REVIEW

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Cellular Ingestible DNA Nanostructures for Biomedical

Applications
Rui Xu, Yujie Li, Chenyou Zhu, Dongsheng Liu,* and Yuhe R. Yang*

(LNPs) have been approved to be an effec-

Delivering functional substances across the cell membrane barriers has been a tive carrier for mRNA vaccines.[3] However,
great challenge in biomedicine. Among various strategies to improve cellular different carrier materials have correspond-
uptake, DNA nanostructures, with different shapes and sizes, supply as suitable ing drawbacks. For example, the potential
toxicity of synthetic nanoparticles cannot
platforms for delivering a wide range of functional substances to cells. With great
be ignored due to their nondegradability
programmability, biocompatibility, and biostability, DNA nanostructure-based in vivo,[4] and the limited DNA packaging
delivery systems have shown and have succeeded in anticancer therapy, anti- capacity and carcinogenesis restricted
bacterial treatment, gene editing, vaccine development, and stem cell bioengi- further biomedical applications of viral
neering. Herein, the development and construction of various cellular ingestible vectors.[5] Thus, it is urgent to expand the
pool of carrier materials with excellent cell
DNA nanostructures are described, the loading strategies of functional sub-
permeability, high loading capacity, and
stances are summarized, current biomedical applications are overviewed, and biocompatibility.
finally the remaining challenges and opportunities are discussed. DNA nanotechnology, an emerging
technique that utilizes the DNA as building
blocks to program well-defined nanostruc-
tures, is considered as a suitable option for
1. Introduction drug delivery due to the intrinsic biocompatibility, programma-
bility, and biostability of DNA nanostructures. Since the concept
While the cell membrane functions as a barrier that separates of DNA nanotechnology was proposed by Seeman in 1982,[6] this
harmful foreign substances from the cytoplasm, it also prevents technology has been rapidly developing. Previous studies have
the therapeutic substances from cellular entry. Functional sub- demonstrated that some DNA nanostructures can be actively
stances (i.e., small molecule drugs, siRNA, Cas9/gRNA, etc.) absorbed by cells, which have attracted extensive attention for
need to penetrate the cell membrane barrier to realize efficient their wide range of applications such as biosensing, tissue cul-
intracellular functions.[1] Popular delivery vehicles for the import ture, drug delivery, etc.[7] In this review, we will focus on cellular
of various functional substances into cells include inorganic ingestible DNA nanostructure-based functional materials and
nanomaterials (i.e., Au nanoparticles, carbon nanomaterials), their biomedical applications. Within this framework, we compre-
polymer materials (i.e., liposome, chitosan), and biomimetic hensively review the development, construction strategy, and
materials (i.e., viral vector).[2] For instance, lipid nanoparticles biomedical applications of DNA nanostructures with particular
emphasis on DNA-functionalized nanoparticles, DNA microgel,
and programmable DNA self-assembly nanostructures. We
R. Xu, Y. Li, C. Zhu, D. Liu mainly discuss their application prospects and current challenges
Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology
and further propose several improvement strategies for broader
(Ministry of Education)
Department of Chemistry therapeutic applications. Owing to space constraints, DNA nano-
Tsinghua University structures working extracellularly or in vitro will not be discussed
Beijing 100084, China in this review but are touched on in companion reviews.[8]
E-mail: liudongsheng@tsinghua.edu.cn
Y. R. Yang
CAS Key Laboratory of Nanosystem and Hierarchical Fabrication 2. Development and Construction
CAS Center for Excellence in Nanoscience
National Center for Nanoscience and Technology
Beijing 100190, China In past decades, numerous efforts have been devoted to the devel-
E-mail: yangyh@nanoctr.cn opment and improvement of manufacture strategies of different
DNA nanostructures. Here we analyze pros and cons for each strat-
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/anbr.202200119. egy and describe the reaction time, yield, scale, cost, etc. as follows.

© 2022 The Authors. Advanced NanoBiomed Research published by

Wiley-VCH GmbH. This is an open access article under the terms of
the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original
work is properly cited.
DOI: 10.1002/anbr.202200119
2.1. DNA-Functionalized Nanoparticles
Au/Ag─S bond is widely employed to mediate ssDNA–metal
nanoparticle conjugates. In 1996, Mirkin and Alivisatos indepen-
dently reported the synthetic approach for DNA-functionalized

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gold nanoparticles (AuNPs) for the first time.[9] Subsequently,
various nanoparticles have been functionalized by DNA and
obtained different applications. Herein, we focused on the syn-
thesis of DNA-functionalized AuNPs for their broad applications
in biomedicine field.
Various efforts have been used to improve the synthesis of
DNA-functionalized AuNPs in efficiency of process and density
of DNA modification (Figure 1A). The high-density DNA-
functionalized AuNP was achieved by Mirkin et al.[10] using a
method referred as salt aging. They successfully promoted higher
surface density of DNA (up to 90 DNA per 13 nm AuNP) by
increasing the concentration of sodium chloride (NaCl) in reac-
tion solution up to
2 mol L1 to prevent the electrostatic exclu-
sion between the AuNPs and DNA strands. However, this salt
aging method requires over 24 h for reaction; it has been opti-
mized by Liu et al. with a low-pH method to enable rapid synthesis
(only 5 min) of high-density DNA-functionalized AuNPs.[11] To
further enhance the surface density and modification efficiency,
Liu et al. additionally introduced a method that froze the reaction
mixture at 20 °C for 2 h and then thawed the mixture at room
temperature.[12] This method can increase the surface density of
DNA up to 110 DNA per 13 nm AuNP. Moreover, based on
DNA-functionalized AuNPs, the DNA with specific functions (i.e.,
i-motif,[13] a structure responding to pH,[14] electric signal,[15]
light,[16] etc.) has been employed to modify AuNPs and
achieved pH-responsive assembly.[17] Number-controlled DNA-
functionalized AuNPs have also been developed for programma-
ble assembly of AuNPs into well-defined superstructures.[18]
Furthermore, similar covalent conjugation approaches have been
established, which utilize dibenzocyclooctyne-modified DNA to
functionalize azide-modified liposomal or protein and apply
tocopherol modified to functionalize liposome via hydrophobic
interaction.[19]
2.2. DNA Microgels
DNA microgels, a nanoscale version of DNA hydrogels, have
been developed to improve cellular applicability due to its

