUC-1 to realize the effective circumvention of
property intrinsic to DNA and the physical properties associated drug resistance.393 Dox was loaded through base-pair inter-
with inorganic nanoparticles, such as plasmonic and magnetic calation, and oligos-conjugated AuNRs were assembled onto the
features. origami through DNA hybridization. The MUC-1 aptamer
In 2014, Mao and co-workers reported that self-assembled, enabled targeted delivery of the origami complex to multidrug
well-defined soft DNA polyhedron can be used for the resistant MCF-7 cells. After the targeted internalization, Ding
encapsulation of rigid AuNPs into their interior spaces through and co-workers shined cells with a near-infrared laser and
a swallow mechanism.389 These AuNPs were conjugated with observed that the multidrug resistance pump protein P-
short DNA oligo strands through thiol linkages. The subsequent glycoprotein was significantly downregulated, indicating the
hybridization of the conjugated DNA oligos with their achievement of the synergistically chemotherapeutic (Dox) and
complementary sequences inside the polyhedron sucked in the photothermal (AuNRs) effects.
AuNPs and positioned them there. Mao and co-workers To further explore the functionality of DNA-assembled
demonstrated that the AuNP guest can be released in a inorganic nanoparticles for navigating complex biological
controllable way from the polyhedron host.389 This work hints environments, Chan and co-workers constructed DNA-con-
at the potential applications in surface engineering and cargo trolled dynamic colloidal nanoparticle systems.394 The systems
delivery of inorganic nanoparticles with DNA nanotechnology. consist of a core nanoparticle surrounded by small satellites,
As described in section 2.6, DNA and inorganic nanoparticles whose conformation can be transformed by a toe-hold DNA
can be blended together to produce core−satellite super- displacement mechanism. Chan and co-workers found that by
structures. Chan and co-workers built such superstructures changing the surface display of targeting ligands (e.g., uncovering
with one or multiple layers of satellite AuNPs surrounding a folic acid conjugated on the core to allow its interaction with the
central core AuNP through the DNA linkages (Figure 23C).208 receptor on the cell membrane), the cellular targeting efficiency
The architecture of the assembled superstructure can be was increased 2.5 times.394 The study indicates that the core−
controlled by adjusting both nanoparticle geometry and DNA satellite DNA−nanoparticle assemblies have the potential for
6492 DOI: 10.1021/acs.chemrev.7b00663
Chem. Rev. 2019, 119, 6459−6506
mimicking the diverse functions of a protein to selectively control the tetrahedron with nucleus-targeting signaling peptides, we
the biological functions of the engineered nanosystem. observed the transport of the DNA nanostructure to nuclei.
Besides caveolae-mediated endocytosis, there are a few other
4. CELLULAR FATE OF DNA NANOSTRUCTURES ways that can be harnessed to avoid the lysosomal degradation of
Within cells, the DNA components of DNA nanostructures are DNA nanostructures. One is to take the retrograde endocytic
believed to be degraded eventually by the natural DNases. pathways to deliver DNA nanostructures to microtubules,
However, when and where the degradation is processed is related endoplasmic reticulum, trans-Golgi network, and/or other
to the uptake pathway of DNA nanostructures, which depends organelles.243,388 An alternative is to use nanoscale needles to
on various factors, including their size, shape, surface chemistry, penetrate the cell membrane and subsequently release surface-
and physical properties, as well as the type of cell for uptake. bound DNA nanostructures directly into cell cytosol.401,402 Chan
Generally, the majority of in vivo-administered nanoparticles, and Lo demonstrated that the silicon nanoneedle (SiNN) arrays
including DNA nanostructures, are sequestered by macrophages enabled the delivery of 3D DNA nanocages into cells with high
or other phagocytes via phagocytosis, wherein various enzymes uptake efficiency, enhanced stability, low cytotoxicity, and little
in phagolysosome eventually degrade the ingested particles. In damage to the cellular membrane.402 By decorating the DNA
2013 Krishnan and co-workers exploited the coelomocytes of the nanocages with organelle-localization signal peptides, Chan and
multicellular model organism Caenorhabditis elegans to study the Lo found that the nanocages could automatically transport to a
lifetime of various DNA nanostructures in vivo.395 Coelomocytes specific subcellular compartment such as the mitochondria or
are phagocytic leukocytes that appear in the bodies of animals nucleus.402
that have a coelom. They found that reducing single-stranded After accomplishing their delivery tasks inside cells, unlike
domains on DNA nanostructures enhanced the nanostructures’ inorganic nanoparticles that can often resist nuclease degradation
in vivo stability: A DNA duplex carrying two single-stranded and result in in vivo persistence, DNA nanostructures are likely
domains was estimated with an in vivo half-life of 8 h, while to be degraded due to their inherent biodegradability. Perhaps
removing some of the single-stranded domains increased its half- the degraded DNA products (nucleotides) are served as
life to 11 h. Moreover, Krishan and co-workers observed that a nutrients and absorbed by cells. Interestingly, according to
DNA icosahedron396 without free termini was able to remain Chan and co-worker’s report,208 inorganic nanoparticle-
intact inside lysosomes over 24 h of investigation.395 An templated DNA nanostructures could be a solution to promote
explanation of the enhanced in vivo stability of DNA icosahedron the exocytosis of inorganic nanoparticles and mitigate their
was that it maintained the structural integrity even at the cellular persistence and the resulting cytotoxicity.
physiological Mg2+ concentration (1−2 mM), and such an
icosahedron conformation protected the DNA nanostructure 5. CHALLENGES AND OUTLOOK
from DNase attack. In this review, we briefly summarized recent advances and
If DNA nanostructures circumvented phagocytosis, they could developments of DNA nanotechnology. The high programm-
be internalized by cells via an active energy-dependent transport ability of DNA enables the design and self-assembly of numerous
process, such as the clathrin- or caveolae-mediated endocytosis. well-defined 2D and 3D nanostructures, many of which have
As discussed earlier in this review, in clathrin-mediated been demonstrated to be elegant drug carriers. The intrinsic
endocytosis, DNA nanostructures are usually encapsulated in biocompatibility and biodegradability of DNA makes DNA−
vesicles and transported to the early endosomes as well as the late nanostructures highly intriguing drug delivery vehicles. Static and
endosomes. They finally arrive at lysosomes for endolysosomal dynamic DNA nanocarriers have been engineered to be able to
degradation.147,268,397 Similar to what happens in phagolyso- either passively or actively release payloads at specific sites. The
somes, the acidic environment and various DNase in lysosomes various practicable chemical modifications on DNA even allow
facilitate the degradation of DNA nanostructures and the release the addition of more functional groups to DNA nanostructures,
of drug molecules. Before reaching lysosomes, according to resulting in the generation of versatile hybrid nanostructures of
Krishan and co-worker’s work,243,388,398−400 there could be up to DNA and other materials. For example, with thiolated end
hours for DNA nanostructures to maintain their structural labeling, DNA can be conjugated with AuNPs111,115−117 and
integrity in vivo and travel through the early endosomes/sorting lipid molecules180,181 for the assembly of AuNP-templated DNA
endosomes and the late endosomes. nanostructures and DNA−nanostructure-guided liposome,
In the above case, lysosomes are the terminal stop of DNA respectively.
nanostructures. However, in some circumstances, drug mole- A number of design strategies, including tile-based bottom-up,
cules need to be delivered to specific subcellular compartments origami-based top-down, nanoparticle-templated, metal-assisted,
for drug functionalization. Several strategies have been and RCA assisted, have been applied for the assembly of DNA
developed to either break DNA nanostructures out of endo- nanostructures to intercalate, conjugate, encapsulate, or non-
somes or train DNA nanostructures to ride through other covalently bind drug molecules to enhance their biostability,
pinocytosis pathway, such as the caveolae-mediated endocytosis, elongate their circulation time, change their surface and
to continuously travel for their destination. For examples, it is mechanical properties, as well as add functions such as
known that through the proton sponge effect, positively charged responding to environmental cues and targeting specific cells.
nanoparticles can induce osmotic lysis upon endosome acid- The unique feature of DNA nanostructure-based drug delivery
ification.252,253 Gu and co-workers took advantage of that and systems is the modularity, meaning that the size of a DNA nano-
decorated a DNA nanoclew vehicle with cationic polyethyleni- object and the positions of modifications (ligands or drug
mine. By doing so, they were able to make the DNA molecules) on or within it can be precisely controlled at
nanostructure escape from the endosomal transportation and nanoscale, as well as the shape and flexibility of the DNA object
deliver the carried Cas9/sgRNA from cytoplasm to nuclei.326 We can be fine tuned. It is the modularity that makes DNA
reported that a DNA tetrahedron could enter live Hela cells nanotechnology a robust platform to bring in a lot of aspects in a
through the caveolae-mediated pathway.199 Also, by decorating controllable manner for targeted drug delivery. In the past
6493 DOI: 10.1021/acs.chemrev.7b00663
Chem. Rev. 2019, 119, 6459−6506
decade, DNA nanostructures have made remarkable transition recently enzymatic digestion of RCA products has also been
from the in vitro to the in vivo environment. Structures such as explored to generate high-quality short single-stranded oligonu-
tetrahedron, octahedron, and origami-based objects have been cleotides. For example, Högberg and co-workers reported using a
demonstrated to be able to carry siRNA, AuNPs, CpG, aptamers, restriction enzyme to digest RCA products to generate desired
antibodies, or small-molecule drugs for targeted delivery. With DNAs with 14−378 bases in length.145 Our team succeeded in
increasing efforts on applying DNA nanotechnology for modern using hydrolytic deoxyribozymes to self-digest RCA products for
drug delivery, we strongly believe that this nascent and desired DNAs with length ranging from 50 to over 100
developing field will have a huge impact on advanced healthcare bases.410,411 Combining these alternative DNA preparation
sciences in the near future. methods, it is likely that we could reduce the cost for DNA−
Despite the advantages, the potential, and expectations, there nanostructure drug vehicles down to that of antibodies ($300 per
are undoubtedly certain obstacles that need to be addressed gram412 or less) or aptamers ($50 per gram227 or less). However,
before making DNA nanostructures competitive with existing still there is a big gap that needs to be filled before being able to
drug vehicles such as polymers, liposomes, viruses, and so on. compete with polymer materials which can cost less than $1 per
First, little is known for the pharmacokinetics (in vivo circulation, gram.
distribution, metabolism, excretion, etc.) of DNA nanostruc- Third, biosafety is another concern. DNA is biodegradable and
tures. Origami-based DNA nanostructures have been reported to biocompatible. However, things could be changed when DNA is
hold the structural integrity for a long time in cell lysate.170 engineered to become a nanostructure. Before using DNA
However, it is unclear how the physical and chemical properties, nanostructures for clinical bioapplications, we must systemati-
including geometry, surface charges, base combinations, and cally investigate their potential immunostimulatory properties. In
oligonucleotide modifications, of DNA nanostructures affect the certain early studies, DNA nanostructures performed very well as
pharmacokinetic bioavailability. From primary targeting to drug vehicles. For example, Anderson and co-workers demon-
secondary targeting, there are a couple of barriersthe strated a well-defined DNA tetrahedron can deliver siRNAs into
blood−brain barrier and the plasma membrane of cellthat cells and silence target genes in tumors.216 In their mouse model,
prevent efficient passage of DNA nanostructures. Little has been they did not observe antibody response against the DNA
studied for penetrating the blood−brain barrier by DNA nanostructure. However, several critical questions remain to be
nanostructures. Also, the current cellular uptake of most DNA
answered, including (a) How do the physiochemical properties
nanostructures mainly relies on the insufficient endocytosis or
of DNA nanostructures affect the renal system, (b) would the
pinocytosis pathway by themselves. Thus, it is very urgent and
foreign DNA sequences in the DNA nanostructures cause any
important to design approaches for selective uptake of DNA
harmful genetic recombination, and (c) for those nanoparticle-
nanostructures by specific organ(s) and diseased cells while
minimizing nonselective uptake by normal organ(s) and cells. templated or metal-assisted DNA nanostructures what is the final
This is a common challenge for any drug vehicle. However, the fate for those metal elements? Are those metal elements toxic to
completely programmable, precisely controllable, and fully cells? We believe these DNA nanostructure-related biosafety
surface-addressable virtue makes DNA nanostructures top questions are potentially attractive topics to be studied in future.
candidates to tackle this problem. Cells evolved to manipulate DNA in a complex yet exquisite
Second, production cost is a concern. For practical biomedical way. It is believed that the total amount of DNA in a cell is strictly
applications, we need to be able to prepare functional DNA maintained at a certain level.413−415 When DNA nanostructures
nanostructures with high purity at a perhaps gram scale. Several penetrate the cell membrane, it must cause a temporary increase
groups have reported convenient and cost-effective purification of DNA level in the cell, which could result in unexpected
methods in the laboratory level, but demonstration of these problems. An alternative approach to carry and deliver drug
methods at a larger scale has not been performed. For example, molecules into cells could be using RNA nanostructure instead of
Shih and co-workers developed an agarose-gel-based separation DNA. As DNA’s biological counterpart, RNA is more versatile
method to purify intact DNA origami nanostructures with high and the total amount of a cell’s RNA can fluctuate in a wide
yields.403 In a following work, Lin and Shih found that range.416 Nanotechnologists have already confirmed that the
ultracentrifugation provides scalable and contamination-free design principles of DNA nanotechnology can be perfectly
means to recover DNA origami nanostructures.404 To reduce applied for assembling RNA nanostructures, some of which were
the cost for scale-up chemical synthesis of DNA, alternative used for drug delivery.417−422 Certain RNA nanostructures have
methods including asymmetric PCR, RCA, fermentation, and in- been demonstrated to self-assemble from the RNA transcripts in
cell production have been explored to produce a large amount of cells423 or in transcription buffer,424 and the former has been
DNA at a relatively cheap cost. For example, Weuster-Botz and harnessed to organize intracellular reactions.423 As a derivative of
co-workers developed high-cell-density bioreactors via fermen- DNA nanotechnology, RNA nanotechnology is definitely
tation to efficiently generate large quantities of M13 phage another field that is worthy to watch and follow, especially
genome,405,406 the major component for most DNA origami when it comes to drug delivery.
nanostructures. Yan and co-workers designed a single-stranded
PX DNA nanostructure for efficient amplification in live cells.407 AUTHOR INFORMATION
To efficiently prepare long single-stranded DNA other than
M13mp18 genome as the scaffold in DNA origami, Pound et al. Corresponding Authors
modified the PCR protocol and succeeded in obtaining single- *E-mail: [email protected].
stranded DNAs up to 10 000 bases in length.408 We further *E-mail: [email protected].
extended the limit of scaffold length up to 26 kilobases with ORCID
asymmetric PCR,409 which resulted in the assembly of supersized
DNA origami with more capacity to carry payloads. Besides the Lihua Wang: 0000-0002-6198-7561
PCR method for the production of long single-stranded DNA, Chunhai Fan: 0000-0002-7171-7338
6494 DOI: 10.1021/acs.chemrev.7b00663
Chem. Rev. 2019, 119, 6459−6506
Author Contributions ABBREVIATIONS

