Rationally Designed DNA‐Origami Nanomaterials for Drug Delivery In Vivo

The recent decades have seen a surge of new nanomaterials designed for efficient drug delivery. DNA nanotechnology has been developed to construct sophisticated 3D nanostructures and artificial molecular devices that can be operated at the nanoscale, giving rise to a variety of programmable functions and fascinating applications. In particular, DNA‐origami nanostructures feature rationally designed geometries and precise spatial addressability, as well as marked biocompatibility, thus providing a promising candidate for drug delivery. Here, the recent successful efforts to employ self‐assembled DNA‐origami nanostructures as drug‐delivery vehicles are summarized. The remaining challenges and open opportunities are also discussed.     

Progress toward Understanding the Interactions between DNA Nanostructures and the Cell

Advances in DNA nanotechnology empower the programmable assembly of DNA building blocks (oligonucleotides and plasmids) into DNA nanostructures with precise architectural control. As DNA nanostructures are biocompatible and can naturally enter mammalian cells without the aid of transfection agents, they have found numerous biological or biomedical applications as delivery carriers of therapeutic and imaging cargoes into mammalian cells for at least a decade. Nevertheless, mechanistic studies on how DNA nanostructures interact with cells have remained limited and incomprehensive until 2–3 years ago. This Review presents the recent progress in elucidating the “cell–nano” interactions of DNA nanostructures, with an emphasis on three key classes of structures commonly utilized in intracellular applications: tile‐based structures, origami‐based structures, and nanoparticle‐templated structures. Structural parameters of DNA nanostructures and strategies of biochemical modification for promoting intracellular delivery are discussed. Biological mechanisms for cellular uptake, including specific pathways and receptors involved, are outlined. Routes of intracellular trafficking and degradation, together with strategies for re‐directing their trafficking, are delineated. This Review concludes with several aspects of the “bio–nano” interactions of DNA nanostructures that warrant future investigations.     

Daunorubicin‐Loaded DNA Origami Nanostructures Circumvent Drug‐Resistance Mechanisms in a Leukemia Model

Many cancers show primary or acquired drug resistance due to the overexpression of efflux pumps. A novel mechanism to circumvent this is to integrate drugs, such as anthracycline antibiotics, with nanoparticle delivery vehicles that can bypass intrinsic tumor drug‐resistance mechanisms. DNA nanoparticles serve as an efficient binding platform for intercalating drugs (e.g., anthracyclines doxorubicin and daunorubicin, which are widely used to treat acute leukemias) and enable precise structure design and chemical modifications, for example, for incorporating targeting capabilities. Here, DNA nanostructures are utilized to circumvent daunorubicin drug resistance at clinically relevant doses in a leukemia cell line model. The fabrication of a rod‐like DNA origami drug carrier is reported that can be controllably loaded with daunorubicin. It is further directly verified that nanostructure‐mediated daunorubicin delivery leads to increased drug entry and retention in cells relative to free daunorubicin at equal concentrations, which yields significantly enhanced drug efficacy. Our results indicate that DNA origami nanostructures can circumvent efflux‐pump‐mediated drug resistance in leukemia cells at clinically relevant drug concentrations and provide a robust DNA nanostructure design that could be implemented in a wide range of cellular applications due to its remarkably fast self‐assembly (≈5 min) and excellent stability in cell culture conditions.     

Stimuli‐Responsive Self‐Assembled DNA Nanomaterials for Biomedical Applications

Stimuli‐responsive DNA‐based materials represent a major class of remarkable functional nanomaterials for nano‐biotechnological applications. In this review, recent progress in the development of stimuli‐responsive systems based on self‐assembled DNA nanostructures is introduced and classified. Representative examples are presented in terms of their design, working principles and mechanisms to trigger the response of the stimuli‐responsive DNA system upon expose to a large variety of stimuli including pH, metal ions, oligonucleotides, small molecules, enzymes, heat, and light. Substantial in vitro studies have clearly revealed the advantages of the use of stimuli‐responsive DNA nanomaterials in different biomedical applications, particularly for biosensing, drug delivery, therapy and diagnostic purposes in addition to bio‐computing. Some of the challenges faced and suggestions for further development are also highlighted.     