Figure 1. DNA-functionalized AuNP and DNA microgels. A) Three main methods for constructing DNA-functionalized AuNP. B) DNA hydrogels con-
structed by Y scaffold and linker. Reproduced with permission.[24] Copyright 2011, Wiley-VCH. C) DNA nanoflower constructed by long DNA chains.
Reproduced with permission.[33] Copyright 2013, American Chemical Society.

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discrete size, high stimuli–response rate, and cellular uptake effi-
ciency.[20] The current design of DNA microgels is built on the
formation strategies of DNA hydrogels. Herein, we reviewed the
development of DNA hydrogels first and discussed the construc-
tion of DNA hydrogels and DNA microgels.
DNA hydrogels can be constructed through chemical or phys-
ical interaction among DNA-based building blocks. In 2006, Luo
et al. initially prepared chemically crosslinked DNA hydrogel by
DNA assembly and ligation.[21] The reformation of duplexes in
rehydration process endowed the hydrogels with amazing shape
memory properties, but also limited their structural dynamic.[22]
Luo et al. elongated DNA chains through rolling circle amplifi-
cation (RCA) and multiprimed chain amplification (MCA) and
then weaved them noncovalently into hydrogels.[23] This strategy
provides high convenience for the synthesis of a large quantity of
DNA hydrogels without the requirement to design specific
sequence. Besides, Liu et al. developed a series of self-
assembled-based DNA hydrogels with a programmable network.
In 2011, they designed self-assembled DNA hydrogels with
designable thermal and enzymatic responsiveness via the hybrid-
ization of two building blocks: a Y-scaffold and a linker
(Figure 1B).[24] Next, they dividedly heated Y-scaffold and linker
DNA strands to 95 °C and cooled down to room temperature after
2 h and then proportionally mixed two solutions, which allowed
to obtain the DNA hydrogels through one-pot reaction. Based on
this strategy, they established a series of stimuli–responsive DNA
hydrogels: photoresponsiveness, thermal responsiveness, and
metal–ion responsiveness.[25,26] Recently, they constructed rein-
forced DNA hydrogels through adding polymeric multiple-unit
linkers to this system, which enabled strong improvement of the
mechanical strength.[27] A similar strategy with Y-scaffold and
i-motif was used to construct a pH-responsive hydrogel.[28] This
method is also suitable for the construction of hybrid DNA hydro-
gels with peptides,[29] proteins,[30] cucurbit[8]uril,[31] etc. The DNA
self-assembly-based strategy possess more specific properties,
including stimuli responsiveness, self-healing, drug sustained
release, which provides DNA hydrogels with high potential
application value, especially in the field of biomedicine.[32]
Based on the generation methods of DNA hydrogels, DNA
microgels similarly can also be composed by the tangle of
long DNA chains and self-assembly of DNA building blocks.
Tan et al. used long DNA chains prepared via RCA to construct
a DNA nanoflower with annealing process (Figure 1C).[33]
Furthermore, Gu et al. reported kinds of DNA microgels called
DNA nanoclews (NCs) which also adopted RCA process.[34] The
sizes of the DNA nanoflower and NC were generally more than
100 nm. More recently, Tan et al. also used Y unit and linker to
synthesize self-assembled DNA microgels with a well-defined net-
work. They employed an aptamer to block the Y-unit and inhibit
the extension of nanostructures, which can regulate the size of
DNA nanohydrogels from 100, 144, to 268 nm through controlling
the proportion of the aptamer-blocked Y-unit and normal Y-unit.[35]
2.3. Programmable DNA Self-Assembly Nanostructures for
Biomedical Applications
2.3.1. DNA Polyhedron
The synthesis of DNA polyhedron has experienced various
evolutions. In 1991, Seeman et al. reported the first DNA poly-
hedron, in which they constructed DNA cube in a DNA ring
nested manner.[36] In 2004, two individual studies using
ssDNA to assemble polyhedron structures in a one-pot annealing
process were reported. Turberfield et al. utilized four ssDNA to
build DNA tetrahedron successfully (Figure 2A),[37] and
Shih et al. created DNA octahedrons with a long ssDNA and five
short ssDNAs.[38] In 2008, Mao et al. described a new method of
constructing various kinds of DNA polyhedrons by linking
three (or four, five)-point-star motifs though sticky-ended
(Figure 2A).[39] They constructed DNA tetrahedrons, DNA

Figure 2. Programable DNA self-assembly nanostructures. A) Two strategies to produce DNA polyhedron. Reproduced with permission.[37,39] Copyright
2004, Royal Society of Chemistry, Copyright 2008, Nature Publishing Group. B) The design and construction of DNA origami. Reproduced with
permission.[41,44,70] Copyright 2006, Nature Publishing Group, Copyright 2009, The American Association for the Advancement of Science,
Copyright 2014, American Chemical Society. C) The method of SST to construct different shapes. Reproduced with permission.[51,52] Copyright 2012,
The American Association for the Advancement of Science, Copyright 2012, Nature Publishing Group.

