DNA-Based Nanostructured Platforms as Drug Delivery Systems

Abstract

DNA nanotechnology has recently provided a novel approach for developing safe, biocompatible, biodegradable, non-immunogenic, and non-toxic drug delivery systems. DNA nanostructures have numerous advantages for deployment as drug delivery platforms, owing to programmable assembly, ease of production, reproducibility, and precise control over size, shape, and function. DNA nanostructures can dramatically improve the delivery of poorly soluble drugs, decreasing cytotoxicity to normal tissues and improving therapeutic effectiveness. Using different conjugation methods, DNA nanostructures can be precisely integrated with a wide range of functional moieties, including proteins, peptides, aptamers, polymers, lipids, inorganic nanoparticles, and targeting groups, to enhance the nanostructure stability, extend circulation, and specify drug delivery. Smart DNA nanostructures with targeting ligands or a stimuli-responsive moiety specify therapeutic delivery to target, minimize drug loss attributable to prior drug release or off-target distribution, improve target accumulation, promote cellular internalization, bypass efflux pumps, and avoid adverse effects. Target-specific delivery by smart DNA nanostructures, in turn, maximizes drug concentration, reaching the target locations at a faster rate and preventing therapeutic failure while also lowering the dose needed for therapeutic effect. This Review provides an overview of DNA nanostructures for drug encapsulation and selective delivery to the desired sites. DNA nanostructures are a novel platform for drug delivery with improved performance that may be used to treat a variety of diseases.

1. Introduction

Many pharmacological APIs have been found for the treatment of various diseases; however, substantial issues like poor absorption, low permeation, non-selective distribution, and instability restrict their clinical usefulness, linked with adverse effects and suboptimal therapeutic effect. DNAs are the biomaterial building blocks that self-assembled to form DNA nanostructures of one-dimensional, two-dimensional, or three-dimensional structures with specifically controlled sizes and shapes.¹ DNA nanostructures are highly accessible and functionally diverse platforms for pharmaceutical and biomedical applications. DNA nanostructures exhibit good self-assembly, mechanical stiffness, structural stability, molecular recognition, biocompatibility, biodegradability, moldability, replicability, and predictability, as well as low-immunogenicity and non-toxicity.^2,3 DNA nanostructures can range from few nanometer to microscale with a predetermined geometries and structure.⁴ The predesigned structural features may include size, shape, and surface of DNA nanostructures that regulate in vivo translation. DNA nanocarriers with spaces or voids can be designed that can effectively hold drug payloads free from interference from the external environment and thus giving a stealth carrier for drug.⁴ Therefore, DNA nanostructures can be widely employed as a safe and effective platform with a higher performance and drug delivery.

DNA nanostructures have recently been designed for numerous drug delivery, imaging, and theranostics applications. DNA nanostructures may considerably improve the therapeutic effectiveness of drugs by enhancing drug solubility, improving systemic circulation and providing better penetration and higher cellular internalization.⁵ DNA nanostructures have numerous benefits, including ease of fabrication, tunable size/shape, and diverse chemical modification avenues. The addition of molecules like metals, polymers, proteins, lipids, or nucleic acid to DNA nanostructures can alter their size, shape, charge, conformation, surface properties, structural stability, therapeutic encapsulation, and cellular uptake efficiency. DNA origami nanostructures may control the release of drug sandwiched in the bilayer structure for improved drug delivery and obtain better therapeutic benefits.⁶ DNA nanostructures come in a range of sizes and forms, which affect the drug distribution in vital organs and target sites. Smaller DNA nanostructures have higher cellular uptake than larger particles due to tumor passive targeting.⁷ Tetrahedral DNA nanocages display “like charge attraction” at the interface of cytoplasmic membranes, preferentially approaching cells with corners to reduce electrostatic repulsion, producing unequal charge redistribution in the membrane, and are endocytosed to enter relying on the caveolin- or clathrin-mediated endocytosis.^8,9

However, lack of specificity may result in non-target distribution, organ toxicity, and therapeutic failure. DNA nanostructures having high surface functionalization avenues are utilized to modify the physicochemical characteristics, specify the drug delivery, increase cellular internalization, and control drug release kinetics in target cells to provide superior therapeutic effects. By integrating the benefits of DNA nanostructures with cell-targeting moiety or stimuli responsive molecules, a new way to overcome limitations of chemotherapeutic drugs such as physiological instability, prior drug loss due to enzymatic degradation or high first pass effect, off target distribution, cytotoxicity, etc., can be obtained.

This Review discusses DNA nanostructures for drug encapsulation and selective distribution to specific locations for better clinical outcomes. First, a brief overview of the design of DNA nanostructures followed by a comprehensive account of DNA nanostructures for drug encapsulation, receptor targeting, stimuli responsive drug delivery, and dual targeted drug delivery is detailed. Finally, the applicability of DNA nanostructures in the therapy of various disorders is discussed.

2. Design of DNA Nanostructures

The fabrication of complex DNA nanostructures for therapeutic applications relies on the self-assembly of DNA strands.¹⁰ The distinct characteristic of DNA as a building block for the synthesis of nanostructures is the dual role that it plays as a biomaterial and as a glue for spontaneous assembly. Approaches most widely employed for the design of complex 2D and 3D nanostructures includes base-pairing, rolling-cycle amplification, tile-based assembly, DNA origami assembly, catalytic-hairpin reaction, hybridization-chain reaction, and composite material assembly.¹¹⁻¹³ The specific Watson–Crick base pairing allows the self-assembly of 2D and 3D DNA nanostructures of various shapes such as cubes, tetrahedrons, octahedrons, hollow cylindrical, rods, etc. Another approach used for the fabrication of DNA nanostructures is an isothermal enzymatic technique which generates of long single-stranded DNA known as rolling cycle amplification (RCA).¹⁴ This RCA-generated ssDNA can self-assemble to form highly stable and multifunctional complex 3D nanostructures such as sponges, flowers, capsules, etc. Li et al. prepared an ultra-long DNA chain via the phi29 polymerase-catalyzed RCA reaction that subsequently captured Cas9/sgRNA RNP via complementary base pairing with sgRNA and assembled into spherical-shaped nanoparticles.¹⁵

In tile-based assembly, the DNA triangular tile (multiarm junction) is formed by sticky end cohesion of circularizing DNA strands which self-assemble in 2D superstructures or lattices upon annealing with staple strands. Due to the obvious helical shape and curvature of the DNA, these 2D superstructures may fold and spin upon themselves, resulting in compact 3D DNA nanostructures such as hydrogels, crystals, dendrimer-like DNA nanostructure, etc.^16,17 The DNA origami technique utilizes scaffold DNA to design the nanostructure with predefined architecture and utilizes staple strands to hold the structure and subsequently endows functional properties necessary for loading therapeutics and diagnostic payloads.¹⁸ The scope of functional modification, controllable morphology, and precision in fabrications has made this technique an ideal approach for the development of compatible and efficient DNA-based nanostructures for drug delivery and theranostic applications.¹⁹ DNA nanostructures due to their characteristic shape and size can readily internalize cells via various uptake mechanisms like lysosomal escape, endocytosis, or active transport.¹³ Therefore, DNA nanostructures can be used as a potent carrier for selective cellular delivery of therapeutics for various biomedical applications.

2.1. DNA Nanostructures

DNA has outstanding chemical and structural features that allow for facile alteration and precision programmability in drug delivery applications. Over the last two decades, tremendous progress has been achieved in the field of DNA nanotechnology toward the fabrication of many types of self-assembled DNA nanostructures (for example, DNA wireframe, DNA origami, DNA tetrahedral nanostructure, DNA hydrogel, DNA nanotube, and so on).¹¹ Originally, the term “scaffold DNA origami” referred to a revolutionary method for transforming a long, single-stranded DNA scaffold into a desired tailored structures.²⁰ DNA origami was first reported by Rothemund in 2006 as DNA-based self-assembly into arbitrary two-dimensional structures. The structures were in the nanorange and of various shapes such as squares, disks, and five-pointed stars. DNA origami can further be programmed into larger assemblies like periodic lattices and a hexamer of triangles (30 MDa molecular complex).¹⁸ Later, by stacking multiple origami, 3D nanostructures can also be fabricated with more rigid structures. DNA origami nanostructures are larger dimension framework structures of various shapes (tube, box, sphere, bundle, cookie, gridiron, 12-tooth-gear, disk, donut) that may provide a high cargo loading and better biostability compared to smaller DNA nanostructures.^13,21,22 However, a large size, complex and time-consuming fabrication process, and compromised cell and tissue penetration ability demand simple and smaller nanostructures. Therefore, smaller origami nanostructures have recently been designed for drug delivery applications.

DNA assembly of small number of short DNA strands can result in the DNA wireframe of various nanostructures, such as tetrahedrons, octahedron, icosahedron, pyramidal, and nanotubes.^13,23 These structures were usually smaller than DNA origami. For example, tetrahedral DNA nanostructure (TDN) is known to have remarkable cell- and tissue-penetrating capacity owing to its size and geometry. TDN can be effectively used for the delivery of various bioactive molecules including antisense peptide nucleic acid, DNA aptamers, or small-molecular-weight drugs. The active delivery can be achieved using TDN modified with targeting or stimuli-sensitive molecule that exhibits receptor-mediated internalization or conformation change for on-site cargo release.²⁴ This could avoid the undetermined cell penetration ability and off-target distribution of DNA-based delivery vehicles. The porous DNA wireframe can be molded into wide range of structures for providing scaffolds for payload.²⁵Figure 1 includes a few forms of DNA wireframe arrays, tubes, and polyhedra.

Figure 1.

