Multivalent Aptamer-modified DNA Origami as Drug Delivery System for Targeted Cancer Therapy

In spite of great development in nanoparticle-based drug delivery systems(DDSs)for improved therapeutic efficacy,it remains challenging for effective delivery of chemotherapeutic drugs to targeted tumor cells.In this work,we report a triangle DNA origami as targeted DDS for cancer therapy.DNA origami shows excellent biocompatibility and stability in cell culture medium for 24 h.In addition,the DNA origami structures conjugated with multivalent aptamers enable for efficient delivery of anticancer drug doxorubicin(Dox)into targeted cancer cell due to their targeting function,reducing side effects associated with nonspecific distribution.Moreover,we also demonstrated that the multivalent aptamer-modified DNA origami loading Dox exhibits prominent therapeutic efficacy in vitro.Accordingly,this work provides a good paradigm for the development of DNA origami nanostructure-based targeted DDS for cancer therapy.     

PEGylated anti-MUC1 aptamer-doxorubicin complex for targeted drug delivery to MCF7 breast cancer cells

Targeted drug delivery is especially important in cancer treatment as many anti-cancer drugs are non-specific and highly toxic to both cancer and normal cells. The targeted drug delivery of DOX to the MUC1-expressing breast cancer cell line (MCF7) was obtained using APT as a carrier. Modification of the APT-DOX complex by PEG increases the survivability of the macrophage control (RAW 264.7) by about six-fold as compared to free DOX treatment without significantly affecting the cytotoxicity toward the target cell line. Thus, PEG-APT-DOX is potentially a new therapeutic agent for targeted drug delivery to MUC1-expressing cell lines.     

可控自组装DNA折纸结构作为药物载体的研究进展

在过去的几十年里, DNA纳米技术作为一种快速发展的可控自组装技术, 使人们能构建出各种复杂的纳米结构. DNA折纸结构具备可编程性、 空间可寻址性、 易修饰性及良好的生物相容性等多种优越的特性, 这些优异的性质使其在药物递送方面具有广阔的应用前景. 本文总结了近年来可控自组装DNA折纸结构作为药物递送系统的研究进展, 展望了DNA折纸纳米载体未来的发展方向, 并讨论了该领域面临的挑战和可能的解决方法.     

DNA Nanostructures for Targeted Antimicrobial Delivery

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram‐positive (Bacillus subtilis) and Gram‐negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme‐functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.     

Spatiotemporal Control over Polynucleotide Brush Growth on DNA Origami Nanostructures

DNA nanotechnology provides an approach to create precise, tunable, and biocompatible nanostructures for biomedical applications. However, the stability of these structures is severely compromised in biological milieu due to their fast degradation by nucleases. Recently, we showed how enzymatic polymerization could be harnessed to grow polynucleotide brushes of tunable length and location on the surface of DNA origami nanostructures, which greatly enhances their nuclease stability. Here, we report on strategies that allow for both spatial and temporal control over polymerization through activatable initiation, cleavage, and regeneration of polynucleotide brushes using restriction enzymes. The ability to site-specifically decorate DNA origami nanostructures with polynucleotide brushes in a spatiotemporally controlled way provides access to “smart” functionalized DNA architectures with potential applications in drug delivery and supramolecular assembly.     

Multiscale Origami Structures as Interface for Cells

A DNA‐based platform was developed to address fundamental aspects of early stages of cell signaling in living cells. By site‐directed sorting of differently encoded, protein‐decorated DNA origami structures on DNA microarrays, we combine the advantages of the bottom‐up self‐assembly of protein–DNA nanostructures and top‐down micropatterning of solid surfaces to create multiscale origami structures as interface for cells (MOSAIC). In a proof‐of‐principle, we use this technology to analyze the activation of epidermal growth factor (EGF) receptors in living MCF7 cells using DNA origami structures decorated on their surface with distinctive nanoscale arrangements of EGF ligand entities. MOSAIC holds the potential to present to adhered cells well‐defined arrangements of ligands with full control over their number, stoichiometry, and precise nanoscale orientation. It therefore promises novel applications in the life sciences, which cannot be tackled by conventional technologies.     