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dodecahedrons, and DNA buckyballs through regulating the
length of the central single-stranded loop from five-base to
three-base long. In the subsequent research, they employed star
motifs to create other polyhedrons, such as DNA cubes, DNA
octahedrons, and DNA triangular prisms. In addition, in
2015, a new method for combination with the motif-based
polyhedron assembly and circular long-stranded DNA origami
strategy was developed by Yan et al., namely, wireframe DNA
origami.[40] Similarly, the length of the central single-stranded
loop also played a vital role in adjusting the size and structure
of the DNA polyhedrons.
At present, DNA tetrahedrons were mainly constructed by
four single-stranded DNA for its simple process and relatively
high yield. With only one annealing step spending about 4 h,
the DNA tetrahedrons could be obtained with a yield of 95%.
DNA polyhedrons with more complex structures were usually
generated using star motifs. Depending on the size and complex-
ity, the annealing time varied from 4 h to more than 48 h whereas
the yield dropped from 90% (i.e., DNA tetrahedrons) to less than
69% (i.e., DNA buckyballs). The process of 12 h annealing was
necessitated for the generation of wireframe DNA origami.
Usually involving a M13 scaffold strand, and the size of DNA
polyhedrons varies from 50 nm to hundreds of nanometers with
yield from 70% to 80%. Based on all the designs of polyhedron,
tradeoffs among the yield, assembly time, and cost, DNA tetra-
hedrons are becoming the most widely used nanostructures in
biomedical field nowadays for its broad applications, relatively
large-scale production, and facile synthesis process.
2.3.2. DNA Origami
DNA origami, a revolutionary method for constructing various
unprecedented DNA structures with fantastic stability and
extensive applications, was proposed by Rothemund in 2006
(Figure 2B).[41] With rigorous design, DNA origami with arbi-
trary shapes can theoretically be formed by a one-step annealing
process which involves a long, ssDNA scaffold (e.g., M13mp18
genomic DNA) and hundreds of short oligonucleotide staple
strands. With the development of user-friendly software (e.g.,
CADnano,[42] Tiamat[43]), a large number of nanostructures from
simple 2D rectangular shapes to complex 3D shapes (e.g., twisted
nanoscale shapes,[44] DNA boxes,[45] flower vase,[46] DNA
origamiþ,[47] square nut[48]) have been constructed using DNA
origami strategy.
The one-step annealing process and purification-free synthe-
sis of staple strands are relatively convenient. Due to the excess
addition of staples to the reaction mixture, stoichiometry reduces
restrictions compared with other methods such as tile-based
assembly.[39] The annealing parameters for different DNA
origami structures vary from 1.5 h for simple structures to more
than 24 h for the complex structures. The yield also varies
dramatically from >70% to <4% with increasing complexity.
DNA origami method has enabled the creation of fancy DNA
structures through simple delicate design that could replicate
art pieces in nanometer scale (e.g., smiley,[41] Mona Lisa,[49]).
However, until now, the DNA origami structures used to carry
materials into cells were limited to relatively simple structures
(i.e., triangle DNA origami, square DNA origami, and tube
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DNA origami). Besides, the assembly procedure, scale, yield,
and other practical factors need to be considered comprehen-
sively when selecting DNA nanostructures as targets in biomed-
ical applications in vivo.
2.3.3. Single-Stranded DNA Tiles
The concept of single-stranded DNA tiles (SSTs) was proposed by
Yin et al. in 2008.[50] An SST consists of a 42-base strand of DNA
entirely composed of concatenated sticky ends, binding to four
local neighbors during self-assembly (Figure 2C). Researchers
have used SSTs to construct various complex shapes (e.g., DNA
triangle, DNA rectangle, and DNA tube). In 2012, Yin et al. also
put forward the concept of DNA bricks which extended the
SST assembly method from 2D to 3D.[51] A canonical DNA brick
consists of a 32-nt ssDNA with four 8-nt binding domains.
Each 32-nt brick is a modular component and binds to four local
neighbors which can be removed or added independently.
A unique feature of SST-based assembly is the unlimited size
of the desired shape which was previously defined by the limited
length of the scaffold DNA in DNA origami strategy. Purification
and exact stoichiometry of DNA components are not essential in
SST-based assembly. The annealing rate varies from 17 to 72 h
depending on the structure of the final product as well as the
yield differing greatly from 80% to 4%. The large range of size
adjustment enables generation of nanostructures to micrometer
scale with this SST method (around 10–200 nm, even up to
1 μm),[52] which compensates for the deficiency of the program-
mable nanomaterials described previously. However, the rela-
tively low yield and high cost of SST-based methods are the
major obstacles. Further development and improvement in these
aspects are required, which will expand their biomedical
applications.
3. Methods of Loading Various Functional
Groups
In addition to the diverse compositions, sizes, and structures of
DNA nanostructures, the unique modifiability of DNA enables
them to carry a variety of target cargos of interest, including small
molecules, nucleic acids, proteins, etc. Depending on the differ-
ent types and properties of the functional groups, various loading
methods are categorized into covalent conjugation, intercalation,
base pairing, and packaging, which will be described as follows.
3.1. Complementary Base Pairing
Nucleic acid-based drugs (i.e., mRNA, miRNA, siRNA, antisense
nucleic acid, aptamer) play an increasingly important role in
modern medicine aiming for cancer treatment, hyperglycemia,
antivirus, and other diseases.[53] All kinds of DNA nanostructures
can potentially be applied as the natural carriers for the
nucleic acid-based drugs through complementary base pairing
(Figure 3A). A large number of studies have reported the appli-
cations of various DNA nanostructures to load nucleic acid-based
drugs including DNA origami assemblies, DNA-functionalized
NPs, DNA microgels, and polyhedrons.[7] These nucleic acid
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Figure 3. Methods of loading various functional groups. A) Complementary base pairing. B) Intercalation. C) Covalent Conjugation. D) FGA strategy for
multiple loading. Reproduced with permission.[64] Copyright 2017, Wiley-VCH.