Wireframe DNA nanostructures: (a) hexagonal, (b) square, (c) triangular, (d) trihexagonal, (e) snub trihexagonal, (f) chimeric pattern, (g & h) two irregular triangular patterns, (i) cobweb-like pattern, (j) straight tube, (k) donut, (l) U-180°-bent tubes, (m) U-135°-bent tubes, (n) U-90°-bent tubes, (o) tetrahedron, (p) octahedron, (q) cuboctahedron, (r) icosahedron, (s) triangulated cube, (t) triangulated buckyball. Reproduced with permission from ref (25). Copyright 2019, The Author(s).

DNA nanocages and hydrogels are other three-dimensional nanostructures with predesigned size and shape. DNA nanocages can be designed as polyhedra (tetrahedra, truncated octahedra, dodecahedra, icosahedra, and truncated icosahedra), cubes, triangular bipyramids, and others. Although DNA nanocages like tetrahedra have good drug carrying capability but poor cellular uptake and nuclease sensitivity limits its drug delivery capability that can be mitigated by surface functionalization.¹¹ DNA hydrogels network can alter as response to microenvironment stimuli (pH, heat, nuclease, or GSH), thereby resulting in water uptake, swelling, and drug release.¹¹ DNA hydrogels have enormous drug delivery potential, making them perfect DDS for topical drug administration.

2.2. DNA-Hybrid Nanostructures

DNA hybrid nanostructures can be created by assembling organic and inorganic materials such as metal ions, proteins, polymers, and lipids, which considerably improves the performance of DNA nanostructures. For example, Xie et al. described the use of an octopus-shaped DNA nanostructure integrated with graphene oxide for the precise detection of hepatitis B virus DNA.²⁶ DNA hybrid nanostructures are reported to have significantly higher drug encapsulation, extended stability, improved cellular internalization capability, and a modulated drug release. Thus, DNA hybrid nanostructures yield drug nanocarriers with superior drug transport potential and minimal side/toxic effects, resulting in improved therapeutic outcomes. A few DNA-hybrid systems that can be used efficiently for drug delivery applications are detailed below.

2.2.1. DNA-Metal Hybrid Nanostructures

Jia et al. reported a metal-DNA nanosphere with larger surface area and high stability. Resultant DNA hybrid structure was biocompatibile and had low toxicity with a high payload efficiency and provided intracellular delivery.²⁷ Another DNA hybrid nanostructure consisting of DNA nanowires conjugated with gold nanoparticles was designed.²⁸ Divalent metal ions (Mg²⁺, Zn²⁺, and Mn²⁺) were observed to favor Mithramycin (MTR) interaction with AS1411-tethered DNA nanotrains (AS1411NTrs) for drug loading and sustained release effect. Mg²⁺, Zn²⁺, and Mn²⁺ interact with the oxygen atoms of MTR molecules to create dimer complexes that enhance the binding of MTR to DNA via hydrogen bonding with the NH₂ of guanine in GC-rich DNA. DNase I was discovered to hasten the release of MTR dimers from the DNA nanostructure, and the order was (MTR)₂Mg²⁺ > (MTR)₂Mn²⁺ > (MTR)₂Zn²⁺. The resulting drug-loaded DNA nanosystems increased cancer cell lethality while decreasing adverse effects. (MTR)₂Zn²⁺ efficiently targets HepG2 cancer cells via AS1411 aptamer binding to nucleolin while reducing harmful side effects on L02 normal cells.²⁹

2.2.2. DNA-Peptide/Protein Hybrid Nanostructures

DNA-peptide hybrid nanostructures combine the properties of peptides and DNA in a single configuration, making them a unique carrier. Peptides can be covalently or non-covalently linked to DNA nanostructures. DNA-peptide hybrid nanostructures have strong structural programmability of DNA and various peptide (oligopeptides/polypeptides) or protein sequence functionality.³⁰ Because of the combined benefits of the DNA nanostructure and peptide activity, the hybrid structure can have a more dramatic effect in drug delivery. Baral et al. constructed W4R4 peptide bound self assembled branched DNA nanostructure (bDNA) to improve the internalization of bDNA in anionic cells.³¹

Wang et al. investigated the potential of cationic amino acids such as arginine and lysine in stimulating DNA assembly to produce Mg²⁺-free 2D DNA origami. When compared with Mg²⁺-assembled DNA nanostructures, DNA self-assembly depends on the neutralization of the negative charge carried by the phosphonate group of DNA and has been reported to have greater structural and serum stability. The diameters of DNA origami and DNA nanotubes were 56.6 ± 0.90 and 14.0 ± 0.86 nm, respectively. The amino acid/DNA complex nanomaterials demonstrated unique cell interface characteristics, allowing for improved cellular localization.³² Kim et al. investigated the function of protein and DNA in the formation of self-assembled linear nanostructures by Watson–Crick base-paring of DNAs. The traptavidin-DNA conjugates combine to form protein-DNA complex polymers, which are then utilized as a one-dimensional template to assemble gold nanoparticles and build linear plasmonic nanostructures with predefined inter-particle spacings. Such plasmonic nanostructures have the potential to boost optical signals for bioimaging applications.³³

2.2.3. DNA-Polymer Hybrid Nanostructures

DNA may be utilized as a blueprint for building nanoplatforms. As DNA is anionic, it may attach to oppositely charged polymers to produce DNA nanostructures with a variety of forms and characteristics. Cationic polymers (like polylysine, polyamidoamine, and polyethylenimine can easily interact with DNA electrostatically to form a unit structure with characteristic morphology.³⁴ Pakornpadungsit et al. demonstrated the development of a foam complex as a result of DNA-chitosan binding. The mechanical properties of the foam were sufficient to provide a scaffold and had no cytotoxicity on cells. Furthermore, chitosan content impacted surface morphology, with high content (ratios 0.25:1, 0.5:1, and 1:1) producing a smooth surface and high DNA content (ratios 1:0.5 and 1:0.25) producing a rough surface. The structures exhibited large-scale holes (100–1000 m), and the percentage porosity decreased as the DNA concentration increased.³⁵ Kasyanenko et al. investigated the usage of DNA for interaction with cationic polymer (poly-N,N-dimethylaminoethyl methacrylate) stabilized silver nanoparticles in order to produce DNA-polycation complexes containing silver nanoparticles, DNA–PDMAEM–AgNPs, as well as dye binding.³⁶

The hybridization chain reaction (HCR) was used to create a DNA-polymer nanoframework (NF) that combines a polymeric nanoframework with DNA nanotechnology for mRNA transfection. NF was made up of polymeric skeleton N-isopropylacrylamide (NIPAM), cross-linker acrydite-DNA, GSH-cleavable linker, N,N′-bis(acryloyl)cysteamine (BACA), and DNA hairpins (H1-polyT and H2-siRNA). Through HCR, siRNA and poly(thymidine acid) (polyT) sequences were absorbed into NF, and the internal space of the DNA nanoframework was expanded at the same time. Below the lower critical solution temperature (LCST, 34 °C), the NF swells to expose polyT sequences to hybridize with the polyadenylic acid (polyA) tail of mRNA due to the phase transition characteristic of polymeric NIPAM and sequence-specific molecular recognition. The NF deswells above the LCST in order to enclose mRNA. (Hsp27 siRNA) augmented the mRNA transfection in macrophages.³⁷ A similar DNA nanoframework was reported by Song et al. made up of N-isopropylacrylamide (NIPAM) as the primary skeleton and N,N-bis(acryloyl)cystamine (BACA) and acrylamide-modified DNA (acDNA) as cross-linkers. Because of dynamic assembly/disassembly, the resulting nanoframework (NF) was capable of enabling regulated release of Cas9 RNP. By overcoming steric hindrance, NF swelled under LCST for the high-capability loading of Cas9 protein and aggregated across LCST to form uniformly sized nanostructures. Disulfide bonds in the BACA cross-linker selectively responded to intracellular GSH and were broken, causing the disintegration of CH-NF. RNase H overexpression in cancer cells particularly digested the RNA in the RNA-DNA complex, resulting in Cas9 RNP controlled release.³⁸

2.2.4. DNA-Lipid Hybrid Nanostructures

DNA nanostructures, although, have good clinical applications. However, their intrinsic properties such as immune system activation, instability in physiological environments, and a short lifespan are major concern. The integration of DNA with lipids, clinically acceptable and safe material for anticancer drug delivery, can improve the translation and therapeutic value, avoiding undesirable responses.³⁹ DNA nanostructures can be used for coating, clustering, and pattern sub-100 nm liposomes to give a robust and versatile vesicle networks, strings, and dimers with characteristic structural feature facilitating functionalization with target molecules and avoided liposome fusion.⁴⁰ Lim and Hwang designed DNA nanostructure immobilized liposomes for co-delivery of mRNA and doxorubicin to cancer cells. Herein, aptamer-modified tetrahedron directed the delivery to nucleolin overexpressing cancer cells via lipid-raft-mediated endocytosis.⁴¹

The DNA nanostructures equipped with lipids can result in a system with a high drug carrying capability. Willem de Vries et al. described lipid-modified DNA-strand-based NPs that are simple to functionalize and can be loaded with drug molecules. DNA aptamer and RNA aptamer were extended at the 3′ end to bind kanamycin B and neomycin B, respectively, and their pairing with DNA nanoparticles results in drug coloaded nanocarrier systems with excellent biocompatibility, higher adhesion to the corneal surface for an extended period responsible for close contact with the corneal surface, and antibiotic activity retention. Under conditions simulating tear fluid discharge, the NPs demonstrated greater bactericidal activity for up to 30 min.⁴²