DNA Origami Disguises Herpes Simplex Virus 1 Particles and Controls Their Virulence

DNA nanostructures are well-established vectors for packaging diversified payloads for targeted cellular delivery. Here, DNA origami rectangular sheets were combined with Herpes Simplex Virus 1 (HSV1) capsids to demonstrate surface coverage of the particle via electrostatic interactions. The optimized origami:HSV1 molar ratios led to characteristic packaging geometries ranging from dispersed “HSV1 pockets” to agglomerated “HSV1 sleeves”. “Pockets” were disguised from cells in HeLa and B16F10 cells and were 44.2% less infective than naked HSV1 particles. However, the pockets were 117% more infective than naked HSV1 particles when the origami sheets were coated with folic acid. We observed infectivity from naked origami, but they are 99.1% less infective with respect to HSV1 and 99.6% less infective with respect to the pocket complexes. This work suggests that DNA origami can selectively modulate virus infectivity.     

A DNA Origami-Based Gene Editing System for Efficient Gene Therapy in Vivo

DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami-based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM-rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM-guided assembly and pre-designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo-loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor-associated gene for gene therapy in vivo. This rationally developed DNA origami-based gene editing system presents a new avenue for the development of gene therapy.     

Single‐Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells

DNA is typically impermeable to the plasma membrane due to its polyanionic nature. Interestingly, several different DNA nanostructures can be readily taken up by cells in the absence of transfection agents, which suggests new opportunities for constructing intelligent cargo delivery systems from these biocompatible, nonviral DNA nanocarriers. However, the underlying mechanism of entry of the DNA nanostructures into the cells remains unknown. Herein, we investigated the endocytotic internalization and subsequent transport of tetrahedral DNA nanostructures (TDNs) by mammalian cells through single‐particle tracking. We found that the TDNs were rapidly internalized by a caveolin‐dependent pathway. After endocytosis, the TDNs were transported to the lysosomes in a highly ordered, microtubule‐dependent manner. Although the TDNs retained their structural integrity within cells over long time periods, their localization in the lysosomes precludes their use as effective delivery agents. To modulate the cellular fate of the TDNs, we functionalized them with nuclear localization signals that directed their escape from the lysosomes and entry into the cellular nuclei. This study improves our understanding of the entry into cells and transport pathways of DNA nanostructures, and the results can be used as a basis for designing DNA‐nanostructure‐based drug delivery nanocarriers for targeted therapy.     

DNA origami: the art of folding DNA

The advent of DNA origami technology greatly simplified the design and construction of nanometer-sized DNA objects. The self-assembly of a DNA-origami structure is a straightforward process in which a long single-stranded scaffold (often from the phage M13mp18) is folded into basically any desired shape with the help of a multitude of short helper strands. This approach enables the ready generation of objects with an addressable surface area of a few thousand nm(2) and with a single "pixel" resolution of about 6 nm. The process is rapid, puts low demands on experimental conditions, and delivers target products in high yields. These features make DNA origami the method of choice in structural DNA nanotechnology when two- and three-dimensional objects are desired. This Minireview summarizes recent advances in the design of DNA origami nanostructures, which open the door to numerous exciting applications.     

DNA纳米结构在药物转运载体和智能载药中的应用进展

纳米材料具有荷载效率高、靶向性能好、半衰期较长等优点, 非常适于作为药物转运载体, 可有效提高药物的水溶性、稳定性和疾病治疗效果.目前, 开发具有良好生物相容性、可控靶向释放能力和精确载药位点的理想药物转运载体, 仍是该领域存在的挑战性问题和当前研究的重点.自组装DNA纳米结构是一类具有精确结构、功能多样的纳米生物材料, 具有良好的生物相容性和稳定性、较高的膜渗透性和可控靶向释放能力等优点, 是理想的药物转运载体和智能载药材料.本文总结了DNA纳米结构的发展历程、DNA纳米结构作为药物转运载体的研究现状、动态DNA纳米结构在智能载药中的应用进展, 并对其发展前景进行了展望.     