nanostructures are mainly loaded with nucleic acid drugs in two
ways. 1) One-step method. By extending the nucleic acid-based
drug sequences as a part of the DNA nanostructures, they can be
loaded into DNA nanostructures through one-pot annealing. In
spite of convenience, the process appears to be destructive for
some RNA-based drugs (i.e., mRNA, siRNA, miRNA) since
the annealing procedure usually involves an initial step of high
temperature to denature all the DNA components. Therefore,
most of the reported works are used for loading more
thermostable DNA-based drugs (i.e., antisense DNA,[35] DNA
aptamer[33]). Some RNA drugs can accommodate assembly con-
ditions with short heating time and fast cooling rate for loading
onto easy-to-form DNA nanostructures. Anderson et al. described
the assembly of tetrahedron–siRNA complex by directly mixing
six ssDNAs with six double-stranded siRNAs, in which the solu-
tion was heated to 90 °C for 2 min and rapidly cooled down to
4 °C.[54] 2) Two-step method. In this way, a short single-stranded
probe is extruded out of the DNA nanoparticle to hybridize with
the part of the nucleic acid-based drugs. This relatively mild con-
dition (usually <37 °C) is not only suitable for RNA-based
drugs,[55] but also enables the coloading of multiple drugs.[56]
However, the sequence of probe needs to be accurately designed
to form stable tethering while minimizing misfolding.[57]
3.2. Intercalation
Intercalation is the most simple and convenient method which
only needs an incubation step of the drugs with corresponding