⊥
Q.H. and H.L.: These authors contributed equally to this work.
Notes
AFM atomic force microscope
AptNA aptamer-based DNA assembly
The authors declare no competing financial interest. aptNTr aptamer-tethered DNA nanotrain
AuNPs gold nanoparticles
Biographies
bp base pairing
Qinqin Hu obtained her Ph.D. degree in Biosystem Engineering from CCMV cowpea chlorotic mottle virus
Zhejiang University in 2016. She now works as a postdoctoral fellow c-di-GMP cyclic-di-GMP
under the supervision of Professor Hongzhou Gu at the Institute of CME clathrin-mediated endocytosis
Biomedical Sciences of Fudan University. Her research focuses on the CP capsid proteins
selection of functional oligonucleotides for biosensing and targeted drug CpG unmethylated cytosine-phosphate-guanine dinucleo-
delivery. tides
CPZ chlorpromazine
Hua Li did his doctoral training under the supervision of Professor CvME caveolae-mediated endocytosis
Junbo Ge at Fudan University. He was awarded his Ph.D. degree in Cy3 cyanine 3
Cardiology in 2013 and then moved to the University of California, Los DNase deoxyribonuclease
Angeles (UCLA) for his postdoctoral training in Professor Peipei Ping’s DNC DNA nanococoon
group. He is now Principle Investigator in Zhongshan Hospital of Fudan Dox doxorubicin
University. His research focuses on novel nanomaterial-based drug dpp diphenylphenanthroline
delivery and cell biology in Cardiology. DX double crossover
Lihua Wang is a Professor at Shanghai Institute of Applied Physics, CAS. ER endoplasmic reticulum
She obtained her Ph.D. degree in Inorganic Chemistry from the FAM fluorescein
Shanghai Institute of Applied Physics, CAS, in 2003. Her research FDA Food and Drug Administration
focuses on the development of novel biosensors using aptamers and FR folate receptor
nanomaterials, high-resolution cell imaging, and multimodality in vivo FRET fluorescent resonance energy transfer
imaging.
GBM glioblastoma multiforme
HA hyaluronic acid
Hongzhou Gu is a Professor in the Institutes of Biomedical Sciences at HBD heparin binding domain
Fudan University and holds a joint faculty appointment at Fudan IL interleukin
University Shanghai Cancer Center. He obtained his Ph.D. degree in JX2 paranemic crossover with two juxtaposed sites
Chemistry from New York University in 2010 and then worked as a MDR multidrug resistance
postdoctoral researcher in Molecular Biology at Yale University. His MMPs matrix metalloproteinases
research focuses on the selection of aptamer-based sensors for molecular MβCD methyl-β-cyclodextrin
probing and imaging, as well as the development of smart DNA or RNA NCa DNase I nanocapsule
nanodevices for targeted drug delivery. NCl DNA nanoclew
Chunhai Fan is Professor and Chief of the Division of Physical Biology
NFs nanoflowers
at SINAP and the Center of Bioimaging at the Shanghai Synchrotron
NKL natural-killer leukemia
NRP neuropilin-1
Radiation Facility (SSRF). He obtained his B.S. and Ph.D. degrees from
PD-1 programmed death 1
Nanjing University in 1996 and 2000, respectively. After postdoctoral
PEI polyethylenimine
research at the University of California, Santa Barbara, he joined the
polyA poly adenine
faculty at Shanghai Institute of Applied Physics (SINAP), Chinese
PT phosphorothioate
Academy of Sciences (CAS), in 2004. He is an elected fellow of the PX paranemic crossover
American Association for the Advancement of Science (AAAS), the RCA rolling-circle amplification
Royal Society of Chemistry (RSC), and the International Society of RNAi RNA interference
Electrochemistry (ISE). He is also an Associate Editor of ACS Applied ROX 6-carboxyl-X-rhodamine
Materials & Interfaces. His research interests are biosensors, bioimaging, SELEX systematic evolution of ligands by exponential
and DNA nanotechnology. enrichment
sgRNA single guide RNA
ACKNOWLEDGMENTS SiNN silicon nanoneedle
siRNA small interfering RNA
The involved research in this work was partially supported by the SNARE soluble N-ethylmaleimide-sensitive factor attach-
China Postdoctoral Science Foundation (2017M611453), the ment protein receptor
Project of Thousand Youth Talents (KHH1340004), the SNAs spherical nucleic acids
National Natural Science Foundation of China (21673050, SR-A class A scavenger receptor
81500229, 21675167, 21390414), the National Key Research SST single-stranded tiles
and Development Program of China (2016YFC1306400, TDNs tetrahedral DNA nanostructures
2016YFA0201200, 2016YFA0400900), and the Key Research TEM transmission electron microscope
Program of Frontier Sciences, CAS, Grant No. QYZDJ-SSW- Tf transferrin
SLH031. We thank Prof. Yimin Wang and Dr. Jiantao Wu from TGMS triglycerol monostearate
Shandong Buchang Pharmaceutical Co., Ltd. for their helpful TGN trans-Golgi network
comments. TLR9 toll-like-receptor 9
6495 DOI: 10.1021/acs.chemrev.7b00663
Chem. Rev. 2019, 119, 6459−6506
TMDs transition metal dichalcogenides (22) Ding, B.; Seeman, N. C. Operation of a DNA Robot Arm Inserted
TNF tumor necrosis factor into a 2D DNA Crystalline Substrate. Science 2006, 314, 1583−1585.
TPP tetraphenylporphyrin (23) Douglas, S. M.; Chou, J. J.; Shih, W. M. DNA-Nanotube-Induced
TRAIL tumor necrosis factor-related apoptosis-inducing Alignment of Membrane Proteins for NMR Structure Determination.
Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 6644−6648.
ligand
(24) Douglas, S. M.; Dietz, H.; Liedl, T.; Högberg, B.; Graf, F.; Shih, W.
TX triple crossover M. Self-Assembly of DNA into Nanoscale Three-Dimensional Shapes.
VEGF vascular endothelial growth factor Nature 2009, 459, 414−418.
(25) Elbaz, J.; Lioubashevski, O.; Wang, F.; Remacle, F.; Levine, R. D.;
REFERENCES Willner, I. DNA Computing Circuits Using Libraries of DNAzyme
Subunits. Nat. Nanotechnol. 2010, 5, 417−422.
(1) Langer, R. New Methods of Drug Delivery. Science 1990, 249,
(26) Fu, T. J.; Seeman, N. C. DNA Double-Crossover Molecules.
1527−1533.
Biochemistry 1993, 32, 3211−3220.
(2) Langer, R. Drug Delivery and Targeting. Nature 1998, 392, 5−10.
(27) Gerling, T.; Wagenbauer, K. F.; Neuner, A. M.; Dietz, H. Dynamic
(3) Steidler, L.; Hans, W.; Schotte, L.; Neirynck, S.; Obermeier, F.;
DNA Devices and Assemblies Formed by Shape-Complementary, Non-
Falk, W.; Fiers, W.; Remaut, E. Treatment of Murine Colitis by
Base Pairing 3D Components. Science 2015, 347, 1446−1452.
Lactococcus Lactis Secreting Interleukin-10. Science 2000, 289, 1352.
(28) Groves, B.; Chen, Y.-J.; Zurla, C.; Pochekailov, S.; Kirschman, J.
(4) Hu, C.-M. J.; Zhang, L.; Aryal, S.; Cheung, C.; Fang, R. H.; Zhang,
L.; Santangelo, P. J.; Seelig, G. Computing in Mammalian Cells with
L. Erythrocyte Membrane-Camouflaged Polymeric Nanoparticles as a
Biomimetic Delivery Platform. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, Nucleic Acid Strand Exchange. Nat. Nanotechnol. 2016, 11, 287−294.
10980−10985. (29) Gu, H.; Chao, J.; Xiao, S.-J.; Seeman, N. C. A Proximity-Based
(5) Felgner, P. L.; Ringold, G. Cationic Liposome-Mediated Programmable DNA Nanoscale Assembly Line. Nature 2010, 465, 202−
Transfection. Nature 1989, 337, 387−388. 205.
(6) Al-Jamal, W. T.; Kostarelos, K. Liposomes: From a Clinically (30) Jung, C.; Allen, P.; Ellington, A. A Stochastic DNA Walker That
Established Drug Delivery System to a Nanoparticle Platform for Traverses a Microparticle Surface. Nat. Nanotechnol. 2016, 11, 157−163.
Theranostic Nanomedicine. Acc. Chem. Res. 2011, 44, 1094−1104. (31) Ke, Y.; Ong, L. L.; Shih, W. M.; Yin, P. Three-Dimensional
(7) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Design and Structures Self-Assembled from DNA Bricks. Science 2012, 338, 1177−
Development of Polymers for Gene Delivery. Nat. Rev. Drug Discovery 1183.
2005, 4, 581−593. (32) Kuzuya, A.; Sakai, Y.; Yamazaki, T.; Xu, Y.; Komiyama, M.
(8) Duncan, B.; Kim, C.; Rotello, V. M. Gold Nanoparticle Platforms as Nanomechanical DNA Origami ’Single-Molecule Beacons’ Directly
Drug and Biomacromolecule Delivery Systems. J. Controlled Release Imaged by Atomic Force Microscopy. Nat. Commun. 2011, 2, 449.
2010, 148, 122−127. (33) Kuzyk, A.; Schreiber, R.; Fan, Z.; Pardatscher, G.; Roller, E. M.; A,
(9) Liu, Z.; Robinson, J. T.; Tabakman, S. M.; Yang, K.; Dai, H. Carbon H.; C, S. F.; O, G. A.; T, L. DNA-Based Self-Assembly of Chiral
Materials for Drug Delivery and Cancer Therapy. Mater. Today 2011, Plasmonic Nanostructures with Tailored Optical Response. Nature
14, 316−323. 2012, 483, 311−314.
(10) Keles, E.; Song, Y.; Du, D.; Dong, W.-J.; Lin, Y. Recent Progress in (34) Kuzyk, A.; Schreiber, R.; Zhang, H.; Govorov, A. O.; Liedl, T.; Liu,
Nanomaterials for Gene Delivery Applications. Biomater. Sci. 2016, 4, N. Reconfigurable 3D Plasmonic Metamolecules. Nat. Mater. 2014, 13,
1291−1309. 862−866.
(11) Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. Toxicity of Cationic (35) Lin, C.; Jungmann, R.; Leifer, A. M.; Li, C.; Levner, D.; Church, G.
Lipids and Cationic Polymers in Gene Delivery. J. Controlled Release M.; Shih, W. M.; Yin, P. Submicrometre Geometrically Encoded
2006, 114, 100−109. Fluorescent Barcodes Self-Assembled from DNA. Nat. Chem. 2012, 4,
(12) Zelphati, O.; Uyechi, L. S.; Barron, L. G.; Szoka, F. C. Effect of 832−839.
Serum Components on the Physico-Chemical Properties of Cationic (36) Liu, H.; Wang, J.; Song, S.; Fan, C.; Gothelf, K. V. A DNA-Based
Lipid/Oligonucleotide Complexes and on Their Interactions with Cells. System for Selecting and Displaying the Combined Result of Two Input
Biochim. Biophys. Acta, Lipids Lipid Metab. 1998, 1390, 119−133. Variables. Nat. Commun. 2015, 6, 10089.
(13) Magrez, A.; Kasas, S.; Salicio, V.; Pasquier, N.; Seo, J. W.; Celio, (37) Mao, C.; Sun, W.; Shen, Z.; Seeman, N. C. A Nanomechanical
M.; Catsicas, S.; Schwaller, B.; Forró, L. Cellular Toxicity of Carbon- Device Based on the B-Z Transition of DNA. Nature 1999, 397, 144−
Based Nanomaterials. Nano Lett. 2006, 6, 1121−1125. 146.
(14) De Jong, W. H.; Hagens, W. I.; Krystek, P.; Burger, M. C.; Sips, A. (38) Marras, A. E.; Zhou, L.; Su, H.-J.; Castro, C. E. Programmable
J.; Geertsma, R. E. Particle Size-Dependent Organ Distribution of Gold Motion of DNA Origami Mechanisms. Proc. Natl. Acad. Sci. U. S. A.
Nanoparticles after Intravenous Administration. Biomaterials 2008, 29, 2015, 112, 713−718.
1912−1919. (39) Meyer, R.; Niemeyer, C. M. Orthogonal Protein Decoration of
(15) Seeman, N. C. Nucleic Acid Junctions and Lattices. J. Theor. Biol. DNA Nanostructures. Small 2011, 7, 3211−3218.
1982, 99, 237−247. (40) Pei, H.; Liang, L.; Yao, G.; Li, J.; Huang, Q.; Fan, C.
(16) Kallenbach, N. R.; Ma, R.-I.; Seeman, N. C. An Immobile Nucleic Reconfigurable Three-Dimensional DNA Nanostructures for the
Acid Junction Constructed from Oligonucleotides. Nature 1983, 305, Construction of Intracellular Logic Sensors. Angew. Chem. 2012, 124,
829−831. 9154−9158.
(17) Acuna, G.; Möller, F.; Holzmeister, P.; Beater, S.; Lalkens, B.; (41) Pei, H.; Lu, N.; Wen, Y.; Song, S.; Liu, Y.; Yan, H.; Fan, C. A. DNA
Tinnefeld, P. Fluorescence Enhancement at Docking Sites of DNA- Nanostructure-Based Biomolecular Probe Carrier Platform for Electro-
Directed Self-Assembled Nanoantennas. Science 2012, 338, 506−510. chemical Biosensing. Adv. Mater. 2010, 22, 4754−4758.
(18) Angelin, A.; Weigel, S.; Garrecht, R.; Meyer, R.; Bauer, J.; Kumar, (42) Puchkova, A.; Vietz, C.; Pibiri, E.; Wünsch, B.; Sanz Paz, M. a.;
R. K.; Hirtz, M.; Niemeyer, C. M. Multiscale Origami Structures as Acuna, G. P.; Tinnefeld, P. DNA Origami Nanoantennas with over
Interface for Cells. Angew. Chem., Int. Ed. 2015, 54, 15813−15817. 5000-Fold Fluorescence Enhancement and Single-Molecule Detection
(19) Bell, N. A.; Engst, C. R.; Ablay, M.; Divitini, G.; Ducati, C.; Liedl, at 25 μM. Nano Lett. 2015, 15, 8354−8359.
T.; Keyser, U. F. DNA Origami Nanopores. Nano Lett. 2012, 12, 512− (43) Rajendran, A.; Endo, M.; Sugiyama, H. Single-Molecule Analysis
517. Using DNA Origami. Angew. Chem., Int. Ed. 2012, 51, 874−890.
(20) Chen, J.; Seeman, N. C. Synthesis from DNA of a Molecule with (44) Ross, M. B.; Ku, J. C.; Blaber, M. G.; Mirkin, C. A.; Schatz, G. C.
the Connectivity of a Cube. Nature 1991, 350, 631−633. Defect Tolerance and the Effect of Structural Inhomogeneity in
(21) Dietz, H.; Douglas, S. M.; Shih, W. M. Folding DNA into Twisted Plasmonic DNA-Nanoparticle Superlattices. Proc. Natl. Acad. Sci. U. S. A.
and Curved Nanoscale Shapes. Science 2009, 325, 725−730. 2015, 112, 10292−10297.
6496 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
(45) Rothemund, P. W. Folding DNA to Create Nanoscale Shapes and (68) Chao, J.; Liu, H.; Su, S.; Wang, L.; Huang, W.; Fan, C. Structural
Patterns. Nature 2006, 440, 297−302. DNA Nanotechnology for Intelligent Drug Delivery. Small 2014, 10,
(46) Seelig, G.; Soloveichik, D.; Zhang, D. Y.; Winfree, E. Enzyme-Free 4626−4635.
Nucleic Acid Logic Circuits. Science 2006, 314, 1585−1588. (69) Chen, Y.-J.; Groves, B.; Muscat, R. A.; Seelig, G. DNA
(47) Seeman, N. C. DNA in a Material World. Nature 2003, 421, 427− Nanotechnology from the Test Tube to the Cell. Nat. Nanotechnol.
431. 2015, 10, 748−760.
(48) Seifert, A.; Göpfrich, K.; Burns, J. R.; Fertig, N.; Keyser, U. F.; (70) Jones, M. R.; Seeman, N. C.; Mirkin, C. A. Programmable
Howorka, S. Bilayer-Spanning DNA Nanopores with Voltage-Switching Materials and the Nature of the DNA Bond. Science 2015, 347, 1260901.
between Open and Closed State. ACS Nano 2015, 9, 1117−1126. (71) Linko, V.; Ora, A.; Kostiainen, M. A. DNA Nanostructures as
(49) Setyawati, M. I.; Kutty, R. V.; Tay, C. Y.; Yuan, X.; Xie, J.; Leong, Smart Drug-Delivery Vehicles and Molecular Devices. Trends Biotechnol.
D. T. Novel Theranostic DNA Nanoscaffolds for the Simultaneous 2015, 33, 586−594.
Detection and Killing of Escherichia Coli and Staphylococcus Aureus. ACS (72) Angell, C.; Xie, S.; Zhang, L.; Chen, Y. DNA Nanotechnology for
Appl. Mater. Interfaces 2014, 6, 21822−21831. Precise Control over Drug Delivery and Gene Therapy. Small 2016, 12,
(50) Shih, W. M.; Quispe, J. D.; Joyce, G. F. A 1.7-Kilobase Single- 1117−1132.