2.3. L-DNA Nanostructures

Three-dimensional DNA nanostructures are often limited in scalability, whereas an L-DNA duplex (L-ds) as a drug carrier may be easily produced due to its scalability. L-DNA is the D-DNA enantiomer that can readily hybridize with the complementary L-DNA sequence via Watson–Crick base-pairing to create an L-DNA duplex as a left-handed B-helix, a mirror structure of D-right-handed DNA’s B-helix. The L-DNA duplex provides improved permeability and retention (EPR) for passive tumor accumulation. L-DNA duplex has adequate cellular uptake and tumor accumulation capability for intracellular tumor delivery of loaded medication, resulting in significant cytotoxicity and tumor growth suppression. Dox@L-ds demonstrated intracellular absorption of L-ds, releasing drug more rapidly and sustainably in endosomes with acidic pH (pH 5–6). The systemic toxicity induced by free Dox was significantly decreased when the medication was delivered with L-ds.⁴³ Kang et al. created an L-DNA tetrahedron (L-Td) that showed increased tumor accumulation of loaded DOX as well as greater cellular penetration via endocytosis. Furthermore, clearance was much lower with DOX-loaded L-Td (DOX@L-Td) than with free DOX, resulting in extended systemic circulation. The half-life of L-Tds was almost double that of free DOX. Furthermore, AUC for DOX@L-Td groups rose 4-fold when compared to the free DOX group, whereas clearance dropped more than 6-fold.⁴⁴

3. Effect of Physicochemical Properties of DNA Nanostructures on Drug Delivery

Tuning the design of the DNA nanostructure may affect the encapsulation efficiency, cell internalization, drug release, and cytotoxicity. DNA origami nanostructures are an effective drug delivery method with high levels of internalization and the ability to customize the drug release kinetics. Straight DNA nanostructures demonstrated comparable cytotoxicity and release kinetics to those of plasmid DNA. Twisted nanostructures, on the other hand, provided better drug delivery. The use of DNA origami nanostructures improves therapeutic effectiveness by offering therapeutic effects at lower concentrations than free drug.⁴⁵ The size, shape, and aspect ratio were also shown to have substantial impacts on endocytosis or cell internalization. Larger particles with higher compactness were preferentially internalized over elongated, high-aspect-ratio particles.⁴⁶

Umemura et al. explored the role of DNA nanostructure size, shape, charge, and binding capacity on cellular uptake of payload. The cellular intake of the ODN-1-loaded polypodna was shown to be dependent on the DNA structure, with hexapodna having the greatest value, followed by pentapodna, tetrapodna, tripodna, dsDNA, and ssDNA. Polypodna interacted more efficiently than ssDNA to enhance uptake. Furthermore, the greater size and increased charge density of DNA-polypodna may improve contact and binding with surface membrane receptors, which are important for effective cellular import. Polypodnas bind to the macrophage scavenger receptor 1 (MSR1) particularly for better performance.⁴⁷ Huang et al. showed the effect of the shape of DNA nanoarchitectures, such as cages, tubes, and plasmids, on drug loading, cellular absorption, and anticancer effectiveness. The results revealed that DNA cages and DNA tubes outperformed plasmids in terms of the drug loading and entrapment effectiveness. The increased surface area of the cages and tubes may have resulted in enhanced interaction between DNA and RuPOP. In addition, the drug loading and trapping effectiveness of DNA cages were greater than that of DNA tubes.⁴⁸ Kang et al. showed the influence of size of the DNA nanostructure on the drug carrying capability and rate of cellular absorption. L-Td30, the long chain L-Td, was found to carry more DOX molecules than L-Td17. While smaller-sized L-Td was shown to accumulate more readily in cancer cells and had higher cellular internalization than L-Td30. DOX@L-Td17 exhibited about 2-fold lower clearance, rapid tumor accumulation, and a higher absorption rate for loaded drug than DOX@L-Td30.⁴⁴

Kim et al. designed distinct forms of wireframe self-assembled DNA cages, including pyramid (D-Py), triangular prism (Tp), cube (Cb), and rugby ball-like construct (Rb) with four different backbones, namely, D-DNA (D), L-DNA (L), 2′-OMe-RNA (M), and 2′-F-RNA (F) (F). Designed nanostructures were studied for tumor specificity, cellular uptake, and antitumor effect. L-cages were more tumor specific than F- and M-cages, whereas Py was the most tumor specific, followed by Tp, Rb, and Cb. After 30 min of injection, the cages of various forms reached the tumor region, with maximal accumulation occurring after 2–6 h. Except for D-cages, all cages accumulated in tumors, with minimal distribution to the kidney, lung, and liver. The shape and backbone of cages were shown to affect cage internalization into macrophages, and the order of uptake was L  ∼  F > M  ≫  D. Cb > Tp > Rb > Py was the sequence of shape-dependent macrophage absorption. The form of the nanostructure impacted tumor delivery. Tp was discovered to have more tumor localization potential than L- and M-cages (L-Tp followed by F-cages, M-cages, and D-cages). L-type backbones should have better serum stability than D-type backbones, including D-RNA backbones (2′-OMe-RNA and 2′-F-RNA). L > F > M > D was the order of tumor accumulation. D-Cb entered cells via energy-dependent endocytosis. Endocytosis processes were involved in the cellular absorption of Dox loaded on L-Py. Dox@Pys had a greater anti-tumor efficacy than Doxil, and the order of potency was Dox@L-Py > Dox@F-Py > Dox@M-Py. Dox@Pys had an IC50 value that was 3–7 times lower than free Dox and 1.5–3 times lower than Doxil. Micropinocytosis was responsible for the cellular absorption of all L-cages. During the cellular absorption of the Py shape in the modified cages, clathrin-mediated endocytosis was implicated. All modified cages had caveolae-mediated endocytosis. The cages were extremely biocompatible, low cytotoxic, and non-immunogenic.⁴⁹ Li et al. reported unimolecular, bimolecular, and tetramolecular G-quadruplex nanostructures grafted onto tumor cell-targeting aptamer Sgc8 (T-GMVs) for targeted drug delivery. Aside from cell recognition specificity and binding affinity of the resultant DNA nanostructures for target cells, high TMPyP4 loading potential and improved cancer treatment effectiveness were found. The unimolecular G-quadruplex-based 4G3-Sgc8 was shown to be more appropriate for targeted drug delivery, with strong tumor cell-targeting ability, effective encapsulation, and the desirable photodynamic treatment effect.⁵⁰

4. Therapeutics Encapsulation by DNA Nanostructures and Associated Advantages

DNA nanostructures can be utilized successfully and efficiently for drug encapsulation. Drug loading in DNA nanostructures can mainly be achieved via intercalation, ligand-based recruitment, DNA strand-modification, hybridization, conjugation, or physical encapsulation,^51,52 for example, binding of DOX to specific base pairs or groups present on DNA nanostructures.⁵³ The interaction results in a stable structure to give a suitable carrier for drug delivery application. The drug loading can be efficiently achieved in various forms of DNA nanostructures. DOX loading into different 2D (triangle, L-shaped, bowtie) and 3D (24 helix bundle, capsule) scaffolded DNA origami nanostructures (DONs) was described by Ijäs et al. The DONs had similar DOX binding capabilities, with one DOX molecule binding for every two to three base pairs and the binding equilibrium being achieved in seconds.¹ He et al. constructed rectangle shape DNA nanostructure for carrying doxorubicin to MCF-7 cells overexpressing cell-specific enzyme (NQO1) and avoided delivery to healthy L-02 cells.⁵⁴ Jiang et al. also designed tubular- and triangular-shaped DNA nanostructures for DOX loading by intercalation (Figure 2). The drug-loaded DNA structures effectively internalized the cancer cells where drug is released as a result of low pH or DNA enzymes induced slow degradation.⁵⁵ Similarly, Zhang et al. exploited DNA nanostructures of triangle, square, and tube shapes for drug loading and determining structure-dependent cellular accumulation (Figure 3). The drug DOX was loaded by intercalation into DNA origami nanovehicles. Triangle-shaped origami exhibited specific and higher tumor accumulation compared to tube- and square-shaped origami that were also distributed in liver and kidney. In an acidic tumor microenvironment, the drug is released from DNA origami to exert its action.⁵⁶

Figure 2.

DNA origami structures for drug loading. AFM images of tubular and triangular DNA origami pre- and post-DOX intercalation. Reproduced with permission from ref (55). Copyright 2012 American Chemical Society.

Figure 3.

Drug-loaded DNA origami structures. (a) ssDNA scaffold hybridizes with helper strands for fabrication of triangular, square, and tube origami structures, used for in vivo biodistribution study. Watson–Crick base pairs in the double helices of DNA origami served as docking sites for doxorubicin intercalation (red triangle origami). (b) Tail-vein injection of origami complexes resulted in accumulation in breast tumor of nude mice via EPR effects where drug is released. Reproduced with permission from ref (56). Copyright 2014 American Chemical Society.