Multifunctional DNA Origami Nanoplatforms for Drug Delivery

DNA nanotechnology has been employed in the construction of self‐assembled nano‐biomaterials with uniform size and shape for various biological applications, such as bioimaging, diagnosis, or therapeutics. Herein, recent successful efforts to utilize multifunctional DNA origami nanoplatforms as drug‐delivery vehicles are reviewed. Diagnostic and therapeutic strategies based on gold nanorods, chemotherapeutic drugs, cytosine–phosphate–guanine, functional proteins, gene drugs, and their combinations for optoacoustic imaging, photothermal therapy, chemotherapy, immunological therapy, gene therapy, and coagulation‐based therapy are summarized. The challenges and opportunities for DNA‐based nanocarriers for biological applications are also discussed.     

Self-assembly of Micrometer-long DNA Nanoribbons with Four Oligonucleotides

DNA nanostructures have found widespread applications in areas including nanoelectronics and biomedicine. However, traditional DNA origami needs a long single‐stranded virus DNA and hundreds of short DNA strands, which make this method complicated and money‐consuming. Here, we present a protocol for the assembly of DNA nanoribbons with only four oligonucleotides. DNA nanoribbons with different dimensions were successfully assembled with a 96‐base scafford strand and three short staples. These biotinylated nanoribbons could also be decorated with streptavidins. This approach suggests that there exist great design spaces for the creation of simple nucleic acid nanostructures which could facilitate their application in plasmonic or drug delivery.     

Multiple-mRNA-controlled and heat-driven drug release from gold nanocages in targeted chemo-photothermal therapy for tumors

Multifunctional drug delivery systems enabling effective drug delivery and comprehensive treatment are critical to successful cancer treatment. Overcoming nonspecific release and off-target effects remains challenging in precise drug delivery. Here, we design triple-interlocked drug delivery systems to perform specific cancer cell recognition, controlled drug release and effective comprehensive therapy. Gold nanocages (AuNCs) comprise a novel class of nanostructures possessing hollow interiors and porous walls. AuNCs are employed as a drug carrier and photothermal transducer due to their unique structure and photothermal properties. A smart triple-interlocked I-type DNA nanostructure is modified on the surface of the AuNCs, and molecules of the anticancer drug doxorubicin (DOX) are loaded as molecular cargo and blocked. The triple-interlocked nanostructure can be unlocked by binding with three types of tumor-related mRNAs, which act as “keys” to the triple locks, sequentially, which leads to precise drug release. Additionally, fluorescence-imaging-oriented chemical–photothermal synergistic treatment is achieved under illumination with infrared light. This drug delivery system, which combines the advantages of AuNCs and interlocked I-type DNA, successfully demonstrates effective and precise imaging, drug release and photothermal therapy. This multifunctional triple-interlocked drug delivery system could be used as a potential carrier for effective cancer-targeting comprehensive chemotherapy and photothermal therapy treatments.

Schematic illustration of the multiple-mRNA-controlled and heat-driven drug release from gold nanocages.     

Designed Intercalators for Modification of DNA Origami Surface Properties

The modification of the backbone properties of DNA origami nanostructures through noncovalent interactions with designed intercalators, based on acridine derivatized with side chains containing esterified fatty acids or oligo(ethylene glycol) residues is reported. Spectroscopic analyses indicate that these intercalators bind to DNA origami structures. Atomic force microscopy studies reveal that intercalator binding does not affect the structural intactness but leads to altered surface properties of the highly negatively charged nanostructures, as demonstrated by their interaction with solid mica or graphite supports. Moreover, the noncovalent interaction between the intercalators and the origami structures leads to alteration in cellular uptake, as shown by confocal microscopy studies using two different eukaryotic cell lines. Hence, the intercalator approach offers a potential means for tailoring the surface properties of DNA nanostructures.     