Mesoporous Silica Nanoparticles: Synthesis,Biocompatibility and Drug Delivery

In the past decade, mesoporous silica nanoparticles (MSNs) have attracted more and more attention for their potential biomedical applications. With their tailored mesoporous structure and high surface area, MSNs as drug delivery systems (DDSs) show significant advantages over traditional drug nanocarriers. In this review, we overview the recent progress in the synthesis of MSNs for drug delivery applications. First, we provide an overview of synthesis strategies for fabricating ordered MSNs and hollow/rattle‐type MSNs. Then, the in vitro and in vivo biocompatibility and biotranslocation of MSNs are discussed in relation to their chemophysical properties including particle size, surface properties, shape, and structure. The review also highlights the significant achievements in drug delivery using mesoporous silica nanoparticles and their multifunctional counterparts as drug carriers. In particular, the biological barriers for nano‐based targeted cancer therapy and MSN‐based targeting strategies are discussed. We conclude with our personal perspectives on the directions in which future work in this field might be focused.     

Gold Nanoparticle–Protein Agglomerates as Versatile Nanocarriers for Drug Delivery

The fabrication of a versatile nanocarrier based on agglomerated structures of gold nanoparticle (Au NP)–lysozyme (Lyz) in aqueous medium is reported. The carriers exhibit efficient loading capacities for both hydrophilic (doxorubicin) and hydrophobic (pyrene) molecules. The nanocarriers are finally coated with an albumin layer to render them stable and also facilitate their uptake by cancer cells. The interaction between agglomerated structures and the payloads is non‐covalent. Cell viability assay in vitro showed that the nanocarriers by themselves are non‐cytotoxic, whereas the doxorubicin‐loaded ones are cytotoxic, with efficiencies higher than that of the free drug. Transmission electron microscopy and fluorescence microscopy along with flow cytometry analysis confirm the uptake of the drug‐loaded nanocarriers by a human cervical cancer HeLa cell line. Field‐emission scanning electron microscopy reveals the formation of apoptotic bodies leading to cell death, confirming the release of the payloads from the nanocarriers into the cell. Overall, the findings suggest the fabrication of novel Au NP–protein agglomerate‐based nanocarriers with efficient drug‐loading and ‐releasing capabilities, enabling them to act as multimodal drug‐delivery vehicles.     

Nucleic Acid Nanostructures for Chemical and Biological Sensing

The nanoscale features of DNA have made it a useful molecule for bottom‐up construction of nanomaterials, for example, two‐ and three‐dimensional lattices, nanomachines, and nanodevices. One of the emerging applications of such DNA‐based nanostructures is in chemical and biological sensing, where they have proven to be cost‐effective, sensitive and have shown promise as point‐of‐care diagnostic tools. DNA is an ideal molecule for sensing not only because of its specificity but also because it is robust and can function under a broad range of biologically relevant temperatures and conditions. DNA nanostructure‐based sensors provide biocompatibility and highly specific detection based on the molecular recognition properties of DNA. They can be used for the detection of single nucleotide polymorphism and to sense pH both in solution and in cells. They have also been used to detect clinically relevant tumor biomarkers. In this review, recent advances in DNA‐based biosensors for pH, nucleic acids, tumor biomarkers and cancer cell detection are introduced. Some challenges that lie ahead for such biosensors to effectively compete with established technologies are also discussed.     

Mesoporous Silica Nanoparticles for Intracellular Controlled Drug Delivery

The application of nanotechnology in the field of drug delivery has attracted much attention in the latest decades. Recent breakthroughs on the morphology control and surface functionalization of inorganic‐based delivery vehicles, such as mesoporous silica nanoparticles (MSNs), have brought new possibilities to this burgeoning area of research. The ability to functionalize the surface of mesoporous‐silica‐based nanocarriers with stimuli‐responsive groups, nanoparticles, polymers, and proteins that work as caps and gatekeepers for controlled release of various cargos is just one of the exciting results reported in the literature that highlights MSNs as a promising platform for various biotechnological and biomedical applications. This review focuses on the most recent progresses in the application of MSNs for intracellular drug delivery. The latest research on the pathways of entry into live mammalian and plant cells together with intracellular trafficking are described. One of the main areas of interest in this field is the development of site‐specific drug delivery vehicles; the contribution of MSNs toward this topic is also summarized. In addition, the current research progress on the biocompatibility of this material in vitro and in vivo is discussed. Finally, the latest breakthroughs for intracellular controlled drug release using stimuli‐responsive mesoporous‐silica‐based systems are described.     