DNA nanostructures can be efficiently used for encapsulation of hydrophobic drugs to improve the aqueous solubility and therapeutic efficacy of payload. Liang et al. reported DNA nanotubes as drug carrier where drug is loaded via π–π interactions. DNA nanotubes of various structure (tetramer, hexamer, and octamer) were constructed for delivery of doxorubicin, daunorubicin, taxol, and vinblastine.⁵⁷ DNA-nanowires (DNA-NWs) produced for loading of CPT found to have encapsulation ranging from 66.85% to 97.35%. Prepared DNA-NWs were homogeneous in shape with lengths ranging from 2 to 4 m and widths ranging from 150 to 300 nm. CPT@DNA-NWs were discovered to have a lower IC50 value of 12.8 nM than free CPT solution (IC50 = 51.2 nM). CPT-DNA-NWs were biocompatible and exhibited strong cytotoxicity at lower concentrations compared to free CPT where the apoptosis increased to 22% compared to placebo DNA-NWs having only 7% apoptosis.¹⁶

DNA nanostructures can improve the stability, transport, and permeation potential of encapsulated drug to desired site, subsiding the toxic and side effects on normal cells or organs in order to improve the safety and therapeutic efficacy of drug. Zhong constructed DNA nanostructures for cisplatin prodrug delivery. Prepared DNA nanostructures provided remarkably enhanced distribution of platinum drugs not only to regular lung cancer cells (A549) but also to cisplatin-resistant cancer cells (A549cisR) and thus increased the anticancer activity. Drug-loaded DNA nanostructures were relatively stable under physiological conditions but actively released the drug from nanostructure inside the cancer cells with high glutathione where prodrug Pt(IV) reduce to its active from Pt(II) through elimination of the axial ligands.⁷ Wang grafted erlotinib onto the azide-modified DNA strands (T15-N3) via a classical Cu(I)-catalyzed azide alkyne cycloaddition-click reaction to give the T15-Er strand, that with A15 strands assembled to form Er-DNA structure for erlotininb delivery to cancer cells. The Er-DNA structure was relatively stable in PBS, released drug in acidic environments due to the decomposition of DNA structures, exhibited enhanced cytotoxicity toward A549 cells, markedly reduced the tumor growth, and demonstrated the low toxicity and side effects in normal cells.⁵ A tetrahedral framework nucleic acid (tFNAs) was used for delivery of Erythromycin into Escherichia coli. The initial particle size and zeta potential of tFNAs-Ery were 18.94 ± 3.087 nm and 5.11 ± 3.21 mV, respectively, which changed after drug loading to 53.94 ± 12.52 nm and 16.0 ± 8.44 mV. tFNAs-Ery improved drug permeation to the bacterial cell wall, reducing the possibility of drug resistance and increasing erythromycin’s antibacterial activity against E. coli when compared to erythromycin alone. Erythromycin administration using tFNAs was more deadly, causing significant damage to the bacterial membrane structure compared to that with free erythromycin. tFNAs-Ery were discovered to be safe and structurally stable in simulated bacterial internal environment where bacteria grow and metabolize.⁵⁸

5. Delivery of DNA Nanostructures via Various Routes of Administration

As stated above, DNA nanostructures can be effectively used for therapeutic encapsulation and delivery to desired site. These nanostructures can be given via various routes of administration including oral, topical, ophthalmic, or systemic for therapeutic payload delivery.

5.1. Oral Delivery

The use of DNA nanostructures for oral drug delivery seeks to shield the loaded payload from the gastrointestinal environment, allowing for safe drug delivery to a suitable absorption region. Such approaches avoid drug wastage while reducing dosage, frequency, and side effects, and they provide improved therapeutic benefits. Baig et al. described the use of Eudragit-coated DNA-Nanospheres for Vildagliptin loading. The prepared nanospheres (Eud-DNA-NS) were stable, smooth, and homogeneous in size with an entrapment effectiveness of up to 92%. Eud-DNA-NS was able to safely circumvent the acidic environment of the stomach and enter the intestine for improved absorption without the risk of pancreatitis. The nanosphere prolonged drug release for up to 15 ± 2 h and improved glycemic control in Db/Db mice or pancreatic cancer after oral administration.⁵⁹ In another work, Baig et al. employed a 3D nanocube to encapsulate Vildagliptin, which binds to DNA owing to the secondary amine in its chemical structure. Negatively charged DNA nanocubes were incubated with PLL to achieve positive charge and then coated with negatively charged Na-alginate through an electrostatic attraction mechanism to produce acid-stable nanospheres for VI oral administration. The spherical nanospheres were homogenous and nanosized (40–150 nm), with high drug entrapment effectiveness (70–83%), prolonged drug release (13 ± 4 h), and superior anti-diabetic actions. The designed nanospheres bypassed the stomach and reached the target location (intestine) for absorption, minimizing side effects and decreasing dose frequency while improving glycemic control.⁶⁰

5.2. Ophthalmic Delivery

The effectiveness of topical eye drops is limited by their short lifespan on the eye surface, necessitating high drug dosages and frequent administration, yet they are rarely bioavailable. To address these limitations, DNA nanocarriers were introduced that remain adhere to the corneal surface for prolonged time, responsible for improved effectiveness.⁴² For drug loading and its ophthalmic delivery, lipid DNA nanoparticles were hybridized with a DNA aptamer bound to the antiglaucoma drug travoprost. Drug-loaded DNA-lipid NPs were found to exhibit inherent affinity for the ocular surface, high retention on the ocular surface, and long-lasting adhesion to the eye for about 60 min following eye drop instillation. Trav-DNA-NPs were more biocompatible, safe, and effective than free medication. Trav-DNA-NPs supplied the drug in twice the quantity of free pristine drug and shown increased effectiveness for the treatment of glaucoma, a severe retinal eye condition.⁶¹ Ren et al. reported a hybrid DNA hydrogel for the delivery of a water-insoluble ophthalmic drug. The DNA hydrogel has spongelike porosity features that aided in the loading and sustained delivery of dexamethasone. DNA hydrogen enhanced drug accumulation by providing extended retention for around 24 h at ocular cells and tissues, which raised cellular uptake by about 8-fold and therefore significantly reduced inflammatory symptoms in allergic conjunctivitis.⁶²

5.3. Dermal Delivery

DNA nanostructures have a high skin penetration efficiency and thus play a pivotal role in dermal drug delivery. For transdermal delivery, framework nucleic acids (FNAs) of various forms and sizes were created. FNAs demonstrated size-dependent skin penetration, with smaller particles of 75 nm successfully reaching the dermis layer. Small tetrahedrons 17 and 44 nm penetrated deeply to 350–400 m beneath the skin, while free DOX reached only 50–75 m beneath the skin surface and was primarily maintained at the skin without penetrating the tumor. When compared to free DOX treatment, the tetrahedron enhanced DOX accumulation by 5.67 times. Doxorubicin-loaded 20 nm FNAs provide for double the drug accumulation and tumor suppression of free doxorubicin or doxorubicin-loaded liposomes and polymeric nanoparticles. DOX-intercalated FNA had a half-life of more than 48 h, which was substantially longer than free DOX (26 h).⁶³ Lee et al. developed double-stranded salmon DNA (SDNA) microneedles to transport loaded drugs directly into the skin using a non-toxic, mechanically robust, well-moldable, bio-absorbable, and biocompatible form. SDNA microneedles enter the skin’s stratum corneum and dissolve, delivering the loaded DOX into the skin. At a 4:1 aspect ratio, SDNA generated structures ranging in size from nano- to microscale.²

5.4. Systemic Delivery

DNA nanostructures can be administered directly to the systemic circulation by intravenous injection. Systemic delivery of DNA nanostructures facilitates rapid distribution of therapeutics to the target site for desired action and can avoid systemic side effects. Zhang et al. revealed triangle-shaped DNA origami for systemic delivery that demonstrated accelerated tumor accumulation by passive targeting and significantly raised anticancer effectiveness without any systemic risk.⁵⁶ Rahman et al. also used the modular DNA brick approach to create rectangular and tubular-shaped DNA nanoparticles for systemic delivery of loaded payloads.⁶⁴

5.5. Intranasal Delivery

To circumvent fast mucociliary clearance and reach the underlying epithelium for pharmacological action, drug delivery systems must cross the highly viscoelastic and adhesive mucus barrier in the airway. Suk et al. described polyethylene glycol (PEG)-PEI coated mucus penetrating DNA nanoparticles for intranasal delivery to the lung. The DNA nanoparticles were distributed uniformly over the airway epithelium, retained for a long time, and quickly penetrated human cystic fibrosis (CF) mucus without exerting acute inflammatory or toxic implications.⁶⁵

6. Receptor Targeted DNA Nanostructures

Actively tailored DNA nanostructures increase treatment effectiveness by specifying the drug delivery in target cells only while preventing off target distribution and thus eliminating any probable side or adverse effects on normal cells or organs. Drug nanostructures owing to their characteristic structural features can undergo non-receptor-mediated endocytosis via caveolin- or clathrin-mediated pathways (Figure 4). Drug-encapsulated DNA nanostructures have been demonstrated to improve the drug permeation and cellular internalization, limiting drug efflux, and increasing drug accumulation, therefore reducing drug resistance and increasing drug effectiveness. CPT@DNA-NWs demonstrated efficient surface binding and internalization in scavenger-receptor-rich HepG2 cells.¹⁶ Abbas et al. used a DNA Nanothread to encapsulate Cisplatin and function as a polyanionic ligand to provide targeted cytotoxicity and improved anticancer treatment. CPT hydrogen-bonded to DNA-NT at certain GC-rich sites. CPT-DNA-NT had a consistent dimension (50–150 nm diameter and 300–600 nm length). DNA-NT demonstrated improved cellular internalization via scavenger-receptor-mediated endocytosis via the clathrin/caveolin pathway, resulting in regulated intracellular drug release in the target. In contrast to free CPT, which underwent paxillin-mediated exocytosis after cellular internalization, culminating in inferior cytotoxicity, CPT-DNA-NT did not efflux out and instead underwent perinuclear localization, where it released CPT via endosomal degradation and the hydrolytic action of lysosomal enzymes. CPT-DNA-NT demonstrated greater cytotoxicity (72.7% apoptosis) than free CPT (64.4% apoptosis), increasing therapeutic effectiveness.⁶⁶

Figure 4.

Mechanism of cellular internalization and intracellular delivery by DNA nanostructures: (A) non-receptor-mediated endocytosis of DNA nanostructures via caveolin- or clathrin-mediated pathways; (B) receptor-mediated endocytosis of DNA nanostructures surface functionalized with targeting groups like peptides, aptamers, antibody, etc.; (C) endosomal escape of DNA nanostructures; (D) formation of endolysosomal complex followed by lysosomal degradation of DNA nanostructure causing release of therapeutic payload; (E) payload (drug, RNA, or DNA) released in cellular microenvironment to exert its action.