Stranded DNA That Folds into a Nanoscale Octahedron. Nature 2004, (73) Lee, D. S.; Qian, H.; Tay, C. Y.; Leong, D. T. Cellular Processing
427, 618−621. and Destinies of Artificial DNA Nanostructures. Chem. Soc. Rev. 2016,
(51) Tan, S. J.; Campolongo, M. J.; Luo, D.; Cheng, W. Building 45, 4199−4225.
Plasmonic Nanostructures with DNA. Nat. Nanotechnol. 2011, 6, 268− (74) Wu, X.; Wu, C.; Zhang, C. Discrete DNA Three-Dimensional
276. Nanostructures: The Synthesis and Applications. Chin. J. Polym. Sci.
(52) Tay, C. Y.; Yuan, L.; Leong, D. T. Nature-Inspired DNA 2017, 35, 1−24.
Nanosensor for Real-Time in Situ Detection of mRNA in Living Cells. (75) LaBean, T. H.; Yan, H.; Kopatsch, J.; Liu, F.; Winfree, E.; Reif, J.
ACS Nano 2015, 9, 5609−5617. H.; Seeman, N. C. Construction, Analysis, Ligation, and Self-Assembly
(53) Voigt, N. V.; Tørring, T.; Rotaru, A.; Jacobsen, M. F.; Ravnsbæk, J. of DNA Triple Crossover Complexes. J. Am. Chem. Soc. 2000, 122,
B.; Subramani, R.; Mamdouh, W.; Kjems, J.; Mokhir, A.; Besenbacher, F. 1848−1860.
Single-Molecule Chemical Reactions on DNA Origami. Nat. Nano- (76) Mathieu, F.; Liao, S.; Kopatsch, J.; Wang, T.; Mao, C.; Seeman, N.
technol. 2010, 5, 200−203. C. Six-Helix Bundles Designed from DNA. Nano Lett. 2005, 5, 661−
(54) Watson, M. A.; Cockroft, S. L. An Autonomously Reciprocating 665.
Transmembrane Nanoactuator. Angew. Chem., Int. Ed. 2016, 55, 1345− (77) Li, X.; Yang, X.; Qi, J.; Seeman, N. C. Antiparallel DNA Double
1349. Crossover Molecules as Components for Nanoconstruction. J. Am.
(55) Wei, B.; Dai, M.; Yin, P. Complex Shapes Self-Assembled from Chem. Soc. 1996, 118, 6131−6140.
Single-Stranded DNA Tiles. Nature 2012, 485, 623−626. (78) Sa-Ardyen, P.; Vologodskii, A. V.; Seeman, N. C. The Flexibility of
(56) Yan, H.; Zhang, X.; Shen, Z.; Seeman, N. C. A Robust DNA DNA Double Crossover Molecules. Biophys. J. 2003, 84, 3829−3837.
Mechanical Device Controlled by Hybridization Topology. Nature (79) Wang, T.; Schiffels, D.; Martinez Cuesta, S.; Kuchnir Fygenson,
2002, 415, 62−65. D.; Seeman, N. C. Design and Characterization of 1D Nanotubes and
(57) Yehl, K.; Mugler, A.; Vivek, S.; Liu, Y.; Zhang, Y.; Fan, M.; Weeks, 2D Periodic Arrays Self-Assembled from DNA Multi-Helix Bundles. J.
E. R.; Salaita, K. High-Speed DNA-Based Rolling Motors Powered by Am. Chem. Soc. 2012, 134, 1606−1616.
Rnase H. Nat. Nanotechnol. 2016, 11, 184−190. (80) Winfree, E.; Liu, F.; Wenzler, L. A.; Seeman, N. C. Design and
(58) Zadegan, R. M.; Jepsen, M. D.; Thomsen, K. E.; Okholm, A. H.; Self-Assembly of Two-Dimensional DNA Crystals. Nature 1998, 394,
Schaffert, D. H.; Andersen, E. S.; Birkedal, V.; Kjems, J. Construction of 539−544.
a 4 Zeptoliters Switchable 3D DNA Box Origami. ACS Nano 2012, 6, (81) Ding, B.; Sha, R.; Seeman, N. C. Pseudohexagonal 2D DNA
10050−10053. Crystals from Double Crossover Cohesion. J. Am. Chem. Soc. 2004, 126,
(59) Zhang, C.; Yang, J.; Jiang, S.; Liu, Y.; Yan, H. DNAzyme-Based 10230−10231.
Logic Gate-Mediated DNA Self-Assembly. Nano Lett. 2016, 16, 736− (82) Park, S. H.; Yin, P.; Liu, Y.; Reif, J. H.; LaBean, T. H.; Yan, H.
741. Programmable DNA Self-Assemblies for Nanoscale Organization of
(60) Zhang, H.; Lai, M.; Zuehlke, A.; Peng, H.; Li, X. F.; Le, X. C. Ligands and Proteins. Nano Lett. 2005, 5, 729−733.
Binding-Induced DNA Nanomachines Triggered by Proteins and (83) Zheng, J.; Constantinou, P. E.; Micheel, C.; Alivisatos, A. P.; Kiehl,
Nucleic Acids. Angew. Chem., Int. Ed. 2015, 54, 14326−14330. R. A.; Seeman, N. C. Two-Dimensional Nanoparticle Arrays Show the
(61) Zheng, J.; Birktoft, J. J.; Chen, Y.; Wang, T.; Sha, R.; Organizational Power of Robust DNA Motifs. Nano Lett. 2006, 6,
Constantinou, P. E.; Ginell, S. L.; Mao, C.; Seeman, N. C. From 1502−1504.
Molecular to Macroscopic Via the Rational Design of a Self-Assembled (84) Kuzuya, A.; Wang, R.; Sha, R.; Seeman, N. C. Six-Helix and Eight-
3D DNA Crystal. Nature 2009, 461, 74−77. Helix DNA Nanotubes Assembled from Half-Tubes. Nano Lett. 2007, 7,
(62) Zhou, C.; Duan, X.; Liu, N. A Plasmonic Nanorod That Walks on 1757−1763.
DNA Origami. Nat. Commun. 2015, 6, 8102. (85) Liu, D.; Wang, M.; Deng, Z.; Walulu, R.; Mao, C. Tensegrity:
(63) Chhabra, R.; Sharma, J.; Liu, Y.; Rinker, S.; Yan, H. DNA Self- Construction of Rigid DNA Triangles with Flexible Four-Arm DNA
Assembly for Nanomedicine. Adv. Drug Delivery Rev. 2010, 62, 617− Junctions. J. Am. Chem. Soc. 2004, 126, 2324−2325.
625. (86) Ma, R.-I.; Kallenbach, N. R.; Sheardy, R. D.; Petrillo, M. L.;
(64) Pinheiro, A. V.; Han, D.; Shih, W. M.; Yan, H. Challenges and Seeman, N. C. Three-Arm Nucleic Acid Junctions Are Flexible. Nucleic
Opportunities for Structural DNA Nanotechnology. Nat. Nanotechnol. Acids Res. 1986, 14, 9745−9753.
2011, 6, 763−772. (87) Wang, Y.; Mueller, J. E.; Kemper, B.; Seeman, N. C. Assembly and
(65) Li, J.; Fan, C.; Pei, H.; Shi, J.; Huang, Q. Smart Drug Delivery Characterization of Five-Arm and Six-Arm DNA Branched Junctions.
Nanocarriers with Self-Assembled DNA Nanostructures. Adv. Mater. Biochemistry 1991, 30, 5667−5674.
2013, 25, 4386−4396. (88) Wang, X.; Seeman, N. C. Assembly and Characterization of 8-Arm
(66) Pei, H.; Zuo, X.; Zhu, D.; Huang, Q.; Fan, C. Functional DNA and 12-Arm DNA Branched Junctions. J. Am. Chem. Soc. 2007, 129,
Nanostructures for Theranostic Applications. Acc. Chem. Res. 2014, 47, 8169−8176.
550−559. (89) Goodman, R. P.; Schaap, I. A.; Tardin, C. F.; Erben, C. M.; Berry,
(67) Zhang, Y.; Chan, H. F.; Leong, K. W. Advanced Materials and R. M.; Schmidt, C. F.; Turberfield, A. J. Rapid Chiral Assembly of Rigid
Processing for Drug Delivery: The Past and the Future. Adv. Drug DNA Building Blocks for Molecular Nanofabrication. Science 2005, 310,
Delivery Rev. 2013, 65, 104−120. 1661−1665.
6497 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
(90) Zhang, Y.; Seeman, N. C. Construction of a DNA-Truncated (113) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Scanometric DNA
Octahedron. J. Am. Chem. Soc. 1994, 116, 1661−1669. Array Detection with Nanoparticle Probes. Science 2000, 289, 1757−
(91) Yan, H.; Park, S. H.; Finkelstein, G.; Reif, J. H.; LaBean, T. H. 1760.
DNA-Templated Self-Assembly of Protein Arrays and Highly (114) Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.;
Conductive Nanowires. Science 2003, 301, 1882−1884. Mirkin, C. A. Nano-Flares: Probes for Transfection and mRNA
(92) He, Y.; Ye, T.; Su, M.; Zhang, C.; Ribbe, A. E.; Jiang, W.; Mao, C. Detection in Living Cells. J. Am. Chem. Soc. 2007, 129, 15477−15479.
Hierarchical Self-Assembly of DNA into Symmetric Supramolecular (115) Mucic, R. C.; Storhoff, J. J.; Mirkin, C. A.; Letsinger, R. L. DNA-
Polyhedra. Nature 2008, 452, 198−201. Directed Synthesis of Binary Nanoparticle Network Materials. J. Am.
(93) Zhang, C.; Su, M.; He, Y.; Zhao, X.; Fang, P.-a.; Ribbe, A. E.; Jiang, Chem. Soc. 1998, 120, 12674−12675.
W.; Mao, C. Conformational Flexibility Facilitates Self-Assembly of (116) Park, S. J.; Lazarides, A. A.; Mirkin, C. A.; Letsinger, R. L.
Complex DNA Nanostructures. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, Directed Assembly of Periodic Materials from Protein and Oligonucleo-
10665−10669. tide-Modified Nanoparticle Building Blocks. Angew. Chem., Int. Ed.
(94) Zhang, C.; Ko, S. H.; Su, M.; Leng, Y.; Ribbe, A. E.; Jiang, W.; 2001, 40, 2909−2912.
Mao, C. Symmetry Controls the Face Geometry of DNA Polyhedra. J. (117) Park, S.-J.; Lazarides, A. A.; Storhoff, J. J.; Pesce, L.; Mirkin, C. A.
Am. Chem. Soc. 2009, 131, 1413−1415. The Structural Characterization of Oligonucleotide-Modified Gold
(95) He, Y.; Su, M.; Fang, P. a.; Zhang, C.; Ribbe, A. E.; Jiang, W.; Mao,
Nanoparticle Networks Formed by DNA Hybridization. J. Phys. Chem. B
C. On the Chirality of Self-Assembled DNA Octahedra. Angew. Chem.
2004, 108, 12375−12380.
2010, 122, 760−763.
(118) Nykypanchuk, D.; Maye, M. M.; Van Der Lelie, D.; Gang, O.
(96) Douglas, S. M.; Marblestone, A. H.; Teerapittayanon, S.; Vazquez,
A.; Church, G. M.; Shih, W. M. Rapid Prototyping of 3D DNA-Origami DNA-Guided Crystallization of Colloidal Nanoparticles. Nature 2008,
Shapes with Cadnano. Nucleic Acids Res. 2009, 37, 5001−5006. 451, 549.
(97) Castro, C. E.; Kilchherr, F.; Kim, D.-N.; Shiao, E. L.; Wauer, T.; (119) Park, S. Y.; Lytton-Jean, A. K.; Lee, B.; Weigand, S.; Schatz, G.
Wortmann, P.; Bathe, M.; Dietz, H. A Primer to Scaffolded DNA C.; Mirkin, C. A. DNA-Programmable Nanoparticle Crystallization.
Origami. Nat. Methods 2011, 8, 221−229. Nature 2008, 451, 553.
(98) Ke, Y.; Douglas, S. M.; Liu, M.; Sharma, J.; Cheng, A.; Leung, A.; (120) Auyeung, E.; Li, T. I.; Senesi, A. J.; Schmucker, A. L.; Pals, B. C.;
Liu, Y.; Shih, W. M.; Yan, H. Multilayer DNA Origami Packed on a De La Cruz, M. O.; Mirkin, C. A. DNA-Mediated Nanoparticle
Square Lattice. J. Am. Chem. Soc. 2009, 131, 15903−15908. Crystallization into Wulff Polyhedra. Nature 2014, 505, 73.
(99) Liedl, T.; Högberg, B.; Tytell, J.; Ingber, D. E.; Shih, W. M. Self- (121) Sun, D.; Gang, O. Binary Heterogeneous Superlattices
Assembly of Three-Dimensional Prestressed Tensegrity Structures from Assembled from Quantum Dots and Gold Nanoparticles with DNA. J.
DNA. Nat. Nanotechnol. 2010, 5, 520−524. Am. Chem. Soc. 2011, 133, 5252−5254.
(100) Han, D.; Pal, S.; Liu, Y.; Yan, H. Folding and Cutting DNA into (122) Zhang, C.; Macfarlane, R. J.; Young, K. L.; Choi, C. H. J.; Hao, L.;
Reconfigurable Topological Nanostructures. Nat. Nanotechnol. 2010, 5, Auyeung, E.; Liu, G.; Zhou, X.; Mirkin, C. A. A General Approach to
712−717. DNA-Programmable Atom Equivalents. Nat. Mater. 2013, 12, 741.
(101) Han, D.; Pal, S.; Nangreave, J.; Deng, Z.; Liu, Y.; Yan, H. DNA (123) Zhang, Y.; Lu, F.; Yager, K. G.; Van Der Lelie, D.; Gang, O. A
Origami with Complex Curvatures in Three-Dimensional Space. Science General Strategy for the DNA-Mediated Self-Assembly of Functional
2011, 332, 342−346. Nanoparticles into Heterogeneous Systems. Nat. Nanotechnol. 2013, 8,
(102) Andersen, E. S.; Dong, M.; Nielsen, M. M.; Jahn, K.; Subramani, 865−872.
R.; Mamdouh, W.; Golas, M. M.; Sander, B.; Stark, H.; Oliveira, C. L. (124) Auyeung, E.; Macfarlane, R. J.; Choi, C. H. J.; Cutler, J. I.; Mirkin,
Self-Assembly of a Nanoscale DNA Box with a Controllable Lid. Nature C. A. Transitioning DNA-Engineered Nanoparticle Superlattices from
2009, 459, 73. Solution to the Solid State. Adv. Mater. 2012, 24, 5181−5186.
(103) Ke, Y.; Sharma, J.; Liu, M.; Jahn, K.; Liu, Y.; Yan, H. Scaffolded (125) Senesi, A. J.; Eichelsdoerfer, D. J.; Macfarlane, R. J.; Jones, M. R.;
DNA Origami of a DNA Tetrahedron Molecular Container. Nano Lett. Auyeung, E.; Lee, B.; Mirkin, C. A. Stepwise Evolution of DNA-
2009, 9, 2445−2447. Programmable Nanoparticle Superlattices. Angew. Chem., Int. Ed. 2013,
(104) Benson, E.; Mohammed, A.; Gardell, J.; Masich, S.; Czeizler, E.; 52, 6624−6628.
Orponen, P.; Högberg, B. DNA Rendering of Polyhedral Meshes at the (126) Huo, F.; Lytton-Jean, A. K.; Mirkin, C. A. Asymmetric
Nanoscale. Nature 2015, 523, 441−444. Functionalization of Nanoparticles Based on Thermally Addressable
(105) Veneziano, R.; Ratanalert, S.; Zhang, K.; Zhang, F.; Yan, H.; DNA Interconnects. Adv. Mater. 2006, 18, 2304−2306.
Chiu, W.; Bathe, M. Designer Nanoscale DNA Assemblies Programmed (127) Xu, X.; Rosi, N. L.; Wang, Y.; Huo, F.; Mirkin, C. A. Asymmetric
from the Top-Down. Science 2016, 352, 1534−1534. Functionalization of Gold Nanoparticles with Oligonucleotides. J. Am.
(106) Fujibayashi, K.; Hariadi, R.; Park, S. H.; Winfree, E.; Murata, S.
Chem. Soc. 2006, 128, 9286−9287.
Toward Reliable Algorithmic Self-Assembly of DNA Tiles: A Fixed-
(128) Millstone, J. E.; Georganopoulou, D. G.; Xu, X.; Wei, W.; Li, S.;
Width Cellular Automaton Pattern. Nano Lett. 2008, 8, 1791−1797.
Mirkin, C. A. DNA-Gold Triangular Nanoprism Conjugates. Small
(107) Barish, R. D.; Schulman, R.; Rothemund, P. W.; Winfree, E. An
Information-Bearing Seed for Nucleating Algorithmic Self-Assembly. 2008, 4, 2176−2180.
Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 6054−6059. (129) Maye, M. M.; Nykypanchuk, D.; Cuisinier, M.; Van Der Lelie,
(108) Liu, W.; Zhong, H.; Wang, R.; Seeman, N. C. Crystalline Two- D.; Gang, O. Stepwise Surface Encoding for High-Throughput
Dimensional DNA-Origami Arrays. Angew. Chem. 2011, 123, 278−281. Assembly of Nanoclusters. Nat. Mater. 2009, 8, 388.
(109) Ke, Y.; Ong, L. L.; Sun, W.; Song, J.; Dong, M.; Shih, W. M.; Yin, (130) Xu, L.; Kuang, H.; Xu, C.; Ma, W.; Wang, L.; Kotov, N. A.
P. DNA Brick Crystals with Prescribed Depths. Nat. Chem. 2014, 6, Regiospecific Plasmonic Assemblies for in Situ Raman Spectroscopy in
994−1002. Live Cells. J. Am. Chem. Soc. 2012, 134, 1699−1709.
(110) Woo, S.; Rothemund, P. W. Programmable Molecular (131) Jones, M. R.; Mirkin, C. A. Materials Science: Self-Assembly
Recognition Based on the Geometry of DNA Nanostructures. Nat. Gets New Direction. Nature 2012, 491, 42−43.
Chem. 2011, 3, 620−627. (132) Wang, Y.; Wang, Y.; Breed, D. R.; Manoharan, V. N.; Feng, L.;
(111) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A Hollingsworth, A. D.; Weck, M.; Pine, D. J. Colloids with Valence and
DNA-Based Method for Rationally Assembling Nanoparticles into Specific Directional Bonding. Nature 2012, 491, 51−U61.
Macroscopic Materials. Nature 1996, 382, 607−609. (133) Mitra, D.; Di Cesare, N.; Sleiman, H. F. Self-Assembly of Cyclic
(112) Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K.; Metal-DNA Nanostructures Using Ruthenium Tris (Bipyridine)-
Han, M. S.; Mirkin, C. A. Oligonucleotide-Modified Gold Nanoparticles Branched Oligonucleotides. Angew. Chem., Int. Ed. 2004, 43, 5804−
for Intracellular Gene Regulation. Science 2006, 312, 1027−1030. 5808.
6498 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
(134) Kieltyka, R.; Fakhoury, J.; Moitessier, N.; Sleiman, H. F. (155) Enochs, W. S.; Harsh, G.; Hochberg, F.; Weissleder, R. Improved
Platinum Phenanthroimidazole Complexes as G-Quadruplex DNA Delineation of Human Brain Tumors on Mr Images Using a Long-
Selective Binders. Chem. - Eur. J. 2008, 14, 1145−1154. Circulating, Superparamagnetic Iron Oxide Agent. J. Magn. Reson.