Assembling Defined DNA Nanostructure with Nitrogen‐Enriched Carbon Dots for Theranostic Cancer Applications

DNA nanostructures as scaffolds for drug delivery, biosensing, and bioimaging are hindered by its vulnerability in physiological settings, less favorable of incorporating arbitrary guest molecules and other desirable functionalities. Noncanonical self‐assembly of DNA nanostructures with small molecules in an alternative system is an attractive strategy to expand their applications in multidisciplinary fields and is rarely explored. This work reports a nitrogen‐enriched carbon dots (NCDs)‐mediated DNA nanostructure self‐assembly strategy. Given the excellent photoluminescence and photodynamic properties of NCDs, the obtained DNA/NCDs nanocomplex holds great potential for bioimaging and anticancer therapy. NCDs can mediate DNA nanoprism (NP^NCD) self‐assembly isothermally at a large temperature and pH range in a magnesium‐free manner. To explore the suitability of NP^NCD in potential biomedical applications, the cytotoxicity and cellular uptake efficiency of NP^NCD are evaluated. NP^NCD with KRAS siRNA (NP^NCDK) is further conjugated for KRAS‐mutated nonsmall cell lung cancer therapy. The NP^NCDK shows excellent gene knockdown efficiency and anticancer effect in vitro. The current study suggests that conjugating NCDs with programmable DNA nanostructures is a powerful strategy to endow DNA nanostructures with new functionalities, and NP^NCD may be a potential theranostic platform with further fine‐tuned properties of CDs such as near‐red fluorescence or photothermal activities.     

DNA Nanostructures as Pt(IV) Prodrug Delivery Systems to Combat Chemoresistance

Cisplatin is a first‐line drug in clinical cancer treatment but its efficacy is often hindered by chemoresistance in cancer cells. Reduced intracellular drug accumulation is revealed to be a major mechanism of cisplatin resistance. Nanoscale drug delivery systems could help to overcome this problem because of their more active cellular uptake and more accurate tumor localization. DNA nanostructures have emerged as promising drug delivery systems because of their intrinsic biocompatibility and structural programmability. Herein, three diverse DNA nanostructures are constructed and their potential for cisplatin prodrug delivery is investigated. Results found that these DNA nanostructures could remarkably enhance the cellular internalization of platinum drugs and thus increase the anticancer activity, not only to regular lung cancer cells (A549), but more importantly to cisplatin‐resistant cancer cells (A549cisR). Further, in vivo studies also demonstrate that cisplatin prodrug loaded DNA nanostructures could effectively suppress tumor growth in both regular and cisplatin‐resistant tumor models. This study suggests that DNA nanostructures are effective carriers for platinum prodrug delivery to combat chemoresistance.     

Nucleic Acid–Based Functional Nanomaterials as Advanced Cancer Therapeutics

Nucleic acid–based functional nanomaterials (NAFN) have been widely used as emerging drug delivery nanocarriers for cancer therapeutics. Considerable works have demonstrated that NAFN can effectively load and protect therapeutic agents, and particularly enable targeting delivery to the tumor site and stimuli‐responsive release. These outstanding performances are due to NAFN's unique properties including inherent biological functions and sequence programmability as well as biocompatibility and biodegradability. In this Review, the recent progress on NAFN as advanced cancer therapeutics is highlighted. Three main cancer therapy approaches are categorized including chemo‐, immuno‐, and gene‐therapy. Examples are presented to show how NAFN are rationally and exquisitely designed to address problems in cancer therapy. The challenges and future development of NAFN are also discussed toward future more practical biomedical applications.     