Targeted drug delivery for DNA nanostructures may be accomplished using targeting ligands such as antibodies, affibodies, aptamers, folates, peptides, polysaccharides, and many more (Table 1). The targeting moiety can be feasibility incorporated to the surface of DNA nanostructures.⁴ These ligands recognize the specific receptors present on target cells and are internalized via receptor-mediated endocytosis (Figure 4). Ligand conjugated DNA nanostructures precise the drug delivery, eliminating the drug wastage and in turn reducing the dose required for similar response obtained with free drug. Vindigni et al. reported octahedral DNA nanocage linked to AS1411 aptamer for selective delivery in cancer cells. Aptamer functionalization improved the anti-cancer effect by 200-fold compared to aptmer-free DNA nanocage.⁶⁷ Li et al. also designed AS1411-modified triangle DNA origami for doxorubicin delivery to cancer cells.⁶⁸ Udomprasert et al. reported MUC1 aptamer functionalized DNA nanostructures tailored to disk (2D), donut (3D), and sphere (3D) shapes for loading of doxorubicin via intercalation and enhanced intracellular internalization via clathrin-mediated endocytosis in MCF-7 and MDA-MB-231 cancer cells (Figure 5) The aptamer-modified DNA nanostructures exhibited excellent drug loading and high targeting potential, providing an improved anticancer effect.²²

Table 1. Surface Functionalized DNA Nanostructures for Targeted and Intracellular Drug Delivery.

DNA Nanostructures	Drug	Targeting mechanism	Observations	References
Tetrahedral framework nucleic acid (tFNA)	Typhaneoside (Typ)		Typ was embedded in tFNA for intracellular delivery, exhibited caveolin-mediated endocytosis and precise mitochondrial targeting, restored kidney function in acute kidney injury.	(83)
TDN	Small molecules, peptides, dsDNA, siRNA	DNA strand (AS1411 aptamer) and pH-sensitive DNA apparatuses, i.e., a cytosine-rich sequence {CCC(TAACCC)3} at one edge of the TDN structure	Dynamic conformation change based on the target and environmental stimuli	(24)
Polyphenol-DNA nanocomplex	Tannic acid (TA)	Disassembly at acidic pH	Lysosomal acidic microenvironment induces the nanocomplex disassembly to release TA and branched-DNA followed by release of precise release of genes from branched-DNA as response to glutathione and DNase I in cytoplasm.	(84)
2D dsDNA nanospindels constituting of Neuregulin-1 (NRG-1) ligand functionalized short circular scaffolds (circular DNA strand)	Dox, DR, and cisplatin	EGFR-mediated cell internalization via NRG-1-mediated Her2/neu receptor positive MCF-7 cell targeting	The nanospindles were DNA-NS were biocompatibile and of 50 to 70 nm diameter and 500 to 700 nm length. DNA nanospindels found to efficiently load DNA intercalating drugs. DR loaded DNA-NS showed enhanced apoptosis than free DR due to increased EGFR-mediated cell internalization via NRG-1-mediated Her2/neu receptor positive MCF-7 cell targeting and decreased paxillin-mediated exocytosis. DNA-NS post lysosomal degradation inside the cells selectively released the drug inside the cells due to hydrolysis of DNA-NS by lysosomes leading to apoptosis of cancer cells.	(85)
Affibody-DNA hybrid strand-modified AuNPs	5-Fluorodeoxyuridine and doxorubicin	Dox@affi-F/AuNPs bind to HER2 overexpressing cancer cells, enter the cell, where DNase II degrades foreign DNA, and trigger the simultaneous release of FUdR and Dox.	Provided more suppression of HER2 overexpressing breast cancer cells and showed superior anticancer efficacy when compared to a mere combination of the two drugs	(86)
DNA nanotrains tethered with AS1411 aptamer	Anthracyclines (DOX, EP, and DAU)	Receptor-mediated endocytosis	Resultant AS1411NTrs was found to have significantly higher cytotoxicity against target HeLa cells than normal human liver cells. The DOX, EPI, or DAU binding to DNA and interaction with AS1411NTrs was observed. AS1411NTrs exhibited a high drug payload due to the large number of binding sites (∼20) and provided targeted release of DOX, EPI, and DAU to specific cancer cells. The binding constant was in the order DOX > EP > DAU. AS1411NTrs targeted the cancer cells and reduced the cytotoxicity of the anticancer drugs.	(87)
Aptamer functionalized DNA origami nanostructures	Antibacterial enzyme lysozyme	Receptor-mediated endocytosis	Delivered the lysozyme as a site-specific and efficient manner. The nanostructures showed potent activity against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) targets, slowing the bacterial growth more effectively than free lysozyme.	(88)
Aptamer attached TDN	Paclitaxel and wogonin	Receptor-mediated endocytosis	Fabricated the TDN-based delivery system with four molecules, a) an antisense peptide nucleic acid to replace a short sDNA strand sequence, b) an aptamer attached to the vertex, c) paclitaxel and wogonin, and d) polymer ethylene imine (PEI) or PEGylated-protamine as protective coating. Modified TDN complexes were revealed to promote the cellular uptake of loaded payload into bacteria or targeted living cells and to lengthen the biostability and may provide targeted antibacterial, anticancer, and tissue regeneration activity.	(89)
Aptamer sgc8c-modified tetrahedron	DOX	Targeted protein tyrosine kinase 7 (PTK7) positive cells (human T-cell ALL), uptaken by the caveolin-dependent pathway	TDNs were internalized. Trapped within the lysosome, TDNs degraded to release DOX, which then diffused to the cytosol and finally to the nucleus, and exhibited specific toxic effect on PTK7-positive cells.	(90)
DNA toehold-switch engineered nucleic acid hydrogel	Dox	ATP triggered drug release due to structural change of toehold switches and dissociation of DNA shell	SNAgel consists of nanoparticle cores (∼20 nm AuNP) as templates and a DNA polymer shell as the shield. Domain Apt1 was an ATP-specific DNA toehold switch for SNAgel, precisely controlled the drug release, maximized drug efficacy, and minimized cytotoxicity.	(91)
Triplex-DNA nanoswitch	Cisplatin, doxorubicin, antisense DNA	Acidic intracellular pH changes the conformation from linear to triplex.	Provided pH responsive drug release	(92)
Aptamer-functionalized cancer-Mucin 1 protein (MUC1)-specific DNA nanosphere	Gold nanoparticles and fluorescent dyes	The nanostructure changed its conformation upon binding to cancer-specific membrane proteins that trigger opening of the nanostructure.	Obtained DNA nanostructure was reported to have high payload capacity and was responsive to cancer cells. The prepared DNA nanosphere was used for encapsulation of gold nanoparticles inside the cavity, implicating its loading ability. In addition, conjugation of fluorescent dyes, (Cy3-blue and Cy5-pink) to the 3′ and 5′ ends resulted in a closed conformation and generated the FRET signal while the open conformation did not.	(93)
Nanoplatform consisting of DNA aptamer-sgc8 integrated polydopamine reduced graphene oxide nanosheets.	Doxorubicin	Selectively delivered the DOX to protein tyrosine kinase 7 overexpressing cancer cells and responded to both acidic pH and heat	DNA aptamer served as a carrier for DOX loading as well as targeting moiety/gatekeeper for specific cellular recognition and possessed good targeting ability, intelligent and controllable release as a response to the acidic intracellular pH, and photothermal heating on exposure to NIR irradiation.	(94)
Folic-acid-modified DNA tetrahedral structure	Doxorubicin	Recognize folate receptors that are overexpressed in cancer cells and may undergo receptor-mediated endocytosis-based cellular uptake	The size of the DNA tetrahedral monomer was 15 nm, which rose to 20 nm for folic acid-DNA tetra-Dox, increased the cellular uptake due to DNA tetra-facilitated penetration across the cellular membrane, and dramatically increased the anticancer effect.	(95)
DNA tetrahedron	Dox	SL2B aptamer and folic acid for mediated endocytosis	Enhanced intracellular delivery, small diameter (6 nm) facilitated entry into nucleus, released drug in cytoplasm and nucleus for inhibiting cell proliferation and enhancing cell death for superior anticancer effect to combat colorectal cancer	(96)
DNA magnetic nanoparticles (silica network with iron oxide nanocrystals)	Fluorescein	Thermosensitive uncapping and release of drug	DNA melting due to increased temperature (42–47 °C) under an alternating magnetic field, opening the mesopores and triggering drug release	(97)

Figure 5.

Disk-, donut-, and sphere-shaped Dox-loaded DNA origami nanostructures with and without targeting moiety (MUC1 aptamer) for cellular internalization. (a) MCF-7 cells and (b) MDA-MB-231 cells internalized by DNA nnaostructures. Reproduced with permission from ref (22). Copyright 2022 American Chemical Society.