(135) Yang, H.; Sleiman, H. F. Templated Synthesis of Highly Stable, Imaging 1999, 9, 228−232.
Electroactive, and Dynamic Metal-DNA Branched Junctions. Angew. (156) Minagar, A.; Alexander, J. S. Blood-Brain Barrier Disruption in
Chem., Int. Ed. 2008, 47, 2443−2446. Multiple Sclerosis. Mult. Scler. 2003, 9, 540−549.
(136) Yang, H.; McLaughlin, C. K.; Aldaye, F. A.; Hamblin, G. D.; Rys, (157) Kizelsztein, P.; Ovadia, H.; Garbuzenko, O.; Sigal, A.; Barenholz,
A. Z.; Rouiller, I.; Sleiman, H. F. Metal-Nucleic Acid Cages. Nat. Chem. Y. Pegylated Nanoliposomes Remote-Loaded with the Antioxidant
2009, 1, 390−396. Tempamine Ameliorate Experimental Autoimmune Encephalomyelitis.
(137) Yang, H.; Rys, A. Z.; McLaughlin, C. K.; Sleiman, H. F. J. Neuroimmunol. 2009, 213, 20−25.
Templated Ligand Environments for the Selective Incorporation of (158) Veiseh, O.; Sun, C.; Fang, C.; Bhattarai, N.; Gunn, J.; Kievit, F.;
Different Metals into DNA. Angew. Chem., Int. Ed. 2009, 48, 9919− Du, K.; Pullar, B.; Lee, D.; Ellenbogen, R. G. Specific Targeting of Brain
9923. Tumors with an Optical/Magnetic Resonance Imaging Nanoprobe
(138) Wen, Y.; McLaughlin, C. K.; Lo, P. K.; Yang, H.; Sleiman, H. F. across the Blood-Brain Barrier. Cancer Res. 2009, 69, 6200−6207.
Stable Gold Nanoparticle Conjugation to Internal DNA Positions: (159) Calvo, P.; Gouritin, B.; Chacun, H.; Desmaële, D.; D’angelo, J.;
Facile Generation of Discrete Gold Nanoparticle-DNA Assemblies. Noel, J.-P.; Georgin, D.; Fattal, E.; Andreux, J. P.; Couvreur, P. Long-
Bioconjugate Chem. 2010, 21, 1413−1416. Circulating Pegylated Polycyanoacrylate Nanoparticles as New Drug
(139) Yang, H.; Altvater, F.; de Bruijn, A. D.; McLaughlin, C. K.; Lo, P. Carrier for Brain Delivery. Pharm. Res. 2001, 18, 1157−1166.
K.; Sleiman, H. F. Chiral Meta-DNA Four-Arm Junctions and Metalated (160) Kreuter, J.; Alyautdin, R. N.; Kharkevich, D. A.; Ivanov, A. A.
Nanotubular Structures. Angew. Chem., Int. Ed. 2011, 50, 4620−4623. Passage of Peptides through the Blood-Brain Barrier with Colloidal
(140) Liu, J.; Cao, Z.; Lu, Y. Functional Nucleic Acid Sensors. Chem. Polymer Particles (Nanoparticles). Brain Res. 1995, 674, 171−174.
Rev. 2009, 109, 1948−1998. (161) Rousselle, C.; Clair, P.; Lefauconnier, J.-M.; Kaczorek, M.;
(141) Chidchob, P.; Edwardson, T. G.; Serpell, C. J.; Sleiman, H. F. Scherrmann, J.-M.; Temsamani, J. New Advances in the Transport of
Synergy of Two Assembly Languages in DNA Nanostructures: Self- Doxorubicin through the Blood-Brain Barrier by a Peptide Vector-
Assembly of Sequence-Defined Polymers on DNA Cages. J. Am. Chem. Mediated Strategy. Mol. Pharmacol. 2000, 57, 679−686.
Soc. 2016, 138, 4416−4425. (162) Lockman, P. R.; Koziara, J. M.; Mumper, R. J.; Allen, D. D.
(142) Li, B. L.; Setyawati, M. I.; Chen, L.; Xie, J.; Ariga, K.; Lim, C.-T.; Nanoparticle Surface Charges Alter Blood-Brain Barrier Integrity and
Garaj, S.; Leong, D. T. Directing Assembly and Disassembly of 2D MoS2 Permeability. J. Drug Target. 2004, 12, 635−641.
Nanosheets with DNA for Drug Delivery. ACS Appl. Mater. Interfaces (163) Conway, J. W.; McLaughlin, C. K.; Castor, K. J.; Sleiman, H.
2017, 9, 15286−15296. DNA Nanostructure Serum Stability: Greater Than the Sum of Its Parts.
(143) Ouyang, X.; Li, J.; Liu, H.; Zhao, B.; Yan, J.; Ma, Y.; Xiao, S.; Chem. Commun. 2013, 49, 1172−1174.
Song, S.; Huang, Q.; Chao, J.; et al. Rolling Circle Amplification-Based (164) Van Kasteren, S. I.; Campbell, S. J.; Serres, S.; Anthony, D. C.;
DNA Origami Nanostructrures for Intracellular Delivery of Immunos- Sibson, N. R.; Davis, B. G. Glyconanoparticles Allow Pre-Symptomatic
timulatory Drugs. Small 2013, 9, 3082−3087. in Vivo Imaging of Brain Disease. Proc. Natl. Acad. Sci. U. S. A. 2009, 106,
(144) Chen, G.; Liu, D.; He, C.; Gannett, T. R.; Lin, W.; Weizmann, Y. 18−23.
Enzymatic Synthesis of Periodic DNA Nanoribbons for Intracellular pH (165) Hengst, V.; Oussoren, C.; Kissel, T.; Storm, G. Bone Targeting
Sensing and Gene Silencing. J. Am. Chem. Soc. 2015, 137, 3844−3851. Potential of Bisphosphonate-Targeted Liposomes: Preparation, Char-
(145) Ducani, C.; Kaul, C.; Moche, M.; Shih, W. M.; Högberg, B. acterization and Hydroxyapatite Binding in Vitro. Int. J. Pharm. 2007,
Enzymatic Production of ’Monoclonal Stoichiometric’ Single-Stranded 331, 224−227.
DNA Oligonucleotides. Nat. Methods 2013, 10, 647−652. (166) Raz, A.; Bucana, C.; Fogler, W. E.; Poste, G.; Fidler, I. J.
(146) Hamblin, G. D.; Carneiro, K. M.; Fakhoury, J. F.; Bujold, K. E.; Biochemical, Morphological, and Ultrastructural Studies on the Uptake
Sleiman, H. F. Rolling Circle Amplification-Templated DNA Nanotubes of Liposomes by Murine Macrophages. Cancer Res. 1981, 41, 487−494.
Show Increased Stability and Cell Penetration Ability. J. Am. Chem. Soc. (167) Hsu, M.; Juliano, R. Interactions of Liposomes with the
2012, 134, 2888−2891. Reticuloendothelial System: II. Nonspecific and Receptor-Mediated
(147) Sun, W.; Jiang, T.; Lu, Y.; Reiff, M.; Mo, R.; Gu, Z. Cocoon-Like Uptake of Liposomes by Mouse Peritoneal Macrophages. Biochim.
Self-Degradable DNA Nanoclew for Anticancer Drug Delivery. J. Am. Biophys. Acta, Mol. Cell Res. 1982, 720, 411−419.
Chem. Soc. 2014, 136, 14722−14725. (168) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.;
(148) Kim, M. G.; Park, J. Y.; Shim, G.; Choi, H. G.; Oh, Y. K. Torchilin, V.; Langer, R. Biodegradable Long-Circulating Polymeric
Biomimetic DNA Nanoballs for Oligonucleotide Delivery. Biomaterials Nanospheres. Science 1994, 263, 1600−1603.
2015, 62, 155−163. (169) Torchilinl, V.; Papisov, M. Why Do Polyethylene Glycol-Coated
(149) Yan, J.; Hu, C.; Wang, P.; Zhao, B.; Ouyang, X.; Zhou, J.; Liu, R.; Liposomes Circulate So Long?: Molecular Mechanism of Liposome
He, D.; Fan, C.; Song, S. Growth and Origami Folding of DNA on Steric Protection with Polyethylene Glycol: Role of Polymer Chain
Nanoparticles for High-Efficiency Molecular Transport in Cellular Flexibility. J. Liposome Res. 1994, 4, 725−739.
Imaging and Drug Delivery. Angew. Chem., Int. Ed. 2015, 54, 2431− (170) Mei, Q.; Wei, X.; Su, F.; Liu, Y.; Youngbull, C.; Johnson, R.;
2435. Lindsay, S.; Yan, H.; Meldrum, D. Stability of DNA Origami Nanoarrays
(150) Schroeder, A.; Heller, D. A.; Winslow, M. M.; Dahlman, J. E.; in Cell Lysate. Nano Lett. 2011, 11, 1477−1482.
Pratt, G. W.; Langer, R.; Jacks, T.; Anderson, D. G. Treating Metastatic (171) Choi, H. S.; Ashitate, Y.; Lee, J. H.; Kim, S. H.; Matsui, A.; Insin,
Cancer with Nanotechnology. Nat. Rev. Cancer 2012, 12, 39. N.; Bawendi, M. G.; Semmler-Behnke, M.; Frangioni, J. V.; Tsuda, A.
(151) Eichler, A. F.; Chung, E.; Kodack, D. P.; Loeffler, J. S.; Fukumura, Rapid Translocation of Nanoparticles from the Lung Airspaces to the
D.; Jain, R. K. The Biology of Brain MetastasesTranslation to New Body. Nat. Biotechnol. 2010, 28, 1300−1303.
Therapies. Nat. Rev. Clin. Oncol. 2011, 8, 344−356. (172) Allen, T.; Hansen, C.; Guo, L. Subcutaneous Administration of
(152) Lesniak, M. S.; Brem, H. Targeted Therapy for Brain Tumours. Liposomes: A Comparison with the Intravenous and Intraperitoneal
Nat. Rev. Drug Discovery 2004, 3, 499. Routes of Injection. Biochim. Biophys. Acta, Biomembr. 1993, 1150, 9−
(153) Sharp, P. A.; Langer, R. Promoting Convergence in Biomedical 16.
Science. Science 2011, 333, 527−527. (173) Reddy, S. T.; Van Der Vlies, A. J.; Simeoni, E.; Angeli, V.;
(154) Jain, R. K. Physiological Barriers to Delivery of Monoclonal Randolph, G. J.; O’Neil, C. P.; Lee, L. K.; Swartz, M. A.; Hubbell, J. A.
Antibodies and Other Macromolecules in Tumors. Cancer Res. 1990, 50, Exploiting Lymphatic Transport and Complement Activation in
814s−819s. Nanoparticle Vaccines. Nat. Biotechnol. 2007, 25, 1159−1164.
6499 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
(174) Sadauskas, E.; Wallin, H.; Stoltenberg, M.; Vogel, U.; Doering, tures for Noninvasive Intracellular Delivery of Immunostimulatory CpG
P.; Larsen, A.; Danscher, G. Kupffer Cells Are Central in the Removal of Oligonucleotides. ACS Nano 2011, 5, 8783−8789.
Nanoparticles from the Organism. Part. Fibre Toxicol. 2007, 4, 10. (198) Walsh, A. S.; Yin, H.; Erben, C. M.; Wood, M. J. A.; Turberfield,
(175) Akinc, A.; Zumbuehl, A.; Goldberg, M.; Leshchiner, E. S.; Busini, A. J. DNA Cage Delivery to Mammalian Cells. ACS Nano 2011, 5,
V.; Hossain, N.; Bacallado, S. A.; Nguyen, D. N.; Fuller, J.; Alvarez, R. A 5427−5432.
Combinatorial Library of Lipid-Like Materials for Delivery of RNAi (199) Liang, L.; Li, J.; Li, Q.; Huang, Q.; Shi, J.; Yan, H.; Fan, C. Single-
Therapeutics. Nat. Biotechnol. 2008, 26, 561. Particle Tracking and Modulation of Cell Entry Pathways of a
(176) Edwards, D. A.; Hanes, J.; Caponetti, G.; Hrkach, J.; Ben-Jebria, Tetrahedral DNA Nanostructure in Live Cells. Angew. Chem., Int. Ed.
A.; Eskew, M. L.; Mintzes, J.; Deaver, D.; Lotan, N.; Langer, R. Large 2014, 53, 7745−7750.
Porous Particles for Pulmonary Drug Delivery. Science 1997, 276, 1868− (200) Choi, C. H.; Hao, L.; Narayan, S. P.; Auyeung, E.; Mirkin, C. A.
1872. Mechanism for the Endocytosis of Spherical Nucleic Acid Nanoparticle
(177) Azarmi, S.; Roa, W. H.; Löbenberg, R. Targeted Delivery of Conjugates. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 7625−7630.
Nanoparticles for the Treatment of Lung Diseases. Adv. Drug Delivery (201) Cutler, J. I.; Auyeung, E.; Mirkin, C. A. Spherical Nucleic Acids. J.
Rev. 2008, 60, 863−875. Am. Chem. Soc. 2012, 134, 1376−1391.
(178) Polach, K. J.; Matar, M.; Rice, J.; Slobodkin, G.; Sparks, J.; (202) Narayan, S. P.; Choi, C. H. J.; Hao, L.; Calabrese, C. M.;
Congo, R.; Rea-Ramsey, A.; McClure, D.; Brunhoeber, E.; Krampert, M. Auyeung, E.; Zhang, C.; Goor, O. J.; Mirkin, C. A. The Sequence-
Delivery of siRNA to the Mouse Lung Via a Functionalized Specific Cellular Uptake of Spherical Nucleic Acid Nanoparticle
Lipopolyamine. Mol. Ther. 2012, 20, 91−100. Conjugates. Small 2015, 11, 4173−4182.
(179) Iinuma, R.; Ke, Y.; Jungmann, R.; Schlichthaerle, T.; Woehrstein, (203) Lim, K. S.; Lee, D. Y.; Valencia, G. M.; Won, Y. W.; Bull, D. A.
J. B.; Yin, P. Polyhedra Self-Assembled from DNA Tripods and Nano-Self-Assembly of Nucleic Acids Capable of Transfection without a
Characterized with 3D DNA-Paint. Science 2014, 344, 65−69. Gene Carrier. Adv. Funct. Mater. 2015, 25, 5445−5451.
(180) Perrault, S. D.; Shih, W. M. Virus-Inspired Membrane (204) Zhang, Y.; Ali, S. F.; Dervishi, E.; Xu, Y.; Li, Z.; Casciano, D.;
Encapsulation of DNA Nanostructures to Achieve in Vivo Stability. Biris, A. S. Cytotoxicity Effects of Graphene and Single-Wall Carbon
ACS Nano 2014, 8, 5132−5140. Nanotubes in Neural Phaeochromocytoma-Derived PC12 Cells. ACS
(181) Yang, Y.; Wang, J.; Shigematsu, H.; Xu, W.; Shih, W. M.; Nano 2010, 4, 3181−3186.
Rothman, J. E.; Lin, C. Self-Assembly of Size-Controlled Liposomes on (205) Longmire, M. R.; Ogawa, M.; Choyke, P. L.; Kobayashi, H.
DNA Nanotemplates. Nat. Chem. 2016, 8, 476. Biologically Optimized Nanosized Molecules and Particles: More Than
(182) Voigt, J.; Christensen, J.; Shastri, V. P. Differential Uptake of Just Size. Bioconjugate Chem. 2011, 22, 993−1000.
Nanoparticles by Endothelial Cells through Polyelectrolytes with (206) Owens, D. E., III; Peppas, N. A. Opsonization, Biodistribution,
Affinity for Caveolae. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 2942− and Pharmacokinetics of Polymeric Nanoparticles. Int. J. Pharm. 2006,
2947. 307, 93−102.
(183) Yu, G.; Zhou, J.; Shen, J.; Tang, G.; Huang, F. Cationic Pillar [6] (207) He, C.; Hu, Y.; Yin, L.; Tang, C.; Yin, C. Effects of Particle Size
Arene/ATP Host-Guest Recognition: Selectivity, Inhibition of ATP and Surface Charge on Cellular Uptake and Biodistribution of Polymeric
Hydrolysis, and Application in Multidrug Resistance Treatment. Chem.
Nanoparticles. Biomaterials 2010, 31, 3657−3666.
Sci. 2016, 7, 4073−4078.
(208) Chou, L. Y. T.; Zagorovsky, K.; Chan, W. C. W. DNA Assembly
(184) Lee, A. G. Endocytosis and Exocytosis; Elsevier: Richmond, VA,
of Nanoparticle Superstructures for Controlled Biological Delivery and
1996.
Elimination. Nat. Nanotechnol. 2014, 9, 148−155.
(185) Bareford, L. M.; Swaan, P. W. Endocytic Mechanisms for
(209) Ma, Y.; Nolte, R. J. M.; Cornelissen, J. J. L. M. Virus-Based
Targeted Drug Delivery. Adv. Drug Delivery Rev. 2007, 59, 748−758.
Nanocarriers for Drug Delivery. Adv. Drug Delivery Rev. 2012, 64, 811−
(186) Oh, N.; Park, J.-H. Endocytosis and Exocytosis of Nanoparticles
825.
in Mammalian Cells. Int. J. Nanomed. 2014, 9, 51−63.
(210) Kostiainen, M. A.; Hiekkataipale, P.; Laiho, A.; Lemieux, V.;
(187) Aderem, A.; Underhill, D. M. Mechanisms of Phagocytosis in
Macrophages. Annu. Rev. Immunol. 1999, 17, 593−623. Seitsonen, J.; Ruokolainen, J.; Ceci, P. Electrostatic Assembly of Binary
(188) Fernández, A.; Vendrell, M. Smart Fluorescent Probes for Nanoparticle Superlattices Using Protein Cages. Nat. Nanotechnol.
Imaging Macrophage Activity. Chem. Soc. Rev. 2016, 45, 1182−1196. 2013, 8, 52−56.
(189) McMahon, H. T.; Boucrot, E. Molecular Mechanism and (211) Mukherjee, S.; Pfeifer, C. M.; Johnson, J. M.; Liu, J.; Zlotnick, A.
Physiological Functions of Clathrin-Mediated Endocytosis. Nat. Rev. Redirecting the Coat Protein of a Spherical Virus to Assemble into
Mol. Cell Biol. 2011, 12, 517−533. Tubular Nanostructures. J. Am. Chem. Soc. 2006, 128, 2538−2539.
(190) Banerjee, A.; Berezhkovskii, A.; Nossal, R. Kinetics of Cellular (212) Mikkilä, J.; Eskelinen, A. P.; Niemelä, E. H.; Linko, V.; Frilander,
Uptake of Viruses and Nanoparticles Via Clathrin-Mediated Endocy- M. J.; Törmä, P.; Kostiainen, M. A. Virus-Encapsulated DNA Origami
tosis. Phys. Biol. 2016, 13, 016005. Nanostructures for Cellular Delivery. Nano Lett. 2014, 14, 2196−2200.
(191) Strømhaug, P.; Berg, T.; Gjøen, T.; Seglen, P. Differences (213) Bujold, K. E.; Fakhoury, J.; Edwardson, T. G. W.; Carneiro, K. M.
between Fluid-Phase Endocytosis (Pinocytosis) and Receptor-Medi- M.; Briard, J. N.; Godin, A. G.; Amrein, L.; Hamblin, G. D.; Panasci, L.
ated Endocytosis in Isolated Rat Hepatocytes. Eur. J. Cell Biol. 1997, 73, C.; Wiseman, P. W.; et al. Sequence-Responsive Unzipping DNA Cubes