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DNA nanostructures. Some small molecules, especially the
anthracyclines drugs containing an anthracene ring connected
to an amino sugar, (e.g., doxorubicin, daunorubicin), have been
found to be able to intercalate between two base pairs of the DNA
(Figure 3B). Various DNA nanostructures carrying Dox have
been widely studied and applied to treat cancers benefiting from
their high efficiency to be loaded onto DNA nanostructures by
simply mixing.[58] Besides doxorubicin, a highly potent platinum-
based DNA intercalator, 56MESS, can also be loaded into the
double-stranded DNA (dsDNA) with this method.[59]
3.3. Covalent Conjugation
Covalent conjugation is a universal method for loading
small-molecular compounds (e.g., cisplatin drugs, paclitaxel),
nucleic acid drugs, and proteinaceous drugs on all types of
DNA nanostructures. 3’ end and 5’ end of ssDNA are the two
common sites for modification. Commonly the used chemical
groups modified to the 3’end and 5’ end of DNA include amino
(–NH3), sulfhydryl (–SH), azide (–N3), and alkyne (–C≡CH)
(Figure 3C). Mirkin et al. applied 28mer oligonucleotides con-
taining a terminal dodecyl amine to conjugate with a platinum
(IV) complex.[60] Besides the modification on 3’ end and 5’ end of
DNA, the phosphodiester linker between each nucleotide can
also be modified with phosphorothioate (PS) in which one of
the oxygen atoms (O) is replaced by sulfur atom (S) to improve
the reactivity of DNA with other compounds. For example, the
carbonethyl bromide-modified drug molecules (e.g., camptothe-
cin[61]) can be employed to directly react with PS-modified DNAs.
In addition to the methods described above, using modified
deoxynucleotide to synthesize base-modified DNA through
solid-phase synthesis is also an effective method to load medici-
nally valuable base analogs (e.g., 5-bromouracil, mercaptopurine,
2,6-diaminopurine, etc.). Zhu et al. replaced thymine with flox-
uridine (an anticancer drug for blocking DNA synthesis and
inhibiting RNA formation) to synthesize DNA strands and con-
structed different DNA polyhedrons for anticancer therapy.[62]
However, the modification of different functional substances
may increase the complexity, which will lead to the arduous
assembly, limited yield, and unpredictable cost of DNA
nanostructures.
3.4. Encapsulation
Encapsulation is generally applicable to DNA hydrogels for
loading drugs (Figure 3D). Depending on the mesh size of
the hydrogels, it can be used to immobilize small-molecule
drugs, nucleic acids, and proteins through encapsulation.
Liu et al. employed the DNA nanoflowers to load both TMPyP4
(a porphyrin-based photosensitizer) and catalase (an enzyme).[63]
Similar to DNA microgels, DNA-functionalized liposomes are
also capable of loading drugs through encapsulation. Using
encapsulation to deliver various functional substances possesses
two distinctive advantages. First, it can achieve coloading of mul-
tiple drugs, including small molecules, proteins, and other poly-
mers. Second, the drugs are embedded in the nanostructures
instead of merely displaying on the surface of nanostructures
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which will avoid drug leakage and realize the gradual release
of drugs.
3.5. Combination of Multiple Methods
The loading methods described above can be further combined
to codeliver various functional materials to specific sites. Liu et al.
employed the frame-guided assembly (FGA) strategy to develop a
modularly designable vesicle for sequential multiple loading
(Figure 3E).[64] With this vesicle, they accomplished programmat-
ically loading of four different functional substances through
layer-by-layer assembly. The FGA strategy provided a potential
approach for the delivery of multiple functional groups and
realized the combination of therapeutic function and tracking
purpose.
4. Biomedical Applications
DNA nanostructures have become a widely used material in bio-
medicine due to their programmability, biocompatibility, and
bio-stability. 1) Programmability: As described above, the size
of DNA nanostructures ranges from a few nanometers to micro-
meters, with diverse shape, which endows DNA nanomaterials
with the different pathways of cellular uptake as well as distinct
targeting and trafficking behavior in animal models.[7,65]
2) Biocompatibility: DNA nanostructures inherit the low toxicity
from ssDNA and dsDNA and the excellent biocompatibility ena-
bles them to be applied in vivo, including cells, tissues, and
organisms.[66] 3) Biostability: With significantly less exposed free
ssDNA, the densely packed DNA nanostructures usually exhibit
better resistance to degradation by nucleases.[67] Resulting from
improved stability in physiological environment, the longer
retention time in animal models expands the applications in
long-term drug release, targeted drug delivery, etc. These excel-
lent properties of DNA nanostructures open up new frontiers of
biomedical applications which are summarized as follows:
cancer treatment, antibacterial treatment, gene editing, vaccine
carrier, and cell differentiation.
4.1. DNA Nanostructures for Anticancer Treatment
Great progress has been made in the development of anticancer
therapy in recent years. However, even for the most successful
and effective anticancer drugs, both innate and acquired mech-
anisms of resistance are increasingly commonplace. An increas-
ing number of reports found that the resistance of some tumor
cells originated from inhibiting the uptake of drugs in cancer
cells.[68] Furthermore, direct injection of anticancer drugs into
the body may cause serious side effects due to the leakage of anti-
cancer drugs to normal tissues. DNA nanostructures exhibit
superior properties in addressing two serious problems
mentioned above, which improve the efficacy of drugs and avoid
damage to healthy tissue cells.
Due to the enhanced permeability and retention effect (EPR
effect) of nanostructures, DNA nanostructures can passively tar-
get cancer tissues in vivo without any auxiliary assistance.[69]
Some reports have illustrated the relationship between the struc-
ture of DNA nanostructures and cellular uptake efficiency.[7]
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In 2007, Mirkin et al. found that uptake efficiency depends on the
density of DNA on the surface of AuNPs. Ding and Du et al.
demonstrated that the triangle-shaped DNA origami possessed
enhanced passive targeting of tumor and long-lasting properties
at the tumor region compared with square and tube DNA
origami (Figure 4A).[70] Based on the EPR effect, various drug
molecules can also be directly taken up by cells after being immo-
bilized on DNA nanostructures, thereby destroying the drug
resistance of cancer cells. Mirkin et al. first reported that connect-
ing platinum (IV) drug to the surface of DNA-functionalized
AuNPs through amide linkages can facilitate the cellular uptake
of platinum complex to increase cytotoxicity significantly.[60]
Subsequently, it has been demonstrated that various DNA nano-
structures exhibited their ability to carry drug molecules into
cancer cells and significantly increase the cytotoxicity to drug-
resistant cancer cells (Figure 4B).[71]
In addition, the modification of some targeting agents can pro-
vide DNA nanostructures with the ability of active targeting
(Figure 4B). Aptamer, an oligonucleotide selected by systematic
evolution of ligands by exponential enrichment (SELEX)

Figure 4. DNA nanostructures for anticancer treatment. A) Passive targeting to various tissues of different structures and sizes of DNA origami.
Reproduced with permission.[70] Copyright 2014, American Chemical Society. B) Various DNA nanostructures can be uptaken by cancer cells.
Reproduced with permission.[70] Copyright 2014, American Chemical Society. C) Recognition methods between DNA nanostructures and cells.
D) Responsive release of drugs in cancer cells.