Ge et al. described the use of an antibody to modify the DNA origami nanostructure (DON) for targeted prostate cancer treatment. In this study, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) was utilized as a targeted ligand against prostate-specific membrane antigen (PSMA). Doxorubicin (Dox) was then intercalated to dsDNA, resulting in a drug-loaded platform with a high loading capacity. The resulting method delivered Dox selectively to PSMA+ cancer cells, exhibiting high fluorescence in the LNCaP cells. Dox was released from DONs in the cytoplasm and spread into the nucleus. Dox-DUPA-DONs therefore enabled very effective selective transport and Dox release in PSMA positive cells.⁶⁹

Zhang et al. built a DNA tetrahedron using four 41-mer DNA strands (A13, B13, C13, and D13). FUdR was first converted to its phosphoramidite form, which was then attached to the 5 end of each 41mer DNA strand. Then, at 5 A13F-NH2, the affibody was connected to FUdR, and all four strands self-assembled to form affibody-FUdR-tetrahedral DNA nanostructures (affi-F/TDNs) as a targeted DNA nanocarrier. affi-F/TDNs demonstrated strong drug loading capacity in the DNA tetrahedral core, good systemic stability, great selectivity for drug accumulation in the tumor area, high tumor inhibition (81.2%) for breast cancer BT474 cells overexpressing HER2, and excellent biocompatibility. The intracellular nucleases degraded FUdRs that enabled sustained drug release in cancer cells. The in vitro and in vivo studies demonstrated a high capability to selectively target HER2-overexpressing breast cancer cells, which were internalized by receptor-mediated endocytosis and had outstanding anticancer effectiveness.⁷⁰

Bleomycin, a hydrophobic DNA-intercalating agent, was utilized to load onto hydrophilic DNA-nanotubes functionalized with FR targeting antibodies. Obtained BM@NTs with diameters of 3 to 5 m and lengths of 200 to 600 nm not only boosted BM aqueous miscibility/dispersibility but also improved therapeutic effectiveness owing to selectivity to the folate receptors/alpha (FR) overexpressed in resistant prostate cancer xenograft CWR22R cells. In comparison with the control cell line, the investigation validated the targeted cytotoxicity and time-dependent transfection of BM@D-NTs into CWR22R cells. D-NTs were stable, biocompatible, had a high loading efficiency ranging from 92.52% to 47.12%, maintained in vitro drug release, targeted cytotoxic effects, reduced dosage, and avoided possible cytotoxic effects on normal cells.¹⁷ Raniolo et al. functionalized octahedral DNA nanocages with folic acid in order to increase internalization into folate receptor positive HeLa cells. Functionalized DNA nanocages were extremely stable, non-cytotoxic, effectively loaded doxorubicin, and selectively delivered the drug to isoform folate receptor (FR) positive cells 40 times more efficiently than non-positive cells, improving uptake efficiency via receptor-mediated strategy. After entering cells, doxorubicin-loaded DNA nanocages were confined in vesicular structures. The Dox was released within the cells due to the selective breaking of DNA cages by the acidic pH of endocytic vesicles, which was responsible for a higher cytotoxic impact than free Dox at the same dosage. The intracellular breakdown of the DNA nanocage also avoids the issue of nanocarrier buildup in vivo.⁷¹

Meng et al. developed Mucin1-aptamer coupled DNA tetrahedral nanostructures to target Mucin 1 protein overexpressing cancer cells. Because of its strong affinity for DNA, MUC1-TDNs was utilized to load the photosensitizer, TMPyP4. The obtained system specified the distribution, enabled effective cell penetration by endocytosis, and enhanced cytotoxicity in MUC1-positive cells, while killing MUC1-negative cells less. This active targeting permitted focused eradication of MUC1-overexpressing tumor cells while minimizing unwanted side effects on normal cells.⁷² Chaithongyot et al. also found that MUC1 aptamer-functionalized nanospheres preferentially delivered encapsulated dox to MFC-7 cells and displayed greater cytotoxicity at lower Dox concentrations. The prepared DNA nanospheres were homogeneous, with a diameter of around 50 nm, moderately stable, and a number of Dox molecules per structure of about 104. MUC1 Apt-DNA nanospheres were internalized into cells by endocytosis and released the drug via lysosomal breakdown to improve the therapeutic benefits of Dox while also reducing drug adverse effects.⁷³

Liu et al. described dual ligand-modified DNA tetrahedron nanocarriers (MUC1-Td-AS1411) for targeted delivery, improved therapeutic effectiveness, and real-time imaging of cancer cells. MUC1 aptamer (MUC1-probe) hybridized to fluorophore extended from one vertex (complementary sequence with quencher) was utilized for cytomembrane targeting and imaging, whereas the AS1411 aptamer hybridized to three vertexes was employed for nucleolin binding. MUC1-Td-AS1411 may be used to efficiently load Dox (Dox@MUC1-Td-AS1411), with about 44 Dox molecules intercalating into a single MUC1-Td-AS1411. Dox@MUC1-Td-AS1411 inhibited drug-resistant breast cancer cells more effectively than did Dox@MUC1-Td or free Dox. First, the MUC1-probe was directed at MUC1-positive cells, inducing a conformational rearrangement of the MUC1 aptamer, releasing a complementary sequence with a quencher and resulting in fluorescence. The MUC1 probe would not be triggered in MUC1-negative cells, distinguishing MUC1-positive cells from MUC1-negative cells. Furthermore, due to the AS1411 aptamer, MUC1-Td-AS1411 bypassed the lysosomal route and avoided lysosomal acidification. AS1411 aptamer bound to nucleolin after internalization and was responsible for selectively targeting and releasing Dox into the nucleus. Dox@MUC1-Td-AS1411 demonstrated enhanced intracellular Dox accumulation and hence fluorescence intensity more than Dox@MUC1-Td. Dox@MUC1-Td-AS1411 was more effective against doxorubicin-resistant MCF-7 cells than against free Dox. Dox@MUC1-Td-AS1411 effectively penetrated the MCF-7/ADR cells, as seen by the spatial co-localization of red, green, and blue fluorescence around the nucleus. The spatial colocalization of green and blue fluorescence revealed that Dox was released within the nucleus. Dox@MUC1-Td-AS1411 increased cancer cell lethality by preferentially delivering drugs into the nucleus, therefore contending with drug resistance.⁷⁴

7. Stimuli Responsive DNA Nanostructures

Stimuli responsive drug delivery systems primarily make use of tumor microenvironmental or cellular physiological characteristics such as lower pH, higher redox potential, overexpressed enzymes, increased levels of reactive oxygen species, and higher temperature to provide drug release at the target site. DNA nanostructures responsive to various external (magnetic or electric field, ultrasound, light and thermal energy) as well as internal (enzyme, pH, GSH, ATP, antigen, and metal ions) stimulus can be designed for target-specific drug delivery applications.^11,13 The stimuli responsive moiety is incorporated in DNA nanostructures that experiences physicochemical changes on reaching the target microenvironment. The drug release occurs mainly via DNA nanostructures conformational change or disassembly or degradation as response to target stimuli (Figure 6).

Figure 6.

Stimuli responsiveness of DNA nanostructures to release the payload at the target site. (I) Drug release as a response to intracellular microenvironment stimuli. DNA nanostructures get internalized to cells via endocytosis (I.A) and undergo endosomal escape (I.B) followed by drug release due to degradation (I.C) of the structure by DNAzymes, or acidic pH, or ROS or GSH overexpressed in the cellular microenvironment. (II.) Drug release as a response to the extracellular microenvironment or external stimuli. DNA nanostructures reach near the target cell or attach to its surface where the payload is released. (III) Drug release as a response to intracellular (III.C) or external (III.D) stimuli. (IV) Endolysosomal-complex-mediated drug release. DNA nanostructures attach to the cell membrane, enter the cell via endocytosis (IV.A), form an endolysosomal complex (IV.B), and then release the payload due to lysosomal degradation (IV.C) of the structure.

Li et al. developed stimuli responsive DNA nanohydrogel incorporated with a disulfide linkage integrated monomer A (3 ssDNA assembly with two sticky ends to hybridize the linker), monomer B (3 ssDNA assembly with a sticky end and a aptamer hybridized strand), and DNA linker. The nanohydrogels were stable in systemic circulation, had efficient cellular uptake, and were cleaved by GSH in cytosol to facilitate the release inside the target cells.⁷⁵ Yan et al. utilized redox sensitive polyethylenimine with disulfide cross-linker to produce cationic polymer as protective covering for doxorubicin-loaded tetrahedral DNA (TDNs) (TDNs). The resulting nanocomplexes (PSP/TDNs@DOX NCs) had a diameter of about 240 nm and disintegrated at the tumor site owing to disulfide breakdown in response to intracellular glutathione. The disassembled NCs formed had a smaller size of 50 nm, which improved the penetration to deep tumor tissues and hence the therapeutic effectiveness. PSP/TDNs@DOX NCs allowed for strong tumor cell/tissue penetration and internalization, allowing them to skip endocytosis and overcome MDR. The NCs also responded to glutathione and DNase I in the cell, causing the assembly to disassemble and the DOX to be released. The investigation of transwell and 3D tumor models indicated that NCs may easily leave cells through holes that leak and infect adjacent cells, allowing them to reach deep tumor tissues. The PSP/TDNs@DOX NCs demonstrated improved tumor penetration and therapeutic effectiveness in drug-resistant tumor mouse models MCF-7/R and SKOV3/R, which reflect MDR with DOX efflux and impermeability, respectively. PSP/TDNs NCs responded to reductive stimuli, caused size reduction by dissociation to small-sized NCs, and provided deep tumor penetration in even DOX-resistant cells in an efficient manner than free DOX.⁷⁶

A novel ATP-responsive system composed of graphene oxide (GO), two single-stranded DNA, and an ATP aptamer was used for targeted delivery of doxorubicin (DOX). Hybridization of ssDNA1 and ssDNA2 with an ATP aptamer as linkers resulted in the formation of GO nanosheets. The resulting layered DNA-GO nanoaggregates loaded DOX with great efficiency thanks to supramolecular stacking between GO and DOX. DOX release from the GO nanosheets was successfully prevented by nanoaggregates with reduced specific surface area toward the surrounding media. However, it released the drug into tumor cells with a high ATP level owing to aggregate dissociation, which reduced the aggregate size and increased the surface area exposed to the medium. When compared to the ATP-deficient extracellular fluid, this enhanced the release of DOX from the GO surface in the tumor environment with a high ATP content, such as cytosol. As predicted, after incubation with ATP, the particle size of DNA-GA decreased as the ATP concentration increased. In the presence of 3 mM ATP, DNA-GA had an average particle size of 270 nm, compared to 550 nm for DNA-GA without ATP.⁷⁷