28−39. with Tunable Cellular Uptake Profiles. Chem. Sci. 2014, 5, 2449−2455.
(192) Pelkmans, L.; Helenius, A. Endocytosis Via Caveolae. Traffic (214) Sudimack, J.; Lee, R. J. Targeted Drug Delivery Via the Folate
2002, 3, 311−320. Receptor. Adv. Drug Delivery Rev. 2000, 41, 147−162.
(193) Nabi, I. R.; Le, P. U. Caveolae/Raft-Dependent Endocytosis. J. (215) Ko, S.; Liu, H.; Chen, Y.; Mao, C. DNA Nanotubes as
Cell Biol. 2003, 161, 673−677. Combinatorial Vehicles for Cellular Delivery. Biomacromolecules 2008,
(194) Peters, P. J.; Mironov, A.; Peretz, D.; van Donselaar, E.; Leclerc, 9, 3039−3043.
E.; Erpel, S.; DeArmond, S. J.; Burton, D. R.; Williamson, R. A.; Vey, M. (216) Lee, H.; Lytton-Jean, A. K.; Chen, Y.; Love, K. T.; Park, A. I.;
Trafficking of Prion Proteins through a Caveolae-Mediated Endosomal Karagiannis, E. D.; Sehgal, A.; Querbes, W.; Zurenko, C. S.; Jayaraman,
Pathway. J. Cell Biol. 2003, 162, 703−717. M.; et al. Molecularly Self-Assembled Nucleic Acid Nanoparticles for
(195) Parton, R. G.; Simons, K. The Multiple Faces of Caveolae. Nat. Targeted in Vivo siRNA Delivery. Nat. Nanotechnol. 2012, 7, 389−393.
Rev. Mol. Cell Biol. 2007, 8, 185−194. (217) Shao, Z.; Shao, J.; Tan, B.; Guan, S.; Liu, Z.; Zhao, Z.; He, F.;
(196) Doherty, G. J.; McMahon, H. T. Mechanisms of Endocytosis. Zhao, J. Targeted Lung Cancer Therapy: Preparation and Optimization
Annu. Rev. Biochem. 2009, 78, 857−902. of Transferrin-Decorated Nanostructured Lipid Carriers as Novel
(197) Li, J.; Pei, H.; Zhu, B.; Liang, L.; Wei, M.; He, Y.; Chen, N.; Li, Nanomedicine for Co-Delivery of Anticancer Drugs and DNA. Int. J.
D.; Huang, Q.; Fan, C. Self-Assembled Multivalent DNA Nanostruc- Nanomed. 2015, 10, 1223−1233.
6500 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
(218) Teesalu, T.; Sugahara, K. N.; Kotamraju, V. R.; Ruoslahti, E. C- (237) Kozlov, M. M.; McMahon, H. T.; Chernomordik, L. V. Protein-
End Rule Peptides Mediate Neuropilin-1-Dependent Cell, Vascular, and Driven Membrane Stresses in Fusion and Fission. Trends Biochem. Sci.
Tissue Penetration. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 16157− 2010, 35, 699−706.
16162. (238) Frolov, V. A.; Shnyrova, A. V.; Zimmerberg, J. Lipid
(219) Haspel, N.; Zanuy, D.; Nussinov, R.; Teesalu, T.; Ruoslahti, E.; Polymorphisms and Membrane Shape. Cold Spring Harbor Perspect.
Aleman, C. Binding of a C-End Rule Peptide to the Neuropilin-1 Biol. 2011, 3, a004747.
Receptor: A Molecular Modeling Approach. Biochemistry 2011, 50, (239) Xu, W.; Wang, J.; Rothman, J. E.; Pincet, F. Accelerating
1755−1762. SNARE-Mediated Membrane Fusion by DNA-Lipid Tethers. Angew.
(220) Gribova, V.; Gauthier-Rouviere, C.; Albiges-Rizo, C.; Auzely- Chem., Int. Ed. 2015, 54, 14388−14392.
Velty, R.; Picart, C. Effect of Rgd Functionalization and Stiffness (240) Xu, W.; Nathwani, B.; Lin, C.; Wang, J.; Karatekin, E.; Pincet, F.;
Modulation of Polyelectrolyte Multilayer Films on Muscle Cell Shih, W.; Rothman, J. E. A Programmable DNA Origami Platform to
Differentiation. Acta Biomater. 2013, 9, 6468−6480. Organize SNAREs for Membrane Fusion. J. Am. Chem. Soc. 2016, 138,
(221) Nasarre, C.; Roth, M.; Jacob, L.; Roth, L.; Koncina, E.; Thien, A.; 4439−4447.
Labourdette, G.; Poulet, P.; Hubert, P.; Cremel, G.; et al. Peptide-Based (241) Niemeyer, C. M.; Sano, T.; Smith, C. L.; Cantor, C. R.
Interference of the Transmembrane Domain of Neuropilin-1 Inhibits Oligonucleotide-Directed Self-Assembly of Proteins: Semisynthetic
Glioma Growth in Vivo. Oncogene 2010, 29, 2381−2392. DNA–Streptavidin Hybrid Molecules as Connectors for the Generation
(222) Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; of Macroscopic Arrays and the Construction of Supramolecular
Agemy, L.; Greenwald, D. R.; Ruoslahti, E. Coadministration of a Bioconjugates. Nucleic Acids Res. 1994, 22, 5530−5539.
Tumor-Penetrating Peptide Enhances the Efficacy of Cancer Drugs. (242) Rosen, C. B.; Kodal, A. L. B.; Nielsen, J. S.; Schaffert, D. H.;
Science 2010, 328, 1031−1035. Scavenius, C.; Okholm, A. H.; Voigt, N. V.; Enghild, J. J.; Kjems, J.;
(223) Yang, Y.; Yan, Z.; Wei, D.; Zhong, J.; Liu, L.; Zhang, L.; Wang, F.; Tørring, T.; et al. Template-Directed Covalent Conjugation of DNA to
Wei, X.; Xie, C.; Lu, W.; et al. Tumor-Penetrating Peptide Native Antibodies, Transferrin and Other Metal-Binding Proteins. Nat.
Functionalization Enhances the Anti-Glioblastoma Effect of Doxor- Chem. 2014, 6, 804−809.
ubicin Liposomes. Nanotechnology 2013, 24, 405101. (243) Modi, S.; Nizak, C.; Surana, S.; Halder, S.; Krishnan, Y. Two
(224) Yan, Z.; Yang, Y.; Wei, X.; Zhong, J.; Wei, D.; Liu, L.; Xie, C.; DNA Nanomachines Map pH Changes Along Intersecting Endocytic
Wang, F.; Zhang, L.; Lu, W.; et al. Tumor-Penetrating Peptide Pathways inside the Same Cell. Nat. Nanotechnol. 2013, 8, 459−467.
Mediation: An Effective Strategy for Improving the Transport of (244) Schaffert, D. H.; Okholm, A. H.; Sørensen, R. S.; Nielsen, J. S.;
Liposomes in Tumor Tissue. Mol. Pharmaceutics 2014, 11, 218−225. Tørring, T.; Rosen, C. B.; Kodal, A. L. B.; Mortensen, M. R.; Gothelf, K.
(225) Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; V.; Kjems, J. Intracellular Delivery of a Planar DNA Origami Structure
Agemy, L.; Girard, O. M.; Hanahan, D.; Mattrey, R. F.; Ruoslahti, E. by the Transferrin-Receptor Internalization Pathway. Small 2016, 12,
2634−2640.
Tissue-Penetrating Delivery of Compounds and Nanoparticles into
(245) Sun, P.; Zhang, N.; Tang, Y.; Yang, Y.; Chu, X.; Zhao, Y. SL2B
Tumors. Cancer Cell 2009, 16, 510−520.
Aptamer and Folic Acid Dual-Targeting DNA Nanostructures for
(226) Xia, Z.; Wang, P.; Liu, X.; Liu, T.; Yan, Y.; Yan, J.; Zhong, J.; Sun,
Synergic Biological Effect with Chemotherapy to Combat Colorectal
G.; He, D. Tumor-Penetrating Peptide-Modified DNA Tetrahedron for
Cancer. Int. J. Nanomed. 2017, 12, 2657−2672.
Targeting Drug Delivery. Biochemistry 2016, 55, 1326−1331.
(246) Song, L.; Hobaugh, M. R.; Shustak, C.; Cheley, S.; Bayley, H.;
(227) Keefe, A. D.; Pai, S.; Ellington, A. Aptamers as Therapeutics. Nat.
Gouaux, J. E. Structure of Staphylococcal α-Hemolysin, a Heptameric
Rev. Drug Discovery 2010, 9, 537−550.
Transmembrane Pore. Science 1996, 274, 1859−1865.
(228) Iliuk, A. B.; Hu, L.; Tao, W. A. Aptamer in Bioanalytical
(247) Langecker, M.; Arnaut, V.; Martin, T. G.; List, J.; Renner, S.;
Applications. Anal. Chem. 2011, 83, 4440−4452. Mayer, M.; Dietz, H.; Simmel, F. C. Synthetic Lipid Membrane
(229) Meng, H.-M.; Zhang, X.; Lv, Y.; Zhao, Z.; Wang, N.-N.; Fu, T.;
Channels Formed by Designed DNA Nanostructures. Science 2012, 338,
Fan, H.; Liang, H.; Qiu, L.; Zhu, G. DNA Dendrimer: An Efficient 932−936.
Nanocarrier of Functional Nucleic Acids for Intracellular Molecular (248) Burns, J. R.; Stulz, E.; Howorka, S. Self-Assembled DNA
Sensing. ACS Nano 2014, 8, 6171−6181. Nanopores That Span Lipid Bilayers. Nano Lett. 2013, 13, 2351−2356.
(230) Wang, Y.-M.; Wu, Z.; Liu, S.-J.; Chu, X. Structure-Switching (249) Burns, J. R.; Gopfrich, K.; Wood, J. W.; Thacker, V. V.; Stulz, E.;
Aptamer Triggering Hybridization Chain Reaction on the Cell Surface Keyser, U. F.; Howorka, S. Lipid-Bilayer-Spanning DNA Nanopores
for Activatable Theranostics. Anal. Chem. 2015, 87, 6470−6474. with a Bifunctional Porphyrin Anchor. Angew. Chem., Int. Ed. 2013, 52,
(231) Hu, R.; Zhang, X.; Zhao, Z.; Zhu, G.; Chen, T.; Fu, T.; Tan, W. 12069−12072.
DNA Nanoflowers for Multiplexed Cellular Imaging and Traceable (250) Burns, J. R.; Seifert, A.; Fertig, N.; Howorka, S. A Biomimetic
Targeted Drug Delivery. Angew. Chem., Int. Ed. 2014, 53, 5821−5826. DNA-Based Channel for the Ligand-Controlled Transport of Charged
(232) Xie, N.; Huang, J.; Yang, X.; Yang, Y.; Quan, K.; Ou, M.; Fang, Molecular Cargo across a Biological Membrane. Nat. Nanotechnol. 2016,
H.; Wang, K. Competition-Mediated FRET-Switching DNA Tetrahe- 11, 152−156.
dron Molecular Beacon for Intracellular Molecular Detection. ACS (251) Rajendran, L.; Knölker, H.-J.; Simons, K. Subcellular Targeting
Sensors 2016, 1, 1445−1452. Strategies for Drug Design and Delivery. Nat. Rev. Drug Discovery 2010,
(233) Walter, H.-K.; Bauer, J.; Steinmeyer, J.; Kuzuya, A.; Niemeyer, C. 9, 29−42.
M.; Wagenknecht, H.-A. DNA Origami Traffic Lights” with a Split (252) Schroeder, A.; Levins, C. G.; Cortez, C.; Langer, R.; Anderson,
Aptamer Sensor for a Bicolor Fluorescence Readout. Nano Lett. 2017, D. G. Lipid-Based Nanotherapeutics for siRNA Delivery. J. Intern. Med.
17, 2467−2472. 2010, 267, 9−21.
(234) Zhu, G.; Zheng, J.; Song, E.; Donovan, M.; Zhang, K.; Liu, C.; (253) Goldberg, M.; Langer, R.; Jia, X. Nanostructured Materials for
Tan, W. Self-Assembled, Aptamer-Tethered DNA Nanotrains for Applications in Drug Delivery and Tissue Engineering. J. Biomater. Sci.,
Targeted Transport of Molecular Drugs in Cancer Theranostics. Proc. Polym. Ed. 2007, 18, 241−268.
Natl. Acad. Sci. U. S. A. 2013, 110, 7998−8003. (254) Bonifacino, J. S.; Rojas, R. Retrograde Transport from
(235) Li, J.; Zheng, C.; Cansiz, S.; Wu, C.; Xu, J.; Cui, C.; Liu, Y.; Hou, Endosomes to the Trans-Golgi Network. Nat. Rev. Mol. Cell Biol.
W.; Wang, Y.; Zhang, L.; et al. Self-Assembly of DNA Nanohydrogels 2006, 7, 568−579.
with Controllable Size and Stimuli-Responsive Property for Targeted (255) Thomas, G. Furin at the Cutting Edge: From Protein Traffic to
Gene Regulation Therapy. J. Am. Chem. Soc. 2015, 137, 1412−1415. Embryogenesis and Disease. Nat. Rev. Mol. Cell Biol. 2002, 3, 753−766.
(236) Graham, T. R.; Kozlov, M. M. Interplay of Proteins and Lipids in (256) McCafferty, J.; Griffiths, A. D.; Winter, G.; Chiswell, D. J. Phage
Generating Membrane Curvature. Curr. Opin. Cell Biol. 2010, 22, 430− Antibodies: Filamentous Phage Displaying Antibody Variable Domains.
436. Nature 1990, 348, 552−554.
6501 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
(257) Shen, X.; Jiang, Q.; Wang, J.; Dai, L.; Zou, G.; Wang, Z.-G.; Immunostimulation by CpG-Sequence-Coated DNA Origami Struc-
Chen, W.-Q.; Jiang, W.; Ding, B. Visualization of the Intracellular tures. ACS Nano 2011, 5, 9696−9702.
Location and Stability of DNA Origami with a Label-Free Fluorescent (277) Zhang, L.; Zhu, G.; Mei, L.; Wu, C.; Qiu, L.; Cui, C.; Liu, Y.;
Probe. Chem. Commun. 2012, 48, 11301−11303. Teng, I. T.; Tan, W. Self-Assembled DNA Immunonanoflowers as
(258) Carvalho, C.; Santos, R. X.; Cardoso, S.; Correia, S.; Oliveira, P. Multivalent CpG Nanoagents. ACS Appl. Mater. Interfaces 2015, 7,
J.; Santos, M. S.; Moreira, P. I. Doxorubicin: The Good, the Bad and the 24069−24074.
Ugly Effect. Curr. Med. Chem. 2009, 16, 3267−3285. (278) Wei, M.; Chen, N.; Li, J.; Yin, M.; Liang, L.; He, Y.; Song, H.;
(259) Tacar, O.; Sriamornsak, P.; Dass, C. R. Doxorubicin: An Update Fan, C.; Huang, Q. Polyvalent Immunostimulatory Nanoagents with
on Anticancer Molecular Action, Toxicity and Novel Drug Delivery Self-Assembled CpG Oligonucleotide-Conjugated Gold Nanoparticles.
Systems. J. Pharm. Pharmacol. 2013, 65, 157−170. Angew. Chem., Int. Ed. 2012, 51, 1202−1206.
(260) Yang, T.; Li, B.; Qi, S.; Liu, Y.; Gai, Y.; Ye, P.; Yang, G.; Zhang, (279) Chen, N.; Wei, M.; Sun, Y.; Li, F.; Pei, H.; Li, X.; Su, S.; He, Y.;
W.; Zhang, P.; He, X. Co-Delivery of Doxorubicin and Bmi1 siRNA by Wang, L.; Shi, J.; et al. Self-Assembly of Poly-Adenine-Tailed CpG
Folate Receptor Targeted Liposomes Exhibits Enhanced Anti-Tumor Oligonucleotide-Gold Nanoparticle Nanoconjugates with Immunosti-
Effects in Vitro and in Vivo. Theranostics 2014, 4, 1096−1111. mulatory Activity. Small 2014, 10, 368−375.
(261) Unsoy, G.; Khodadust, R.; Yalcin, S.; Mutlu, P.; Gunduz, U. (280) Mohri, K.; Kusuki, E.; Ohtsuki, S.; Takahashi, N.; Endo, M.;
Synthesis of Doxorubicin Loaded Magnetic Chitosan Nanoparticles for Hidaka, K.; Sugiyama, H.; Takahashi, Y.; Takakura, Y.; Nishikawa, M.
pH Responsive Targeted Drug Delivery. Eur. J. Pharm. Sci. 2014, 62, Self-Assembling DNA Dendrimer for Effective Delivery of Immunos-
243−250. timulatory CpG DNA to Immune Cells. Biomacromolecules 2015, 16,
(262) Ke, X.-Y.; Lin Ng, V. W.; Gao, S.-J.; Tong, Y. W.; Hedrick, J. L.; 1095−1101.
Yang, Y. Y. Co-Delivery of Thioridazine and Doxorubicin Using (281) Qu, Y.; Yang, J.; Zhan, P.; Liu, S.; Zhang, K.; Jiang, Q.; Li, C.;
Polymeric Micelles for Targeting Both Cancer Cells and Cancer Stem Ding, B. Self-Assembled DNA Dendrimer Nanoparticle for Efficient
Cells. Biomaterials 2014, 35, 1096−1108. Delivery of Immunostimulatory CpG Motifs. ACS Appl. Mater. Interfaces
(263) Zhao, Y.-X.; Shaw, A.; Zeng, X.; Bensön, E.; Nystrom, A. M.; 2017, 9, 20324−20329.
Högberg, B. R. DNA Origami Delivery System for Cancer Therapy with (282) Paik, S.; Shak, S.; Tang, G.; Kim, C.; Baker, J.; Cronin, M.;
Tunable Release Properties. ACS Nano 2012, 6, 8684−8691. Baehner, F. L.; Walker, M. G.; Watson, D.; Park, T.; et al. A Multigene
(264) Jiang, Q.; Song, C.; Nangreave, J.; Liu, X.; Lin, L.; Qiu, D.; Wang, Assay to Predict Recurrence of Tamoxifen-Treated, Node-Negative
Z. G.; Zou, G.; Liang, X.; Yan, H.; et al. DNA Origami as a Carrier for Breast Cancer. N. Engl. J. Med. 2004, 351, 2817−2826.
Circumvention of Drug Resistance. J. Am. Chem. Soc. 2012, 134, 13396− (283) Turajlic, S.; Swanton, C. Metastasis as an Evolutionary Process.
13403. Science 2016, 352, 169−175.
(265) Zhang, Q.; Jiang, Q.; Li, N.; Dai, L.; Liu, Q.; Song, L.; Wang, J.; (284) Grivennikov, S. I.; Greten, F. R.; Karin, M. Immunity,
Li, Y.; Tian, J.; Ding, B. DNA Origami as an in Vivo Drug Delivery Inflammation, and Cancer. Cell 2010, 140, 883−899.
Vehicle for Cancer Therapy. ACS Nano 2014, 8, 6633−6643. (285) Stephan, S. B.; Taber, A. M.; Jileaeva, I.; Pegues, E. P.; Sentman,
(266) Wu, C.; Han, D.; Chen, T.; Peng, L.; Zhu, G.; You, M.; Qiu, L.; C. L.; Stephan, M. T. Biopolymer Implants Enhance the Efficacy of
Sefah, K.; Zhang, X.; Tan, W. Building a Multifunctional Aptamer-Based Adoptive T-Cell Therapy. Nat. Biotechnol. 2015, 33, 97−101.
DNA Nanoassembly for Targeted Cancer Therapy. J. Am. Chem. Soc. (286) Wang, C.; Sun, W.; Wright, G.; Wang, A. Z.; Gu, Z.