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targeting to different aptamer targets, can be designed into the
sequence of DNA chain directly. Liu et al. encoded the sequence
of sgc8c aptamer to the DNA templates of RCA and achieved the
targeted delivery of DNA nanosponges in mouse models. Within
step-wise strategy, aptamer can be connected to DNA nanostruc-
tures through covalent conjugation or base pairing.[72] Liu and
Ding et al. used triangle DNA origami to link with MUC-1
aptamer and achieve codelivery of Dox and Au nanorods into
cancer cells.[73] Other targeted agents such as folic acid[54,74]
and antibodies[59] have also been employed on DNA nanostruc-
tures for targeting cancer cells(Figure 4C).
Besides the passive and active targeting strategy, another
method is to design switchable devices to achieve controlled
release of drugs in cancer cells. The first example is the
telomerase-response release (Figure 4D). Gu and Qian et al. com-
bined FITC-labeled hairpin DNA sequences hybridized with
telomerase primers to construct DNA-functionalized AuNPs.[75]
It would precisely release doxorubicin in response to the telome-
rase existing only in the tumor cells and prevent toxicity to
normal organs. In addition, disulfide linkages endow DNA nano-
structures the ability to be degraded by glutathione (GSH), which
has realized the controlled release of ASO,[35] shRNA,[76] etc.
The responsiveness of light,[77] pH,[56] and other factors[78] was
also used for the selective release of anticancer drugs in cancer
cells, which reduced the lethality of normal tissue cells.
4.2. DNA Nanostructures for Antibacterial
One of the most important issues of antibacterial is that some
antibacterial drugs such as ASO and antibiotics are unable to
penetrate bacterial cell wall and membrane directly into drug-
resistant bacteria.[79] Several studies have reported that certain
DNA nanostructures such as tetrahedrons had the ability to accel-
erate the ingestion process of bacterial cells. In 2014, Leong et al.
first discovered that DNA tetrahedrons were able to delivery red-
emissive glutathione-protected gold nanoclusters (GSH-Au NCs)
and actinomycin D (AMD) into bacterial cells and achieved
bacterial detection and treatment at the same time.[80] In addition
to the DNA tetrahedrons, other bacteria ingestible DNA nano-
structures have been discovered. Kaminski et al. adopted DNA
origami as the delivering vehicle for the antibacterial enzyme
lysozyme.[81] Li et al. reported DNA six-helix bundle (6HB) nano-
tubes to codeliver ASO and Agþ into bacterium with high
antibacterial efficacy (Figure 5).[82] They employed Agþ to destroy
the cell wall of the bacterium and the ASO to inhibit the expres-
sion of the ftsZ gene. However, only a small amount of DNA
nanostructures has been proved to be able to enter the bacterial
cells, and the mechanism is not revealed, which needs further
investigation.
4.3. DNA Nanostructures for Gene Editing
Gene editing is a powerful strategy to modify genes and treat
genetic diseases. The Clustered regularly interspaced short
palindromic repeats (CRISPR) gene editing system is the most
efficient and widely used next-generation technology involving
CRISPR-associated nuclease (Cas) proteins, guide-RNA (gRNA),
donor DNA, and other factors.[83] It has been a long-existing
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Figure 5. DNA 6HB nanotubes for codelivering antisense oligonucleotide
(ASO) and silver ions into bacterium. Reproduced with permission.[82]
Copyright 2021, American Chemical Society.
challenge for codelivery of all the components to realize complete
function of this large complex with high homology-directed
repair (HDR) efficiency and low off-target effect.[84] DNA nano-
structures have been proved to be a scaffold with controllable
sizes to load multiple components, which match the requirement
of delivering CRISPR-Cas systems. Gu et al. designed a DNA NC
constructed by RCA-generated ssDNA and loaded the NC with
Cas9/sgRNA ribonucleoprotein (RNP) complexes by hybridizing
partial sequence of the NC and sgRNA (Figure 6A).[85] They suc-
cessfully delivered the complexes to the nucleus of human cells
with the assistance of nuclear-localization-signal (NLS) peptides
fused to Cas9 protein. They also employed polyethylenimine
(PEI) to coat the complex for inducing endosomal escape.
After cells were treated by the Cas9/sgRNA/NC-12/PEI, the assay
revealed mutation frequencies of
28%. The similar strategies
have been applied to the successful delivery of CRISPR/
Cas12a complex.[86] Ding et al. constructed the sgRNA/Cas9/
Antisense complex to codeliver CRISPR Cas9 system and
ASO through coassembly of branched DNA, linker (antisense
with two disulfide linkage), and 3’ terminal extended sgRNA
(Figure 6B).[87] With such smart design, these DNA nanostruc-
tures achieved the dual function of gene editing in nucleus and
gene silencing in cytoplasm with
80% loss of EGFP fluorescent
signal. They also combined sgRNA/Cas9 system with gold nano-
rods to achieve the combination of gene editing and photother-
mal therapy for efficient tumor therapy.[88] Recently, Ding et al.
construct a double-stranded DNA–ribonucleoprotein (RNP)
hybrid nanostructure to serve as the platform of CRIPSR/dead
Cas (dCas) system. They fold double-stranded DNA with a
covalently bivalent between dCas9 RNPs and dCas12a RNPs
to codeliver the dCas9 and dCas12a systems.[89]
4.4. DNA Nanostructures for Vaccine Carrier
There is a critical need for delivery vehicles to enhance the
immune response due to the deficient immunogenicity of some
antigens in vivo. DNA nanostructures provide plenty of alterna-
tive platforms for loading antigens. Chang and Yan et al. first
used DNA tetrahedron as a scaffold to load a model antigen,
streptavidin (STV), and CpG adjuvants together, which resulted
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Figure 6. DNA nanostructures for gene editing. A) DNA NC loading Cas9/sgRNA complexes by through partially complementary to the sgRNA.
Reproduced with permission.[85] Copyright 2015, Wiley-VCH. B) Branched DNA structure loading a sgRNA/Cas9/antisense complex to achieve the
combination of gene editing and gene silencing. Reproduced with permission.[87] Copyright 2019, American Chemical Society.