Intracellular Labeling with Extrinsic Probes: Delivery Strategies and Applications

Extrinsic probes have outstanding properties for intracellular labeling to visualize dynamic processes in and of living cells, both in vitro and in vivo. Since extrinsic probes are in many cases cell‐impermeable, different biochemical, and physical approaches have been used to break the cell membrane barrier for direct delivery into the cytoplasm. In this Review, these intracellular delivery strategies are discussed, briefly explaining the mechanisms and how they are used for live‐cell labeling applications. Methods that are discussed include three biochemical agents that are used for this purpose—purpose‐different nanocarriers, cell penetrating peptides and the pore‐foraming bacterial toxin streptolysin O. Most successful intracellular label delivery methods are, however, based on physical principles to permeabilize the membrane and include electroporation, laser‐induced photoporation, micro‐ and nanoinjection, nanoneedles or nanostraws, microfluidics, and nanomachines. The strengths and weaknesses of each strategy are discussed with a systematic comparison provided. Finally, the extrinsic probes that are reported for intracellular labeling so‐far are summarized, together with the delivery strategies that are used and their performance. This combined information should provide for a useful guide for choosing the most suitable delivery method for the desired probes.     

Graphene‐Based Smart Platforms for Combined Cancer Therapy

The extensive research of graphene and its derivatives in biomedical applications during the past few years has witnessed its significance in the field of nanomedicine. Starting from simple drug delivery systems, the application of graphene and its derivatives has been extended to a versatile platform of multiple therapeutic modalities, including photothermal therapy, photodynamic therapy, magnetic hyperthermia therapy, and sonodynamic therapy. In addition to monotherapy, graphene‐based materials are widely applied in combined therapies for enhanced anticancer activity and reduced side effects. In particular, graphene‐based materials are often designed and fabricated as “smart” platforms for stimuli‐responsive nanocarriers, whose therapeutic effects can be activated by the tumor microenvironment, such as acidic pH and elevated glutathione (termed as “endogenous stimuli”), or light, magnetic, or ultrasonic stimuli (termed as “exogenous stimuli”). Herein, the recent advances of smart graphene platforms for combined therapy applications are presented, starting with the principle for the design of graphene‐based smart platforms in combined therapy applications. Next, recent advances of combined therapies contributed by graphene‐based materials, including chemotherapy‐based, photothermal‐therapy‐based, and ultrasound‐therapy‐based synergistic therapy, are outlined. In addition, current challenges and future prospects regarding this promising field are discussed.     

Remote Light‐Responsive Nanocarriers for Controlled Drug Delivery: Advances and Perspectives

Engineering of smart photoactivated nanomaterials for targeted drug delivery systems (DDS) has recently attracted considerable research interest as light enables precise and accurate controlled release of drug molecules in specific diseased cells and/or tissues in a highly spatial and temporal manner. In general, the development of appropriate light‐triggered DDS relies on processes of photolysis, photoisomerization, photo‐cross‐linking/un‐cross‐linking, and photoreduction, which are normally sensitive to ultraviolet (UV) or visible (Vis) light irradiation. Considering the issues of poor tissue penetration and high phototoxicity of these high‐energy photons of UV/Vis light, recently nanocarriers have been developed based on light‐response to low‐energy photon irradiation, in particular for the light wavelengths located in the near infrared (NIR) range. NIR light‐triggered drug release systems are normally achieved by using two‐photon absorption and photon upconversion processes. Herein, recent advances of light‐responsive nanoplatforms for controlled drug release are reviewed, covering the mechanism of light responsive small molecules and polymers, UV and Vis light responsive nanocarriers, and NIR light responsive nanocarriers. NIR‐light triggered drug delivery by two‐photon excitation and upconversion luminescence strategies is also included. In addition, the challenges and future perspectives for the development of light triggered DDS are highlighted.     

Cation‐Induced Stabilization and Denaturation of DNA Origami Nanostructures in Urea and Guanidinium Chloride

The stability of DNA origami nanostructures under various environmental conditions constitutes an important issue in numerous applications, including drug delivery, molecular sensing, and single‐molecule biophysics. Here, the effect of Na⁺ and Mg²⁺ concentrations on DNA origami stability is investigated in the presence of urea and guanidinium chloride (GdmCl), two strong denaturants commonly employed in protein folding studies. While increasing concentrations of both cations stabilize the DNA origami nanostructures against urea denaturation, they are found to promote DNA origami denaturation by GdmCl. These inverse behaviors are rationalized by a salting‐out of Gdm⁺ to the hydrophobic DNA base stack. The effect of cation‐induced DNA origami denaturation by GdmCl deserves consideration in the design of single‐molecule studies and may potentially be exploited in future applications such as selective denaturation for purification purposes.     