2013, 135, 18644−18650. Inflammation-Triggered Cancer Immunotherapy by Programmed
(267) Zhang, H.; Ma, Y.; Xie, Y.; An, Y.; Huang, Y.; Zhu, Z.; Yang, C. J. Delivery of CpG and Anti-PD1 Antibody. Adv. Mater. 2016, 28,
A Controllable Aptamer-Based Self-Assembled DNA Dendrimer for 8912−8920.
High Affinity Targeting, Bioimaging and Drug Delivery. Sci. Rep. 2015, (287) Gajanayake, T.; Olariu, R.; Leclère, F. M.; Dhayani, A.; Yang, Z.;
5, 10099. Bongoni, A. K.; Banz, Y.; Constantinescu, M. A.; Karp, J. M.; Vemula, P.
(268) Mei, L.; Zhu, G.; Qiu, L.; Wu, C.; Chen, H.; Liang, H.; Cansiz, S.; K. A Single Localized Dose of Enzyme-Responsive Hydrogel Improves
Lv, Y.; Zhang, X.; Tan, W. Self-Assembled Multifunctional DNA Long-Term Survival of a Vascularized Composite Allograft. Sci. Transl.
Nanoflowers for the Circumvention of Multidrug Resistance in Med. 2014, 6, 249ra110.
Targeted Anticancer Drug Delivery. Nano Res. 2015, 8, 3447−3460. (288) Hannon, G. J. RNA Interference. Nature 2002, 418, 244−251.
(269) Krieg, A. M.; Yi, A.-K.; Matson, S.; Waldschmidt, T. J. CpG (289) Bernstein, E.; Caudy, A. A.; Hammond, S. M.; Hannon, G. J.
Motifs in Bacterial DNA Trigger Direct B-Cell Activation. Nature 1995, Role for a Bidentate Ribonuclease in the Initiation Step of RNA
374, 546−549. Interference. Nature 2001, 409, 363−366.
(270) Kanzler, H.; Barrat, F. J.; Hessel, E. M.; Coffman, R. L. (290) Reynolds, A.; Leake, D.; Boese, Q.; Scaringe, S.; Marshall, W. S.;
Therapeutic Targeting of Innate Immunity with Toll-Like Receptor Khvorova, A. Rational siRNA Design for RNA Interference. Nat.
Agonists and Antagonists. Nat. Med. 2007, 13, 552−559. Biotechnol. 2004, 22, 326−330.
(271) Klinman, D. M. Immunotherapeutic Uses of CpG Oligodeox- (291) Whitehead, K. A.; Langer, R.; Anderson, D. G. Knocking Down
ynucleotides. Nat. Rev. Immunol. 2004, 4, 249−259. Barriers: Advances in siRNA Delivery. Nat. Rev. Drug Discovery 2009, 8,
(272) Heidegger, S.; Gößl, D.; Schmidt, A.; Niedermayer, S.; Argyo, C.; 129−138.
Endres, S.; Bein, T.; Bourquin, C. Immune Response to Functionalized (292) Lee, J.-S.; Green, J. J.; Love, K. T.; Sunshine, J.; Langer, R.;
Mesoporous Silica Nanoparticles for Targeted Drug Delivery. Nanoscale Anderson, D. G. Gold, Poly (B-Amino Ester) Nanoparticles for Small
2016, 8, 938−948. Interfering RNA Delivery. Nano Lett. 2009, 9, 2402−2406.
(273) Morishita, M.; Takahashi, Y.; Matsumoto, A.; Nishikawa, M.; (293) Davis, M. E.; Zuckerman, J. E.; Choi, C. H. J.; Seligson, D.;
Takakura, Y. Exosome-Based Tumor Antigens-Adjuvant Co-Delivery Tolcher, A.; Alabi, C. A.; Yen, Y.; Heidel, J. D.; Ribas, A. Evidence of
Utilizing Genetically Engineered Tumor Cell-Derived Exosomes with RNAi in Humans from Systemically Administered siRNA Via Targeted
Immunostimulatory CpG DNA. Biomaterials 2016, 111, 55−65. Nanoparticles. Nature 2010, 464, 1067.
(274) Mutwiri, G. K.; Nichani, A. K.; Babiuk, S.; Babiuk, L. A. Strategies (294) Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.; Cho,
for Enhancing the Immunostimulatory Effects of CpG Oligodeox- C. K.; Sah, D. W.; Stebbing, D.; Crosley, E. J.; Yaworski, E. Rational
ynucleotides. J. Controlled Release 2004, 97, 1−17. Design of Cationic Lipids for siRNA Delivery. Nat. Biotechnol. 2010, 28,
(275) Heikenwalder, M.; Polymenidou, M.; Junt, T.; Sigurdson, C.; 172−176.
Wagner, H.; Akira, S.; Zinkernagel, R.; Aguzzi, A. Lymphoid Follicle (295) Oh, Y.-K.; Park, T. G. siRNA Delivery Systems for Cancer
Destruction and Immunosuppression after Repeated CpG Oligodeox- Treatment. Adv. Drug Delivery Rev. 2009, 61, 850−862.
ynucleotide Administration. Nat. Med. 2004, 10, 187. (296) Fakhoury, J. J.; McLaughlin, C. K.; Edwardson, T. W.; Conway, J.
(276) Schüller, V. J.; Heidegger, S.; Sandholzer, N.; Nickels, P. C.; W.; Sleiman, H. F. Development and Characterization of Gene Silencing
Suhartha, N. A.; Endres, S.; Bourquin, C.; Liedl, T. Cellular DNA Cages. Biomacromolecules 2014, 15, 276−282.
6502 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
(297) Bujold, K. E.; Hsu, J. C.; Sleiman, H. F. Optimized DNA Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.
″Nanosuitcases″ for Encapsulation and Conditional Release of siRNA. J. Science 2014, 343, 84−87.
Am. Chem. Soc. 2016, 138, 14030−14038. (318) Doudna, J. A.; Charpentier, E. The New Frontier of Genome
(298) Jensen, S. A.; Day, E. S.; Ko, C. H.; Hurley, L. A.; Luciano, J. P.; Engineering with CRISPR-Cas9. Science 2014, 346, 1258096.
Kouri, F. M.; Merkel, T. J.; Luthi, A. J.; Patel, P. C.; Cutler, J. I. Spherical (319) Ran, F. A.; Cong, L.; Yan, W. X.; Scott, D. A.; Gootenberg, J. S.;
Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Kriz, A. J.; Zetsche, B.; Shalem, O.; Wu, X.; Makarova, K. S. In Vivo
Glioblastoma. Sci. Transl. Med. 2013, 5, 209ra152. Genome Editing Using Staphylococcus Aureus Cas9. Nature 2015, 520,
(299) Dunn, G. P.; Rinne, M. L.; Wykosky, J.; Genovese, G.; Quayle, S. 186−191.
N.; Dunn, I. F.; Agarwalla, P. K.; Chheda, M. G.; Campos, B.; Wang, A. (320) Platt, R. J.; Chen, S.; Zhou, Y.; Yim, M. J.; Swiech, L.; Kempton,
Emerging Insights into the Molecular and Cellular Basis of H. R.; Dahlman, J. E.; Parnas, O.; Eisenhaure, T. M.; Jovanovic, M.
Glioblastoma. Genes Dev. 2012, 26, 756−784. CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer
(300) Furnari, F. B.; Fenton, T.; Bachoo, R. M.; Mukasa, A.; Stommel, Modeling. Cell 2014, 159, 440−455.
J. M.; Stegh, A.; Hahn, W. C.; Ligon, K. L.; Louis, D. N.; Brennan, C. (321) Kotterman, M. A.; Schaffer, D. V. Engineering Adeno-Associated
Malignant Astrocytic Glioma: Genetics, Biology, and Paths to Viruses for Clinical Gene Therapy. Nat. Rev. Genet. 2014, 15, 445−451.
Treatment. Genes Dev. 2007, 21, 2683−2710. (322) Pattanayak, V.; Lin, S.; Guilinger, J. P.; Ma, E.; Doudna, J. A.; Liu,
(301) Wen, P. Y.; Kesari, S. Malignant Gliomas in Adults. N. Engl. J. D. R. High-Throughput Profiling of Off-Target DNA Cleavage Reveals
Med. 2008, 359, 492−507. RNA-Programmed Cas9 Nuclease Specificity. Nat. Biotechnol. 2013, 31,
(302) Lee, J. B.; Hong, J.; Bonner, D. K.; Poon, Z.; Hammond, P. T. 839−843.
Self-Assembled RNA Interference Microsponges for Efficient siRNA (323) Fu, Y.; Foden, J. A.; Khayter, C.; Maeder, M. L.; Reyon, D.;
Delivery. Nat. Mater. 2012, 11, 316−322. Joung, J. K.; Sander, J. D. High-Frequency Off-Target Mutagenesis
(303) Roh, Y. H.; Deng, J. Z.; Dreaden, E. C.; Park, J. H.; Yun, D. S.; Induced by CRISPR-Cas Nucleases in Human Cells. Nat. Biotechnol.
Shopsowitz, K. E.; Hammond, P. T. A Multi-RNAi Microsponge 2013, 31, 822−826.
Platform for Simultaneous Controlled Delivery of Multiple Small (324) Zuris, J. A.; Thompson, D. B.; Shu, Y.; Guilinger, J. P.; Bessen, J.
Interfering RNAs. Angew. Chem., Int. Ed. 2016, 55, 3347−3351. L.; Hu, J. H.; Maeder, M. L.; Joung, J. K.; Chen, Z.-Y.; Liu, D. R. Cationic
(304) Dalby, B.; Cates, S.; Harris, A.; Ohki, E. C.; Tilkins, M. L.; Price, Lipid-Mediated Delivery of Proteins Enables Efficient Protein-Based
P. J.; Ciccarone, V. C. Advanced Transfection with Lipofectamine 2000 Genome Editing in Vitro and in Vivo. Nat. Biotechnol. 2015, 33, 73−80.
Reagent: Primary neurons, si RNA, and High-Throughput Applications. (325) Sun, W.; Ji, W.; Hall, J. M.; Hu, Q.; Wang, C.; Beisel, C. L.; Gu, Z.
Methods 2004, 33, 95−103. Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR-
(305) Sharei, A.; Zoldan, J.; Adamo, A.; Sim, W. Y.; Cho, N.; Jackson, Cas9 for Genome Editing. Angew. Chem., Int. Ed. 2015, 54, 12029−
12033.
E.; Mao, S.; Schneider, S.; Han, M. J.; Lytton-Jean, A.; et al. A Vector-
(326) Varkouhi, A. K.; Scholte, M.; Storm, G.; Haisma, H. J.
Free Microfluidic Platform for Intracellular Delivery. Proc. Natl. Acad.
Endosomal Escape Pathways for Delivery of Biologicals. J. Controlled
Sci. U. S. A. 2013, 110, 2082−2087.
Release 2011, 151, 220−228.
(306) Chen, A. K.; Behlke, M. A.; Tsourkas, A. Efficient Cytosolic
(327) Kang, W. J.; Chae, J. R.; Cho, Y. L.; Lee, J. D.; Kim, S. Multiplex
Delivery of Molecular Beacon Conjugates and Flow Cytometric
Imaging of Single Tumor Cells Using Quantum-Dot-Conjugated
Analysis of Target RNA. Nucleic Acids Res. 2008, 36, e69.
Aptamers. Small 2009, 5, 2519−2522.
(307) Santangelo, P. J.; Nix, B.; Tsourkas, A.; Bao, G. Dual FRET
(328) Kotula, J. W.; Pratico, E. D.; Ming, X.; Nakagawa, O.; Juliano, R.
Molecular Beacons for mRNA Detection in Living Cells. Nucleic Acids L.; Sullenger, B. A. Aptamer-Mediated Delivery of Splice-Switching
Res. 2004, 32, e57. Oligonucleotides to the Nuclei of Cancer Cells. Nucleic Acid Ther. 2012,
(308) Medley, C. D.; Drake, T. J.; Tomasini, J. M.; Rogers, R. J.; Tan, 22, 187−195.
W. Simultaneous Monitoring of the Expression of Multiple Genes Inside (329) Soundararajan, S.; Chen, W.; Spicer, E. K.; Courtenay-Luck, N.;
of Single Breast Carcinoma Cells. Anal. Chem. 2005, 77, 4713−4718. Fernandes, D. J. The Nucleolin Targeting Aptamer AS1411 Destabilizes
(309) Wang, Q.; Cheng, H.; Peng, H.; Zhou, H.; Li, P. Y.; Langer, R. Bcl-2 Messenger RNA in Human Breast Cancer Cells. Cancer Res. 2008,
Non-Genetic Engineering of Cells for Drug Delivery and Cell-Based 68, 2358−2365.
Therapy. Adv. Drug Delivery Rev. 2015, 91, 125−140. (330) Bates, P. J.; Choi, E. W.; Nayak, L. V. G-Rich Oligonucleotides
(310) Sachdeva, M.; Mo, Y. Y. miR-145-Mediated Suppression of Cell for Cancer Treatment. Methods Mol. Biol. 2009, 542, 379−392.
Growth, Invasion and Metastasis. Am. J. Transl. Res. 2010, 2, 170−180. (331) Soundararajan, S.; Wang, L.; Sridharan, V.; Chen, W.;
(311) Fuse, M.; Nohata, N.; Kojima, S.; Sakamoto, S.; Chiyomaru, T.; Courtenay-Luck, N.; Jones, D.; Spicer, E. K.; Fernandes, D. J. Plasma
Kawakami, K.; Enokida, H.; Nakagawa, M.; Naya, Y.; Ichikawa, T.; et al. Membrane Nucleolin Is a Receptor for the Anticancer Aptamer AS1411
Restoration of miR-145 Expression Suppresses Cell Proliferation, in MV4−11 Leukemia Cells. Mol. Pharmacol. 2009, 76, 984−991.
Migration and Invasion in Prostate Cancer by Targeting FSCN1. Int. (332) Mongelard, F.; Bouvet, P. As-1411, a Guanosine-Rich
J. Oncol. 2011, 38, 1093−1101. Oligonucleotide Aptamer Targeting Nucleolin for the Potential
(312) Qian, H.; Tay, C. Y.; Setyawati, M. I.; Chia, S. L.; Lee, D. S.; Treatment of Cancer, Including Acute Myeloid Leukemia. Curr. Opin.
Leong, D. T. Protecting microRNAs from RNase Degradation with Mol. Ther. 2010, 12, 107−114.
Steric DNA Nanostructures. Chem. Sci. 2017, 8, 1062−1067. (333) Reyes-Reyes, E. M.; Teng, Y.; Bates, P. J. A New Paradigm for
(313) Nahar, S.; Nayak, A. K.; Ghosh, A.; Subudhi, U.; Maiti, S. Aptamer Therapeutic AS1411 Action: Uptake by Macropinocytosis and
Enhanced and Synergistic Downregulation of Oncogenic miRNAs by Its Stimulation by a Nucleolin-Dependent Mechanism. Cancer Res.
Self-Assembled branched DNA. Nanoscale 2018, 10, 195−202. 2010, 70, 8617−8629.
(314) Fu, Z.; Tindall, D. J. FOXOs, Cancer and Regulation of (334) Charoenphol, P.; Bermudez, H. Aptamer-Targeted DNA
Apoptosis. Oncogene 2008, 27, 2312−2319. Nanostructures for Therapeutic Delivery. Mol. Pharmaceutics 2014,
(315) Hsu, P. D.; Lander, E. S.; Zhang, F. Development and 11, 1721−1725.
Applications of CRISPR-Cas9 for Genome Engineering. Cell 2014, 157, (335) Chang, M.; Yang, C.-S.; Huang, D.-M. Aptamer-Conjugated
1262−1278. DNA Icosahedral Nanoparticles as a Carrier of Doxorubicin for Cancer
(316) Ran, F. A.; Hsu, P. D.; Lin, C.-Y.; Gootenberg, J. S.; Konermann, Therapy. ACS Nano 2011, 5, 6156−6163.
S.; Trevino, A. E.; Scott, D. A.; Inoue, A.; Matoba, S.; Zhang, Y.; et al. (336) Brayman, M.; Thathiah, A.; Carson, D. D. MUC1: A
Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Multifunctional Cell Surface Component of Reproductive Tissue
Editing Specificity. Cell 2013, 154, 1380−1389. Epithelia. Reprod. Biol. Endocrinol. 2004, 2, 4.
(317) Shalem, O.; Sanjana, N. E.; Hartenian, E.; Shi, X.; Scott, D. A.; (337) Gendler, S. J. MUC1, the Renaissance Molecule. J. Mammary.
Mikkelsen, T. S.; Heckl, D.; Ebert, B. L.; Root, D. E.; Doench, J. G.; et al. Gland. Biol. 2001, 6, 339−353.
6503 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
(338) Litvinov, S.; Hilkens, J. The Epithelial Sialomucin, Episialin, Is (361) Vitale, C.; Romagnani, C.; Puccetti, A.; Olive, D.; Costello, R.;
Sialylated During Recycling. J. Biol. Chem. 1993, 268, 21364−21371. Chiossone, L.; Pitto, A.; Bacigalupo, A.; Moretta, L.; Mingari, M. C.
(339) Altschuler, Y.; Kinlough, C. L.; Poland, P. A.; Bruns, J. B.; Surface Expression and Function of p75/AIRM-1 or CD33 in Acute
Apodaca, G.; Weisz, O. A.; Hughey, R. P. Clathrin-Mediated Myeloid Leukemias: Engagement of CD33 Induces Apoptosis of
Endocytosis of MUC1 Is Modulated by Its Glycosylation State. Mol. Leukemic Cells. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 5764−5769.
Biol. Cell 2000, 11, 819−831. (362) Ye, Z.; Lee, C. M. L.; Sun, G. W.; Gan, Y.-H. Burkholderia
(340) Ferreira, C. S.; Cheung, M. C.; Missailidis, S.; Bisland, S.; Pseudomallei Infection of T Cells Leads to T-Cell Costimulation
Gariépy, J. Phototoxic Aptamers Selectively Enter and Kill Epithelial Partially Provided by Flagellin. Infect. Immun. 2008, 76, 2541−2550.
Cancer Cells. Nucleic Acids Res. 2009, 37, 866−876. (363) Liu, X.; Xu, Y.; Yu, T.; Clifford, C.; Liu, Y.; Yan, H.; Chang, Y. A
(341) Banerjee, A.; Bhatia, D.; Saminathan, A.; Chakraborty, S.; Kar, S.; DNA Nanostructure Platform for Directed Assembly of Synthetic
Krishnan, Y. Controlled Release of Encapsulated Cargo from a DNA Vaccines. Nano Lett. 2012, 12, 4254−4259.
Icosahedron Using a Chemical Trigger. Angew. Chem., Int. Ed. 2013, 52, (364) Zhang, Z.; Eckert, M. A.; Ali, M. M.; Liu, L.; Kang, D.-K.; Chang,
6854−6857. E.; Pone, E. J.; Sender, L. S.; Fruman, D. A.; Zhao, W. DNA-Scaffolded
(342) Mo, R.; Jiang, T.; DiSanto, R.; Tai, W.; Gu, Z. ATP-Triggered Multivalent Ligands to Modulate Cell Function. ChemBioChem 2014,
Anticancer Drug Delivery. Nat. Commun. 2014, 5, 3364. 15, 1268−1273.
(343) Mo, R.; Jiang, T.; Gu, Z. Enhanced Anticancer Efficacy by ATP- (365) Wu, K.; Liu, J.; Johnson, R. N.; Yang, J.; Kopeček, J. Drug-Free