in strong B cell response (Figure 7A).[90] Bathe et al. applied scaf-
folded DNA origami to carry HIV immunogens with controllable
organization and revealed the impact of antigen spacing.[91]
Mirkin et al. encapsulated the receptor-binding domain (RBD)
subunit into liposomes and decorated the core with a dense shell
of CpG toll-like receptor 9 (TLR9) agonist oligonucleotides. Such
a DNA-functionalized liposome vaccine exhibited high resistance
to the SARS-Cov-2 virus.[92] Tubular DNA origami can serve as a
carrier to encapsulate functional substances and achieve the
stimuli–response.[93] Ding et al. incorporated a lock and key
design into a rectangular DNA nanoplatform coloaded with
peptide antigen and TLR) agonists (i.e., double-stranded RNA
(dsRNA) and CpG DNA) and achieved the protection of the anti-
gen and TLR agonists from degradation (Figure 7B).[56] After
entering CD8þ cytotoxic T lymphocyte (CTL), the nanodevice
would open in low pH (pH 5.5) and expose the antigen and
TLR agonists to activate a strong immune response.
4.5. DNA Nanostructures for Stem Cell Proliferation and
Differentiation
After being ingested by cells, the DNA nanostructures have
shown potential effects on cell behaviors, such as inducing
immunological rejection, stimulating cell signaling pathways,
etc. For example, DNA tetrahedrons have been discovered to
promote the proliferation and differentiation of stem cells
(e.g., embryonic stem cells, endothelial cells, neural stem cells
[NSCs], dental pulp stem cells, etc.).[94]
DNA hydrogel is a kind of unique biomaterial containing a
series of characteristics comparable to natural tissue, (i.e., high
water content (
90%), suitable mechanical properties, and shape
plasticity). DNA hydrogels have been applied to serve as excellent
biomaterials in biological sensing,[95] tissue regeneration,[96] and
stem cell culture [25c,97] for their high permeability, good mechan-
ical strength, and thixotropic properties. Especially, DNA hydro-
gels exhibited excellent properties in regulating the proliferation
and differentiation of stem cells. Liu et al. employed DNA supra-
molecular hydrogel to carry bone marrow mesenchymal stem
cells (BMSCs) and demonstrated that it would protect the
BMSCs from the strong shear force and enhance the therapeutic
effect of BMSCs (Figure 8A).[97a] Liu et al. designed a DNA hydro-
gel encapsulating NSCs to repair a 2 mm spinal cord gap in rats.
The sufficient and tunable mechanical strength, swift thixotropy,
and excellent biocompatibility contributed to the proliferation
and differentiation of the NSCs (Figure 8B).[96a] They demon-
strated that a renascent neural network with basic hindlimb
function was formed within 8 weeks through the test of various
tools, and DNA hydrogels with high mechanical strength could
provide cells with excellent antifriction protection and 3D sup-
port. Therefore, they can serve as a suitable filler between osteo-
articular. More recently, Liu et al. prepared L-DNA hydrogels with

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Figure 7. DNA nanostructures for vaccine carrier. A) DNA tetrahedron as a vaccine carrier to load antigen. Reproduced with permission.[88] Copyright
2012, American Chemical Society. B) Locked DNA origami tube to codeliver peptide antigen, CpG loop, and double-strand RNA. Reproduced with
permission.[56] Copyright 2020, The Author(s), under exclusive licence to Springer Nature Limited.

univariate mechanical strength, improved biostability, and low
inflammation response which greatly promoted the application
of DNA hydrogels in the field of stem cell culture.[98]
5. Conclusion and Future Prospects
This review has focused on the construction methods and bio-
medical applications of multiple cellular ingestible DNA nano-
structures. With numerous efforts in the past decades devoted
in designing artificial DNA structures,[8b] DNA nanostructures
with various shapes, structures, and sizes have been constructed.
Additional modularity has also been achieved through modifica-
tion of DNA strands via covalent connection, complementary
base pairing, or other interactions, which enabled the convenient
loading of functional substances (e.g., drugs, fluorophores). The
development of DNA nanotechnology, combined with excellent
biocompatibility, and optimal stability of DNA molecules, makes
DNA nanostructures be excellent materials for a wide range of
applications in biomedical field, including anticancer treatment,
antibacterial treatment, gene editing and cellular regulation, etc.
Although DNA nanostructures are becoming the key materials to
realize safe delivery of functional substances in vivo, there are
still many limitations demanding prompt solution.
First, the imperfect monodispersity, high cost, strict storage
conditions, small preparation scale of DNA nanostructures limit
their further biomedical and clinical applications. When drug
carriers are applied in organisms, especially human bodies,
the homogeneity is strictly required to avoid side effects caused
by the uncertainty of drug dosage. However, construction of the
DNA nanostructures via the methods mentioned above
cannot guarantee their sizes and structures 100% identical.
Furthermore, the number of functional molecules loaded onto
DNA nanostructures through various interactions is still uncer-
tain. Therefore, to achieve applications in human bodies, the syn-
thesis and loading process of DNA nanostructures need to be
further improved. Liu et al. developed a FGA to prepare hetero-
vesicles with programmed geometry and dimensions.[99]