Development of biocompatible fluorescent gelatin nanocarriers for cell imaging and anticancer drug targeting

In recent years, fluorescent carbon dots (CDs) have attracted a great deal of attention in imaging and related biomedical applications due to their excellent photoluminescence properties, low cost, high quantum yield and low cytotoxicity in comparison with semiconductor quantum dots based on metallic elements. In this paper, a new and simple design for development of CDs/gelatin nanoparticles (CDs/GNPs) is described which used as a novel methotrexate (MTX) nanocarrier and MCF-7 cell imaging. The obtained fluorescent nanocarriers were characterized using FTIR, SEM, XRD, DLS, PL, TGA, and zeta-potential analysis. Afterward, the performance of developed NPs was investigated through different in vitro tests such as MTT assay, fluorescence microscopy, and flow cytometry analyses. MTX was successfully loaded into the fluorescent NPs at physiological pH (7.4) by ionic interactions between anionic carboxylate groups of MTX and cationic amino groups on the surface of NPs. MTX releasing ability of the obtained nanocarrier was illustrated through the comparison of in vitro drug release at both simulated tumor tissue and physiological environment. The MTT assay revealed that the MTX-loaded nanocarriers have higher cytotoxicity in MCF-7 breast cancer cells than nanocarriers without MTX. Upon the obtained results, our fluorescent nanocarriers hold great potential as drug delivery carriers for the targeted MTX delivery to the cancer cells and biological fluorescent labeling.     

Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin‐Receptor Internalization Pathway

DNA origami provides rapid access to easily functionalized, nanometer‐sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA‐based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA‐directed, site‐selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22‐fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface.     

Motion Manipulation of Micro‐ and Nanomotors

Inspired by the self‐migration of microorganisms in nature, artificial micro‐ and nanomotors can mimic this fantastic behavior by converting chemical fuel or external energy into mechanical motion. These self‐propelled micro‐ and nanomotors, designed either by top‐down or bottom‐up approaches, are able to achieve different applications, such as environmental remediation, sensing, cargo/sperm transportation, drug delivery, and even precision micro‐/nanosurgery. For these various applications, especially biomedical applications, regulating on‐demand the motion of micro‐ and nanomotors is quite essential. However, it remains a continuing challenge to increase the controllability over motors themselves. Here, we will discuss the recent advancements regarding the motion manipulation of micro‐ and nanomotors by different approaches.     

Biomimetic Compartments Scaffolded by Nucleic Acid Nanostructures

The behaviors of living cells are governed by a series of regulated and confined biochemical reactions. The design and successful construction of synthetic cellular reactors can be useful in a broad range of applications that will bring significant scientific and economic impact. Over the past few decades, DNA self‐assembly has enabled the design and fabrication of sophisticated 1D, 2D, and 3D nanostructures, and is applied to organizing a variety of biomolecular components into prescribed 2D and 3D patterns. In this Concept, the recent and exciting progress in DNA‐scaffolded compartmentalizations and their applications in enzyme encapsulation, lipid membrane assembly, artificial transmembrane nanopores, and smart drug delivery are in focus. Taking advantage of these features promises to deliver breakthroughs toward the attainment of new synthetic and biomimetic reactors.     

Engineering Biomimetic Platesomes for pH‐Responsive Drug Delivery and Enhanced Antitumor Activity

Biomimetic camouflage, i.e., using natural cell membranes for drug delivery, has demonstrated advantages over synthetic materials in both pharmacokinetics and biocompatibility, and so represents a promising solution for the development of safe nanomedicine. However, only limited efforts have been dedicated to engineering such camouflage to endow it with optimized or additional properties, in particular properties critical to a “smart” drug delivery system, such as stimuli‐responsive drug release. A pH‐responsive biomimetic “platesome” for specific drug delivery to tumors and tumor‐triggered drug release is described. This platesome nanovehicle is constructed by merging platelet membranes with functionalized synthetic liposomes and exhibits enhanced tumor affinity, due to its platelet membrane–based camouflage, and selectively releases its cargo in response to the acidic microenvironment of lysosomal compartments. In mouse cancer models, it shows significantly better antitumor efficacy than nanoformulations based on a platesome without pH responsiveness or those based on traditional pH‐sensitive liposomes. A convenient way to incorporate stimuli‐responsive features into biomimetic nanoparticles is described, demonstrating the potential of engineered cell membranes as biomimetic camouflages for a new generation of biocompatible and efficient nanocarriers.