Mediated Liposomal Drug Delivery. Angew. Chem. 2014, 126, 5925− Macromolecular Therapeutics: Induction of Apoptosis by Coiled-Coil-
5930. Mediated Cross-Linking of Antigens on the Cell Surface. Angew. Chem.
(344) Mo, R.; Jiang, T.; Sun, W.; Gu, Z. ATP-Responsive DNA- 2010, 122, 1493−1497.
Graphene Hybrid Nanoaggregates for Anticancer Drug Delivery. (366) Chu, T.-W.; Yang, J.; Kopeček, J. Anti-CD20 Multivalent HPMA
Biomaterials 2015, 50, 67−74. Copolymer-Fab′ Conjugates for the Direct Induction of Apoptosis.
(345) Zhou, W.; Saran, R.; Liu, J. Metal Sensing by DNA. Chem. Rev. Biomaterials 2012, 33, 7174−7181.
2017, 117, 8272−8325. (367) Chu, T.-W.; Yang, J.; Zhang, R.; Sima, M.; Kopeček, J. Cell
(346) Torabi, S.-F.; Wu, P.; McGhee, C. E.; Chen, L.; Hwang, K.; Surface Self-Assembly of Hybrid Nanoconjugates Via Oligonucleotide
Zheng, N.; Cheng, J.; Lu, Y. In Vitro Selection of a Sodium-Specific Hybridization Induces Apoptosis. ACS Nano 2014, 8, 719−730.
DNAzyme and Its Application in Intracelular Sensing. Proc. Natl. Acad. (368) Johnson, R. N.; Kopečková, P.; Kopeček, J. Biological Activity of
Sci. U. S. A. 2015, 112, 5903−5908. Anti-CD20 Multivalent HPMA Copolymer-Fab’ Conjugates. Biomacro-
(347) Zhou, W.; Ding, J.; Liu, J. A Highly Specific Sodium Aptamer molecules 2012, 13, 727−735.
Probed by 2-Aminopurine for Robust Na+ Sensing. Nucleic Acids Res. (369) Crawford, R.; Erben, C. M.; Periz, J.; Hall, L. M.; Brown, T.;
2016, 44, 10377−10385. Turberfield, A. J.; Kapanidis, A. N. Non-Covalent Single Transcription
(348) Zhou, W.; Saran, R.; Huang, P-J. J.; Ding, J.; Liu, J. An
Factor Encapsulation inside a DNA Cage. Angew. Chem., Int. Ed. 2013,
Exceptionally Selective DNA Cooperatively Binding Two Ca2+ Ions.
52, 2284−2288.
ChemBioChem 2017, 18, 518−522.
(370) Brodin, J. D.; Sprangers, A. J.; McMillan, J. R.; Mirkin, C. A.
(349) Roth, A.; Breaker, R. R. An Amino Acid as a Cofactor for a
DNA-Mediated Cellular Delivery of Functional Enzymes. J. Am. Chem.
Catalytic Polynucleotide. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 6027−
Soc. 2015, 137, 14838−14841.
6031.
(371) Patel, P. C.; Giljohann, D. A.; Daniel, W. L.; Zheng, D.;
(350) Zhou, W.; Ding, J.; Liu, J. Theranostic DNAzymes. Theranostics
Prigodich, A. E.; Mirkin, C. A. Scavenger Receptors Mediate Cellular
2017, 7, 1010−1025.
(351) Cai, H.; Santiago, F. S.; Prado-Lourenco, L.; Wang, B.; Uptake of Polyvalent Oligonucleotide-Functionalized Gold Nano-
Patrikakis, M.; Davenport, M. P.; Maghzal, G. J.; Stocker, R.; Parish, particles. Bioconjugate Chem. 2010, 21, 2250−2256.
C. R.; Chong, B. H.; et al. DNAzyme Targeting c-jun Suppresses Skin (372) Sun, W.; Ji, W.; Hu, Q.; Yu, J.; Wang, C.; Qian, C.; Hochu, G.;
Cancer Growth. Sci. Transl. Med. 2012, 4, 139ra82. Gu, Z. Transformable DNA Nanocarriers for Plasma Membrane
(352) Wu, P.; Hwang, K.; Lan, T.; Lu, Y. A DNAzyme-Gold Targeted Delivery of Cytokine. Biomaterials 2016, 96, 1−10.
Nanoparticle Probe for Uranyl Ion in Living Cells. J. Am. Chem. Soc. (373) Spiller, K. L.; Nassiri, S.; Witherel, C. E.; Anfang, R. R.; Ng, J.;
2013, 135, 5254−5257. Nakazawa, K. R.; Yu, T.; Vunjak-Novakovic, G. Sequential Delivery of
(353) Yehl, K.; Joshi, J. P.; Greene, B. L.; Dyer, R. B.; Nahta, R.; Salaita, Immunomodulatory Cytokines to Facilitate the M1-to-M2 Transition of
K. Catalytic Deoxyribozyme-Modified Nanoparticles for RNAi- Macrophages and Enhance Vascularization of Bone Scaffolds.
Independent Gene Regulation. ACS Nano 2012, 6, 9150−9157. Biomaterials 2015, 37, 194−207.
(354) Peng, H.; Li, X.-F.; Zhang, H.; Le, X. C. A microRNA-Initiated (374) Gratton, S. E.; Ropp, P. A.; Pohlhaus, P. D.; Luft, J. C.; Madden,
DNAzyme Motor Operating in Living Cells. Nat. Commun. 2017, 8, V. J.; Napier, M. E.; DeSimone, J. M. The Effect of Particle Design on
14378. Cellular Internalization Pathways. Proc. Natl. Acad. Sci. U. S. A. 2008,
(355) Zhou, W.; Liang, W.; Li, D.; Yuan, R.; Xiang, Y. Dual-Color 105, 11613−11618.
Encoded DNAzyme Nanostructures for Multiplexed Detection of (375) Gao, H.; Shi, W.; Freund, L. B. Mechanics of Receptor-Mediated
Intracellular Metal Ions in Living Cells. Biosens. Bioelectron. 2016, 85, Endocytosis. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 9469−9474.
573−579. (376) Tang, L.; Yang, X.; Yin, Q.; Cai, K.; Wang, H.; Chaudhury, I.;
(356) Meng, H.-M.; Zhang, X.; Lv, Y.; Zhao, Z.; Wang, N.-N.; Fu, T.; Yao, C.; Zhou, Q.; Kwon, M.; Hartman, J. A. Investigating the Optimal
Fan, H.; Liang, H.; Qiu, L.; Zhu, G.; et al. DNA Dendrimer: An Efficient Size of Anticancer Nanomedicine. Proc. Natl. Acad. Sci. U. S. A. 2014,
Nanocarrier of Functional Nucleic Acids for Intracellular Molecular 111, 15344−15349.
Sensing. ACS Nano 2014, 8, 6171−6181. (377) Kohlhaas, S. L.; Craxton, A.; Sun, X.-M.; Pinkoski, M. J.; Cohen,
(357) Leader, B.; Baca, Q. J.; Golan, D. E. Protein Therapeutics: A G. M. Receptor-Mediated Endocytosis Is Not Required for Tumor
Summary and Pharmacological Classification. Nat. Rev. Drug Discovery Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced
2008, 7, 21−39. Apoptosis. J. Biol. Chem. 2007, 282, 12831−12841.
(358) Gu, Z.; Biswas, A.; Zhao, M.; Tang, Y. Tailoring Nanocarriers for (378) Hu, Q.; Sun, W.; Lu, Y.; Bomba, H. N.; Ye, Y.; Jiang, T.; Isaacson,
Intracellular Protein Delivery. Chem. Soc. Rev. 2011, 40, 3638−3655. A. J.; Gu, Z. Tumor Microenvironment-Mediated Construction and
(359) Scott, A. M.; Wolchok, J. D.; Old, L. J. Antibody Therapy of Deconstruction of Extracellular Drug-Delivery Depots. Nano Lett. 2016,
Cancer. Nat. Rev. Cancer 2012, 12, 278−287. 16, 1118−1126.
(360) Douglas, S. M.; Bachelet, I.; Church, G. M. A Logic-Gated (379) Brglez, V.; Lambeau, G.; Petan, T. Secreted Phospholipases A2
Nanorobot for Targeted Transport of Molecular Payloads. Science 2012, in Cancer: Diverse Mechanisms of Action. Biochimie 2014, 107, 114−
335, 831−834. 123.
6504 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
(380) Kelly, K. L.; Coronado, E.; Zhao, L. L.; Schatz, G. C. The Optical (402) Chan, M. S.; Lo, P. K. Nanoneedle-Assisted Delivery of Site-
Properties of Metal Nanoparticles: The Influence of Size, Shape, and Selective Peptide-Functionalized DNA Nanocages for Targeting
Dielectric Environment. J. Phys. Chem. B 2003, 107, 668−677. Mitochondria and Nuclei. Small 2014, 10, 1255−1260.
(381) Lu, A. H.; Salabas, E. e. L.; Schüth, F. Magnetic Nanoparticles: (403) Bellot, G.; McClintock, M. A.; Lin, C.; Shih, W. M. Recovery of
Synthesis, Protection, Functionalization, and Application. Angew. Chem., Intact DNA Nanostructures after Agarose Gel-Based Separation. Nat.
Int. Ed. 2007, 46, 1222−1244. Methods 2011, 8, 192−194.
(382) Ghosh, P.; Han, G.; De, M.; Kim, C. K.; Rotello, V. M. Gold (404) Lin, C.; Perrault, S. D.; Kwak, M.; Graf, F.; Shih, W. M.
Nanoparticles in Delivery Applications. Adv. Drug Delivery Rev. 2008, 60, Purification of DNA-Origami Nanostructures by Rate-Zonal Centrifu-
1307−1315. gation. Nucleic Acids Res. 2013, 41, e40.
(383) Boisselier, E.; Astruc, D. Gold Nanoparticles in Nanomedicine: (405) Kick, B.; Praetorius, F.; Dietz, H.; Weuster-Botz, D. Efficient
Preparations, Imaging, Diagnostics, Therapies and Toxicity. Chem. Soc. Production of Single-Stranded Phage DNA as Scaffolds for DNA
Rev. 2009, 38, 1759−1782. Origami. Nano Lett. 2015, 15, 4672−4676.
(384) Weissleder, R.; Nahrendorf, M.; Pittet, M. J. Imaging (406) Kick, B.; Hensler, S.; Praetorius, F.; Dietz, H.; Weuster-Botz, D.
Macrophages with Nanoparticles. Nat. Mater. 2014, 13, 125−138. Specific Growth Rate and Multiplicity of Infection Affect High-Cell-
(385) Dong, H.; Du, S.-R.; Zheng, X.-Y.; Lyu, G.-M.; Sun, L.-D.; Li, L.-
Density Fermentation with Bacteriophage M13 for ssDNA Production.
D.; Zhang, P.-Z.; Zhang, C.; Yan, C.-H. Lanthanide Nanoparticles: From
Biotechnol. Bioeng. 2017, 114, 777−784.
Design toward Bioimaging and Therapy. Chem. Rev. 2015, 115, 10725−
(407) Lin, C.; Rinker, S.; Wang, X.; Liu, Y.; Seeman, N. C.; Yan, H. In
10815.
Vivo Cloning of Artificial DNA Nanostructures. Proc. Natl. Acad. Sci. U.
(386) Lucky, S. S.; Soo, K. C.; Zhang, Y. Nanoparticles in
Photodynamic Therapy. Chem. Rev. 2015, 115, 1990−2042. S. A. 2008, 105, 17626−17631.
(387) Yah, C. S.; Simate, G. S.; Iyuke, S. E. Nanoparticles Toxicity and (408) Pound, E.; Ashton, J. R.; Becerril, H. A.; Woolley, A. T.
Their Routes of Exposures. Pak. J. Pham. Sci. 2012, 25, 477−491. Polymerase Chain Reaction Based Scaffold Preparation for the
(388) Bhatia, D.; Arumugam, S.; Nasilowski, M.; Joshi, H.; Wunder, C.; Production of Thin, Branched DNA Origami Nanostructures of
Chambon, V.; Prakash, V.; Grazon, C.; Nadal, B.; Maiti, P. K.; et al. Arbitrary Sizes. Nano Lett. 2009, 9, 4302−4305.
Quantum Dot-Loaded Monofunctionalized DNA Icosahedra for Single- (409) Zhang, H.; Chao, J.; Pan, D.; Liu, H.; Huang, Q.; Fan, C. Folding
Particle Tracking of Endocytic Pathways. Nat. Nanotechnol. 2016, 11, Super-Sized DNA Origami with Scaffold Strands from Long-Range
1112−1119. PCR. Chem. Commun. 2012, 48, 6405−6407.
(389) Zhang, C.; Li, X.; Tian, C.; Yu, G.; Li, Y.; Jiang, W.; Mao, C. DNA (410) Gu, H.; Breaker, R. R. Production of Single-Stranded DNAs by
Nanocages Swallow Gold Nanoparticles (AuNPs) to Form AuNP@ Self-Cleavage of Rolling-Circle Amplification Products. BioTechniques
DNA Cage Core-Shell Structures. ACS Nano 2014, 8, 1130−1135. 2013, 54, 337.
(390) Albanese, A.; Tang, P. S.; Chan, W. C. The Effect of Nanoparticle (411) Gu, H.; Furukawa, K.; Weinberg, Z.; Berenson, D. F.; Breaker, R.
Size, Shape, and Surface Chemistry on Biological Systems. Annu. Rev. R. Small, Highly Active DNAs That Hydrolyze DNA. J. Am. Chem. Soc.
Biomed. Eng. 2012, 14, 1−16. 2013, 135, 9121−9129.
(391) Raeesi, V.; Chou, L. Y.; Chan, W. C. Tuning the Drug Loading (412) Kelley, B. Industrialization of mAb Production Technology: The
and Release of DNA-Assembled Gold-Nanorod Superstructures. Adv. Bioprocessing Industry at a Crossroads. MAbs 2009, 1, 443−452.
Mater. 2016, 28, 8511−8518. (413) Mirsky, A. E.; Ris, H. The Desoxyribonucleic Acid Content of
(392) Qin, Z.; Bischof, J. C. Thermophysical and Biological Responses Animal Cells and Its Evolutionary Significance. J. Gen. Physiol. 1951, 34,
of Gold Nanoparticle Laser Heating. Chem. Soc. Rev. 2012, 41, 1191− 451−462.
1217. (414) Cavalier-Smith, T. Nuclear Volume Control by Nucleoskeletal
(393) Song, L.; Jiang, Q.; Liu, J.; Li, N.; Liu, Q.; Dai, L.; Gao, Y.; Liu, DNA, Selection for Cell Volume and Cell Growth Rate, and the Solution
W.; Liu, D.; Ding, B. DNA Origami/Gold Nanorod Hybrid of the DNA C-Value Paradox. J. Cell Sci. 1978, 34, 247−278.
Nanostructures for the Circumvention of Drug Resistance. Nanoscale (415) Gregory, T. R. Coincidence, Coevolution, or Causation? DNA
2017, 9, 7750−7754. Content, Cell Size, and the C-Value Enigma. Biol. Rev. 2001, 76, 65−
(394) Ohta, S.; Glancy, D.; Chan, W. C. DNA-Controlled Dynamic 101.
Colloidal Nanoparticle Systems for Mediating Cellular Interaction. (416) Schwanhäusser, B.; Busse, D.; Li, N.; Dittmar, G.; Schuchhardt,
Science 2016, 351, 841−845. J.; Wolf, J.; Chen, W.; Selbach, M. Global Quantification of Mammalian
(395) Surana, S.; Bhatia, D.; Krishnan, Y. A Method to Study in Vivo Gene Expression Control. Nature 2011, 473, 337−342.
Stability of DNA Nanostructures. Methods 2013, 64, 94−100. (417) Afonin, K. A.; Bindewald, E.; Yaghoubian, A. J.; Voss, N.;
(396) Bhatia, D.; Mehtab, S.; Krishnan, R.; Indi, S. S.; Basu, A.; Jacovetty, E.; Shapiro, B. A.; Jaeger, L. In Vitro Assembly of Cubic RNA-
Krishnan, Y. Icosahedral DNA Nanocapsules by Modular Assembly.
Based Scaffolds Designed in Silico. Nat. Nanotechnol. 2010, 5, 676−682.
Angew. Chem., Int. Ed. 2009, 48, 4134−4137.
(418) Grabow, W. W.; Jaeger, L. RNA Self-Assembly and RNA
(397) Halley, P. D.; Lucas, C. R.; McWilliams, E. M.; Webber, M. J.;
Nanotechnology. Acc. Chem. Res. 2014, 47, 1871−1880.
Patton, R. A.; Kural, C.; Lucas, D. M.; Byrd, J. C.; Castro, C. E.
(419) Hao, C.; Li, X.; Tian, C.; Jiang, W.; Wang, G.; Mao, C.
Daunorubicin-Loaded DNA Origami Nanostructures Circumvent
Construction of RNA Nanocages by Re-Engineering the Packaging
Drug-Resistance Mechanisms in a Leukemia Model. Small 2016, 12,
308−320. RNA of Phi29 Bacteriophage. Nat. Commun. 2014, 5, 3890.
(398) Modi, S.; Swetha, M.; Goswami, D.; Gupta, G. D.; Mayor, S.; (420) Li, H.; Lee, T.; Dziubla, T.; Pi, F.; Guo, S.; Xu, J.; Li, C.; Haque,
Krishnan, Y. A. DNA Nanomachine That Maps Spatial and Temporal F.; Liang, X.-J.; Guo, P. RNA as a Stable Polymer to Build Controllable
pH Changes inside Living Cells. Nat. Nanotechnol. 2009, 4, 325−330. and Defined Nanostructures for Material and Biomedical Applications.
(399) Surana, S.; Bhat, J. M.; Koushika, S. P.; Krishnan, Y. An Nano Today 2015, 10, 631−655.
Autonomous DNA Nanomachine Maps Spatiotemporal pH Changes in (421) Yu, J.; Liu, Z.; Jiang, W.; Wang, G.; Mao, C. De Novo Design of
a Multicellular Living Organism. Nat. Commun. 2011, 2, 340. an RNA Tile That Self-Assembles into a Homo-Octameric Nanoprism.
(400) Saha, S.; Prakash, V.; Halder, S.; Chakraborty, K.; Krishnan, Y. A Nat. Commun. 2015, 6, 5724.
pH-Independent DNA Nanodevice for Quantifying Chloride Transport (422) Khisamutdinov, E. F.; Jasinski, D. L.; Li, H.; Zhang, K.; Chiu, W.;
in Organelles of Living Cells. Nat. Nanotechnol. 2015, 10, 645−651. Guo, P. Fabrication of RNA 3D Nanoprisms for Loading and Protection
(401) Shalek, A. K.; Robinson, J. T.; Karp, E. S.; Lee, J. S.; Ahn, D.-R.; of Small RNAs and Model Drugs. Adv. Mater. 2016, 28, 10079−10087.
Yoon, M.-H.; Sutton, A.; Jorgolli, M.; Gertner, R. S.; Gujral, T. S. Vertical (423) Delebecque, C. J.; Lindner, A. B.; Silver, P. A.; Aldaye, F. A.
Silicon Nanowires as a Universal Platform for Delivering Biomolecules Organization of Intracellular Reactions with Rationally Designed RNA
into Living Cells. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 1870−1875. Assemblies. Science 2011, 333, 470−474.
6505 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506
(424) Geary, C.; Rothemund, P. W.; Andersen, E. S. A Single-Stranded