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Figure 8. DNA Nanostructures for stem cell proliferation and differentia-
tion. A) DNA hydrogel loading BMSCs to improve the therapy for
severe osteoarthritis. Reproduced with permission.[97a] Copyright 2021,
Wiley-VCH. B) DNA hydrogel loading NSCs to repair a 2 mm-long spinal
cord cavity. Reproduced with permission.[96a] Copyright 2021,
Wiley-VCH.
Versatile core material such as AuNPs and DNA origami[100]
can be used as the scaffolds to construct well-programmed het-
erovesicles with diverse responsiveness to temperature,[101]
pH,[102] and redox substances.[103] The FGA strategy provided
a generalized platform with improved monodispersity of DNA
nanostructures.[104] Besides monodispersity, the large-scale
synthesis and low cost are the two key factors to realize the indus-
trialization of DNA nanostructures for clinical applications.
Compared with silica nanostructures (cost about $0.1 g1), poly-
mer nanostructures (cost less than $1g1), and carbon nanotubes
(cost less than $50 g1), the expense of DNA molecules is rela-
tively higher for about $50–300 g1, which is too expensive to
achieve the broad clinical application of DNA nanostructures.[105]
Second, the interaction mode and function mechanism of
DNA nanostructures with cells have not been clearly explained,
as well as the mechanism and pathway by which the DNA nano-
structures are internalized by cells. Several researches have
reported the factors influencing the entrance of nanostructures
into cells (e.g., the DNA density of DNA-functionalized
nanoparticles,[106] the shape and size of DNA origami[65a]).
Fan et al. explained the entrance pathways of DNA tetrahedron
and its transportation pathways after entering cell.[65b,107]
However, the detailed pathway of other DNA nanostructures
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has not been reported. In addition, the studies on explaining
the effects of DNA nanostructures on cells (i.e., induction of
immune response, regulation of cytokine expression, and inspi-
ration of cell proliferation, migration, and differentiation) stim-
ulated by the structural properties (e.g., size, shape, loop) or
specific sequence are not sufficient currently, which are signifi-
cantly demanded.
Third, the gene safety and long-term effects of DNA nano-
structures have not been investigated systematically. DNA, as
the main element of DNA nanostructures, remains the probabil-
ity to be integrated into the cellular genome through genetic
recombination. The harmful genetic recombination will lead
to an irreversible change of cellular function and even cell carci-
nogenesis. Furthermore, at present, most of the experiments
were applied in the bodies of mice and the cycle is 3–6 months,
which cannot satisfy the requirement of long-term biosecurity
validation for clinical applications.
To summarize, cellular ingestible DNA nanostructures are
still worth to be explored for potential applications in many fields.
The optimizations and improvements of the synthesis and load-
ing strategies of these DNA nanostructures would make up the
huge gap between the labs and clinical use for perfect monodis-
persity, low cost, simple storage conditions, and mass produc-
tion. While current studies focus on anticancer treatment due
to the excellent permeability of DNA nanostructures to cancer
cells, it has been ignored to apply DNA nanostructures to other
diseases. With the developing research on the interaction and
function mechanism of DNA nanostructures with cells, treating
other cell types is also expected to take the advantage of DNA
nanostructures, but the balance between specific target efficiency
and high permeability should be thoroughly considered. Besides,
combining all the DNA nanotechnology-based delivery system
mentioned above, DNA-functionalized nanostructures can be
an alternative carrier for mRNA vaccine.[108] However, more
studies on strategy to maintain the DNA nanostructures’ integ-
rity in extracellular fluid and to ensure the release of full-length
sequence mRNA after entering the cell are urgently needed to
achieve highly efficient protein expression. Additionally, intact
validation in both cell level and animal models needs to be veri-
fied for further clinical applications.
Acknowledgements
This work was supported by National Natural Science Foundation of China
(nos. 21890731, 21821001, 21971248, 21890730), National Basic Research
Plan of China (2018YFA0208900), and National Center for Nanoscience
and Technology, Chinese Academy of Sciences (E1l0511ZX and
E1XT2611FT).
Conflict of Interest
The authors declare no conflict of interest.
Keywords
biomedical applications, cellular uptake, DNA nanostructures

www.advancedsciencenews.com
Received: August 3, 2022
Revised: October 7, 2022
Published online: December 4, 2022
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Rui Xu graduated at Wuhan University with a B.S. in chemistry in 2021. He is now a Ph.D. student at
Tsinghua University under the supervision of Professor Dongsheng Liu, Yuanchen Dong, and Yuhe
Renee Yang. He focuses on functional biomolecules design and their biomedical applications.

Dongsheng Liu graduated from University of Science and Technology of China in 1993 and did his Ph.D.
study in the Hong Kong Polytechnic University from 1999 to 2002. He then joined the Chemistry
Department of Cambridge University as a postdoc. Dongsheng is now a full professor at Department of
Chemistry, Tsinghua University. He was awarded the 1st “CCS-RSC Young Chemist Award” in 2008 and
the 7th CCS-BASF Youth Innovation Prize in 2014. Dongsheng was invited as FRSC in 2011, became
“Changjiang” Chair Professor in 2015, and fellow of CCS in 2020. His research mainly focuses on the
assembly of DNA and its hybrids.

Yuhe Renee Yang graduated from Tsinghua University in 2007 and started her Ph.D. journey in Arizona
State University. While working with Dr. Hao Yan, she created artificial multienzyme DNA complexes and
networks that addressed important fundamental enzymology issues related to multienzyme catalysis.
During postdoc training in TSRI with Dr. Andrew B. Ward, Renee worked on solving high-resolution
cryoEM structures of HIV/flu surface protein and neutralizing antibody complexes. She started her own
lab in NCNST in 2021. She worked in DNA nanotechnology and structural biology for more than a
decade and continues to find the joy in this interdisciplinary niche.

Adv. NanoBiomed Res. 2023, 3, 2200119 2200119 (14 of 14) © 2022 The Authors. Advanced NanoBiomed Research published by Wiley-VCH GmbH