DNA Nanostructures as Drug Carriers for Cellular Delivery

Drug delivery systems have been widely developed for enhancing target activity and improving drug functions.Liposomes,high-molecular polymer,gold nanoparticles and carbon nanomaterials,etc.,are all the candidates of drug carriers.However,immunotoxicity,heterogeneity and low solubility generally exist and hamper their applications.As a kind of biological materials,DNA has its unique advantages in biomedical applications,including excellent biological compatibility and programmability.DNA nanostructures have been proved to possess high cellular uptake efficiency,which sheds new light on DNA-based drug delivery system.In this review,we summarize the influence factors of DNA nanostructure internalization efficiency,including cell lines,and the size and the shape of DNA nanoparticles.Uniformity of DNA nanostructures in appearance and properties ensures the stability in research,which makes DNA carriers stand out from other nanomaterials.Next,we focus on the functionalization of DNA carriers,which endows DNA nanostructures with the potential to construct integrated drug delivery platforms.We also discuss the internalization pathways of DNA nanostructures and their fate in cells.The deeply understanding about the endocytic pathways provides new sight for the further design strategy on changing the transportation routes of DNA carriers in cells.Finally,the challenges in further applications are discussed,and suggestions are proposed.     

Camptothecin@HMSNs/thermosensitive hydrogel composite for applications in preventing local breast cancer recurrence

The CPT was loaded into the HMSNs with the high loading capacity. Then the CPT@HMSNs were loaded into the PLEL thermosensitive hydrogels for local therapy to prevent the recurrence of breast cancer after the tumor was resected.     

DNA Origami Post‐Processing by CRISPR‐Cas12a

Customizable nanostructures built through the DNA‐origami technique hold tremendous promise in nanomaterial fabrication and biotechnology. Despite the cutting‐edge tools for DNA‐origami design and preparation, it remains challenging to separate structural components of an architecture built from—thus held together by—a continuous scaffold strand, which in turn limits the modularity and function of the DNA‐origami devices. To address this challenge, here we present an enzymatic method to clean up and reconfigure DNA‐origami structures. We target single‐stranded (ss) regions of DNA‐origami structures and remove them with CRISPR‐Cas12a, a hyper‐active ssDNA endonuclease without sequence specificity. We demonstrate the utility of this facile, selective post‐processing method on DNA structures with various geometrical and mechanical properties, realizing intricate structures and structural transformations that were previously difficult to engineer. Given the biocompatibility of Cas12a‐like enzymes, this versatile tool may be programmed in the future to operate functional nanodevices in cells.     

Growth and Origami Folding of DNA on Nanoparticles for High‐Efficiency Molecular Transport in Cellular Imaging and Drug Delivery

A novel three‐dimensional (3D) superstructure based on the growth and origami folding of DNA on gold nanoparticles (AuNPs) was developed. The 3D superstructure contains a nanoparticle core and dozens of two‐dimensional DNA belts folded from long single‐stranded DNAs grown in situ on the nanoparticle by rolling circle amplification (RCA). We designed two mechanisms to achieve the loading of molecules onto the 3D superstructures. In one mechanism, ligands bound to target molecules are merged into the growing DNA during the RCA process (merging mechanism). In the other mechanism, target molecules are intercalated into the double‐stranded DNAs produced by origami folding (intercalating mechanism). We demonstrated that the as‐fabricated 3D superstructures have a high molecule‐loading capacity and that they enable the high‐efficiency transport of signal reporters and drugs for cellular imaging and drug delivery, respectively.     

In situ analysis of cisplatin binding to DNA: the effects of physiological ionic conditions

Platinum-based anti-cancer drugs form a major family of cancer chemotherapeutic agents. Cisplatin, the first member of the family, remains a potent anti-cancer drug and exhibits its clinical effect by inducing local DNA kinks and subsequently interfering with DNA metabolism. Although its mechanism is reasonably well understood, effects of intracellular ions on cisplatin activity are left to be elucidated because cisplatin binding to DNA, thus its drug efficacy, is modified by various ions. One such issue is the effect of carbonate ions: cisplatin binding to DNA is suppressed under physiological carbonate conditions. Here, we examined the role of common cellular ions (carbonate and chloride) by measuring cisplatin binding in relevant physiological buffers via a DNA micromanipulation technique. Using two orthogonal single-molecule methods, we succeeded in detecting hidden monofunctional adducts (kink-free, presumably clinically inactive form) and clearly showed that the major effect of carbonates was to form such adducts and to prevent them from converting to bifunctional adducts (kinked, clinically active). The chloride-rich environment also led to the formation of monofunctional adducts. Our approach is widely applicable to the study of the transient behaviours of various drugs and proteins that bind to DNA in different modes depending on various physical and chemical factors such as tension, torsion, ligands, and ions.