Architecture for Cotranscriptional Folding of RNA Nanostructures.
Science 2014, 345, 799−804.
6506 DOI: 10.1021/acs.chemrev.7b00663

Chem. Rev. 2019, 119, 6459−6506

Hu Et Al 2018 Dna Nanotechnology Enabled Drug Delivery Systems

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Cite This: Chem. Rev. 2019, 119, 6459−6506 pubs.acs.org/CR

DNA Nanotechnology-Enabled Drug Delivery Systems

CONTENTS 3.3.3. Proteins 6491

© 2018 American Chemical Society 6459 DOI: 10.1021/acs.chemrev.7b00663

2. STRUCTURAL DNA NANOTECHNOLOGY of a designed, self-assembled, 3D crystal based on the DNA

Figure 9. Gateways and intracellular fate of nanocarriers.

Author Contributions ABBREVIATIONS

6496 DOI: 10.1021/acs.chemrev.7b00663

6497 DOI: 10.1021/acs.chemrev.7b00663

6498 DOI: 10.1021/acs.chemrev.7b00663

6499 DOI: 10.1021/acs.chemrev.7b00663

6500 DOI: 10.1021/acs.chemrev.7b00663

6501 DOI: 10.1021/acs.chemrev.7b00663

6502 DOI: 10.1021/acs.chemrev.7b00663

6503 DOI: 10.1021/acs.chemrev.7b00663

6504 DOI: 10.1021/acs.chemrev.7b00663

6505 DOI: 10.1021/acs.chemrev.7b00663

(424) Geary, C.; Rothemund, P. W.; Andersen, E. S. A Single-Stranded

6506 DOI: 10.1021/acs.chemrev.7b00663

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