The long ssDNA produced via rolling circle amplification was utilized as a scaffold strand, and it was hybridized to three short staple strands to form a DNA origami belt through Watson–Crick base-pairing. The obtained DNA belts had the highest stability in neutral pH (pH = 7) and the worst resilience to severe pH settings (pH = 3, pH = 11). DNA belts in an extreme pH environment (pH = 3) became shorter and thinner over time, with the hydrogen bond that held the double-stranded structure of the DNA belts breaking first, followed by the phosphodiester linkage between nucleotides and the glycoside bond inside the nucleotide, resulting in broken DNA belts in pieces.⁷⁸

Genomic DNA isolated from kiwifruit was used as a primary component of the NGs, produced by cross-linking amino groups on nitrogen bases (ACG) with genipin, and was biocompatible and biodegradable. The obtained disulfide cross-linked DNA NGs were 100 and 150 nm in size, with pH- and reduction-sensitive characteristics suitable for anticancer drug administration. Furthermore, the phosphate group in the DNA backbone had a negative charge, which favored solubility in bodily fluids and non-toxicity to normal cells. The electrostatic interactions, as well as the preferential intercalation of DOX and the GC bases of DNA, led to dox encapsulation (92.5 ±1.23%) in DNA NGs, but its dissociation in acidic and reductive environments resulted in dox release. The protonation of the phosphate groups (HPO42 and PO43) in acidic environments promoted DOX release, resulting in the breakdown of the DOX/DNA complexes established by electrostatic interactions. The GSH causes the NGs to de-cross-link, leading to increased drug release. The drug delivery capacity of DNA-DOX NGs was superior to that of free DOX. At acidic pH and high glutathione (GSH) concentrations, the amount of DOX released by DOX-loaded DNA (DNA-DOX) NGs was greater, and the IC50 of DNA-DOX NGs in cancer cells was lower than that of free DOX. Furthermore, as compared to free DOX, the DNA-DOX NGs increased DOX uptake and apoptotic death in cancer cells.⁷⁹

8. Dual Targeted DNA Nanostructures

Dual targeted DNA nanostructures make use of two or more or a combination of a targeting ligand or stimulus sensitive group. Such dual targeted nanostructures distribute the drugs in a more precise manner, resulting in maximal therapeutics accumulation in target cells, no drug waste owing to non-target distribution, and therefore no toxicity or side effects in normal organs. Wang et al. reported a multifunctional DNA nanodrug with high payload and biosafety. The DNA nanodrug was photoactivatable and actively targeted to mitochondria for better photodynamic and antitumor therapy (Figure 7). The DNA nanodrug encodes the mucin 1 (MUC1) aptamer and the cytochrome C (CytC) aptamer that facilitated active targeting to the mitochondria in the tumor cell. On exposure of near-infrared (NIR) light irradiation, reactive oxygen species (ROS) are generated that precisely damage the mitochondria and break single-stranded DNA (ssDNA) to activate disassembly of DNA nanodrug to release loaded DOX and P-gp DNAzyme. This results in downregulation of P-gp and ATP, reducing DOX efflux and improving gene therapy.⁸⁰

Figure 7.

Mitochondrial targeted photoactivated DNA nanomedicine for breast cancer therapy. (a) DNA nanodrug synthesis where the straight-stranded DNA template encodes complementary sequences of MUC1 aptamer, CytC aptamer, DNAzyme, and G-quadruplex with primers in the presence of T4 DNA ligase to form a circular DNA template, followed by RCA reaction with the photosensitizer TMPyP₄ and the chemotherapeutic agent DOX to generate the self-assembled DNA nanodrug. (b) DNA nanodrug selectively transported to the mitochondria of MCF-7/ADR cells via MUC1 aptamer and CytC aptamer. Near-infrared light on exposure triggered the photodynamic therapy by releasing ROS to damage mitochondria and retarded ATP generation to inhibit drug-resistant protein P-gp activity. Released ROS promoted the distribution of DNA nanodrug to release the incorporated DNAzyme and DOX. DNAzyme targets the silencing of MDR1 mRNA and suppresses P-gp expression in order to prevent DOX efflux, allowing it to efficiently enter the nucleus and kill tumor cells. Reproduced with permission from ref (80). Copyright 2023 American Chemical Society.

To create another multitargeted drug nanocarrier sensitive to pH changes in the surroundings, MUC1 aptamers as targeting ligand and i-motif DNA as pH-responsive moiety were used to modify gold nanoparticles (AuNPs). When the pH of the environment changes from 7.4 to 5.0, the i-motif DNA folds into four-stranded DNA (C-quadruplex). Furthermore, MUC1 aptamers selectively identify the MUC1 protein overexpressed on the human lung cancer cell line (A549) in order to specify and increase cellular uptake. MUC1 aptamer was covalently linked to strands S2 and S3, which subsequently hybridized with strand S1 on AuNP-S1 to produce a complex structure (Y-shape DNA). The surface modification of AuNP with DNA strand S1 increased its size from 25 to 42 nm and then to 107 nm following hybridization with strands S2 and S3. Doxorubicin was intercalated into GC base pairs of DNA nanostructure to produce drug-loaded nanovehicles (Dox@AuNP-MUC1), which give a synergistic impact between chemotherapy and AuNPs guided photothermal treatment when exposed to 808 nm NIR irradiation. Dox@AuNP-MUC1 treated group had good stability, high photothermal conversion efficiency, and greater fluorescence than the Dox@AuNP-NEG treated group without MUC1. The nanocarrier selectively targeted cancer cells, culminating in no damage to normal cells. Due to combined MUC1 aptamer-mediated cellular internalization and increased temperature induced by NIR irradiation, Dox@AuNP-MUC1 provided combined chemo-photothermal therapy responsible for significant anticancer activity with apoptotic rates 57% ± 3.2% after treatment with Dox@AuNP-MUC1 and 80% ± 2.9% for NIR/Dox@AuNP-MUC1.⁸¹

In another work, Miao et al., created i-motif DNA-conjugated gold nanostars (GNSs) as stimuli-responsive DNA-based nanostructures (A-GNS/DNA/DOX). The i-motif DNA was pH sensitive, and under normal physiological circumstances, the single strands, S2, remained hybridized with S1 to create dsDNA. However, if the pH of the environment drops from neutral (7.4) to acidic (6.0), then it can transition from a single-stranded structure to a C-tetrad (i-motif) structure. Furthermore, to increase cellular uptake and cancer cell selectivity, the aptamer AS1411, which detects nucleolins preferentially, was used as a targeting moiety and was incorporated into the i-motif strands (S2). As a result of conformational changes, the loaded drug might be released. A-GNS/DNA/DOX nanocomposites demonstrated good biocompatibility, high photothermal conversion, and photostability when exposed to near-infrared (NIR) light. The targeting ligands improved the internalization of the A-GNS/DNA/DOX nanocomposite. Doxorubicin (DOX), an anticancer medication, was firmly intercalated into the CG base pairs of dsDNA via hydrophobic and hydrophilic interactions. Because of the targeted recognition, A-GNS/DNA/DOX demonstrated more cellular uptake and therapeutic efficacy than GNS/DNA/DOX. Following endocytosis, pH and NIR irradiation successfully stimulated drug release to 40% at pH 5.0 without laser irradiation. In acidic endosomes/lysosomes, the S2 strands adopt stable i-motif conformations and dehybridize the S1 strands for dsDNA dissociation, resulting in drug release. Furthermore, the GNSs convert external NIR irradiation to heat, increasing drug release to 60% at pH 7.4, and 73% due to combined acidic conditions and NIR heating and offered outstanding photothermal effects. The prepared nanocomposites demonstrated great photothermal conversion efficiency, stability, and biocompatibility, as well as selective cancer cellular uptake stimulus responsive drug release and combination chemo-photothermal treatment.⁸²

9. Applications

DNA nanostructures exhibiting biodegradability, biocompatibility, non-immunogeneicity, and non-cytotoxicity can be successfully utilized for the management of a variety of diseases. DNA nanostructures have been reported for application in kidney disorder, cancer, inflammatory illnesses, fibrosis, infections, anti-aging, mucosa/skin repair, tissue regeneration, immunoregulation, neuroprotection, liver injury, etc.⁹⁸ A little research on the use of DNA nanostructures for drug delivery and theranostics applications is reported below.

9.1. DNA Nanostructures for Cancer Therapy

DNA nanostructures with low bio-toxicity, high stability, and superior adaptability make them an excellent carrier for anticancer drugs to improve therapy and considerably reduce side effects by offering precise, targeted, and multifunctional drug delivery.⁵² Huang et al. demonstrated biocompatible cancer-targeted DNA nanocarriers with increased anticancer efficacy and decreased toxicity. The unique tetrahedral nanostructure of DNA cages substantially improved the interaction and intercalation of ruthenium polypyridyl complexes (RuPOP) with DNA nanostructures, which improved drug loading effectiveness. The bio-cage had a height of 1.1 nm and a diameter of 12.2 nm before loading with RuPOP, which increased the height to 2.0 nm and the diameter to 33.5 nm. As shown in Figure 8, biotin conjugation to the DNA nanosystem (Bio-cage@Ru) at the 5′ terminus of each DNA strand facilitated the selective accumulation in tumor locations, resulting in increased cellular uptake, drug retention, and cytotoxicity against HepG2 cells. After internalization, the bio-cage@Ru enhanced translocation to the cell nucleus, where DNases began cleavage, resulting in drug release and apoptosis. Bio-cage@Ru enhanced the accumulation of RuPOP via biotin/receptor and the subcellular localization from cytosols to the nucleus. The size of Bio-cage@Ru was tiny enough to enter the nucleus, and it was swiftly degraded in the lysosomal environment to liberate RuPOP via DNase I digestion, which may have started an assault on nuclear DNA and eventually induced cancer cell death. Bio-cage@Ru had more than three times the anticancer ability and a 10-fold greater safety index than Cage@Ru.⁴⁸

Figure 8.

Biotin functionalized tetrahedral DNA nanocage for targeted delivery to cancer cells: (a) biotin conjugated to the 5′ terminal of DNA strand used for fabrication of bio-cage and DNA bio-cage as carrier (Bio-cage@Ru) to Ru complex; (b) biotin-receptor-mediated cellular localization of nucleus targeting by Bio-cage@Ru, release RuPOP by DNase I digestion; (c) bio-cage@Ru resulting in enhanced penetration, selective distribution, better anticancer effect, and minimal systemic toxicity. Reproduced with permission from ref (48). Copyright 2016 Elsevier Ltd.

DNA nanobots formed by folding a unique strand of DNA into a systematic structure can precisely detect and destroy cancer cells. These exhibit potent passive tumor-targeting potential as well as active targeting based on modification on DNA strands or incorporated targeting moiety.⁹⁹ A nanorobot with an aptamer of about 90 nm long tube-like structure was reported to recognize tumor-specific target molecules for active targeting, tumor necrosis, and tumor growth inhibition. The triangle-shaped DNA nanobot, in particular, reported for excellent accumulation by passive targeting.⁹⁹ Another study by Wang et al. reported a DNA-based nanobarrel as suitable carrier of PTX and DOX, equipped with targeting aptamer (mucin-1) for precise drug delivery.¹⁰⁰

9.2. DNA Nanostructures for Inflammatory Disorders

A pH-responsive i-motif sequence was used to link modified oligonucleotide-coupled dexamethasone to DNA nanotubes. The resulting dexamethasone-conjugated DNA nanotubes delivered the glucocorticoid dexamethasone into tissue macrophages, which is important in regulating inflammatory responses. Within minutes, dexamethasone nanotubes were internalized and localized in their endosomes. When compared to similar quantities of free dexamethasone, dex nanotubes substantially attenuated LPS-induced TNF production by macrophages without compromising cell survival.¹⁰¹

Wang et al. reported conjugation of NF-B decoy oligodeoxynucleotides (dODNs) and VCAM-1 targeting peptides (P) onto self-assembled DNA tetrahedrons to yield DNA nanodrugs (TDs). When compared to free dODNs, TD-P-dODN had quicker and greater cellular absorption by inflammatory cells. In both cells and adjuvant-induced arthritis (AIA) mice, TD-P-dODN substantially decreased the level of inflammatory markers. TDs was modified with a peptide (P) to selectively target VCAM-1, a glycoprotein overexpressed on inflammatory synovial endothelium cells, RA macrophages, and RA fibroblast-like synoviocytes and thus enhance the specificity of DNA nanodrugs for inflamed joints. For the therapy of inflammatory arthritis, TD-P-dODN was more stable than dODN, TD, and TD-dODN and could reach the cytoplasm to bind to its target. TD-P-dODNs fluoresced the inflamed paws strongly, indicating prime delivery and greater accumulation in inflamed paws by TD-P-dODNs than TD-dODNs.¹⁰²

9.3. DNA Nanostructures for Fibrotic Diseases

DNA nanostructures are promising carriers for selective cellular uptake, owing to their flexible structure. Ito et al. reported DNA nanotubes for higher uptake by fibroblasts and fibroblast-like cells than other DNA nanostructures such as polypod or tetrahedrons, which are easily taken up by macrophages. DNA nanotubes were observed to effectively distribute on cell surface, fold, fragment, and take up by phagocytosis. DNA nanotubes were found to be readily taken up by fibroblasts and myoblasts rather than macrophages and dendritic cells responsible for clearance. Thus, DNA nanotubes emerged as efficient carriers toward the treatment of fibrotic diseases.¹⁰³

Zhang et al. produced tetrahedral framework DNA nanostructures (TFNAs) for the treatment of fibrotic disorders. TFNAs were produced by using four single-stranded DNA molecules that self-assembled to give the nanostructures. TFNAs were effectively formed into triangular particles with a diameter of around 20 nm and showed excellent cellular uptake. Cy5-labeled TFNAs were internalized and distributed in the cytoplasm after entering RLE-6TN cells after 6 and 10 h. Single-stranded oligodeoxynucleotides, unlike TFNAs, are seldom internalized and are destroyed in the cytoplasm. Because of their unusual tetrahedral shape, TFNAs rapidly entered cell membranes via a caveolin-based route and were then degraded by nucleases in the lysosomes. Thus, TFNAs were biodegradable, reduced ROS generation and fibrosis development through regulating the EMT process and ECM formation, and downregulated the expression of collagen I, fibronectin, and Smad2/Smad3 signals.¹⁰⁴

9.4. DNA Nanostructures for Theranostic Applications

For targeted cell imaging and drug delivery, a new beansprout-like aptamer-tethered DNA assembly was created. The DNA bean sprout (DNA BS) was made up of two aptamer-tethered single-stranded DNAs (two “cotyledons”) for attaching to the target cell’s membrane and a double-stranded DNA (“hypocotyl”) intercalated with medicines or fluorescent dyes. The findings indicate that DNA BS has a better targeting ability, higher cellular uptake, and more effective drug delivery to MCF-7 cells than monovalent DNA assembly. The DNA BS’s two aptamers assisted in selective binding to target cells and endocytosis. Intercalated with fluorescent dyes or doxorubicin (DOX) molecules, the hypocotyl of the DNA BS allowed targeted live cell imaging and medication administration. The DOX loading level of DNA BS is predicted to be around 1:35. The fluorescence intensity of MCF-7 cells treated with DNA BS was greater than that of Single-Apt, indicating that bivalent DNA BS had a stronger target ability than monovalent DNA assembly. DNA BS-DOX was more lethal to MCF-7 cells than Single-Apt-DOX, showing that bivalency is preferable to monovalency. DNA BS showed higher binding affinity, increased cellular uptake, and more specific cytotoxicity against the target MCF-7 cells than monovalent DNA assemblies.¹⁰⁵ Yu et al. developed a glutathione-responsive tumor-targeted DNA-Au nanostructure for the encapsulation of photosensitizers chlorin e6 (Ce6) and doxorubicin (Dox). To avoid non-target distribution, the resulting nanostructure preferentially identified the tumor cells led by the AS1411 aptamer. When the loaded payload entered tumor cells, the cellular milieu with high GSH resulted in disulfide bond breakdown in nanostructures, allowing the loaded payload to be released. The release of DOX resulted in chemotherapy, whereas the aggregation of AuNPs resulted in imaging, as well as photothermal treatment. The generation of reactive oxygen species by Ce6 resulted in an improved photodynamic treatment. As a result, a multifunctional theranostics DNA-Au nanostructure developed for imaging guided cancer therapy offered multimodal chemo/photo/pharmacodynamics therapy.¹⁰⁶

A multifunctional DNA-metal hybrid nanostructure was reported by Miao et al. for chemo-photothermal Cancer Therapy (Figure 9). The drug conjugated to DNA was linked to gold nanostars (GNS) to give GNS/DNA/DOX. GNS/DNA/DOX was functionalized with aptamer AS1411 to give A-GNS/DNA/DOX with enhanced cellular uptake in cancer cells. GNSs had a diameter of 35 nm and showed a localized SPR peak in the NIR region at 750 nm, which red shifted to 780 nm after conjugation with DNA for the resulting A-GNS/DNA. A new peak developed at about 500 nm for the drug-loaded A-GNS/DNA/DOX nanocomposites was due to DOX absorption at this area, indicating effective DOX loading. Hydrodynamic diameters rose from 60 to 70 nm after DNA grafting and drug loading, while zeta potentials increased from 40 to 30 mV. The polydispersity value was less than 0.3, showing that the nanocomposites were dispersed well. The pH sensitive i-motif DNA can exhibit conformational change from single stranded structure to C-tetrad (i-motif) as pH change from ∼7.4 (systemic) to <6 (cancer microenvironment) to release the loaded drug. Upon exposure to NIR radiation, a synergistic chemo-photothermal effect was obtained for better cancer therapy.⁸²

Figure 9.

Multifunctional dual-responsive (near-infrared and pH) DNA nanocomposite (A-GNS/DNA/DOX) for combined chemo-photothermal cancer therapy. Reproduced with permission from ref (82). Copyright 2020 American Chemical Society.

10. Conclusion

DNA nanotechnology has attracted enormous attention in the past two decades. The introduction of techniques like DNA origami by Rothemund in 2006, enabling the fabrication of nanostructures with precise morphology and size, has further fueled the research interest in this field. The versatility, biocompatibility, and stability of DNA nanostructures make them an ideal platform for drug delivery and theranostic application. The control over the structural architecture, the site-specific functionality, and programmability of the nanostructure to respond to desired microenvironment bestows these nanostructures with significant superiority over other nanosized carriers in their application as effective and targeted systems for the delivery of drugs, biologicals, gene, RNA, DNA, peptides, etc. These distinct characteristics also indicate their application in numerous inflammatory disorders, injectable targeted chemotherapeutic delivery, fibrotic diseases, and theranostic applications. The versatile nature is also evident in its use as a carrier in formulation for various routes such as dermal, oral, ophthalmic, and parenteral. Although the clinical application of these structures is still far from reality, various aspects of in vivo fate, toxicity, immunogenicity, effect of process parameter on their clinical performance, mechanism of drug release, etc. still requires comprehensive investigations. Furthermore, the major translational challenge for DNA nanostructure will be the large-scale production, and focusing on some lesser explored top-town methods may address this issue. With our ever so increasing knowledge about DNA as a biomaterial, integration of technologies like 3D printing, and development of physiologically relevant screening models, there is a possibility of a clinically relevant DNA-nanostructure for drug delivery being a reality in the near future.

Author Contributions

^# M.K. and A.J. contributed equally to this manuscript

The authors declare no competing financial interest.