Past and recent studies have developed a range of nanocarriers for ocular drug delivery. Each carrier incorporated a functionalization strategy designed to address the specific limitations inherent in drug delivery. By utilizing passive targeting strategies that improve the permeability and retention effect through controlling physical and chemical properties, such as the combined impact of surface charge and surface chemistry on the size and shape of the structure, the diffusion of nanocarriers can be improved. Furthermore, it has been shown that the use of targeting molecules such as proteins, aptamers, and peptides in active targeting can enhance sensitivity toward target cells. This also mitigates the reduction in permeability caused by ocular tissue cell screening. Consequently, this contributes to improving the efficiency of drug delivery. By capitalizing on these complementary strategies, researchers can develop effective ocular drug delivery systems that improve treatment outcomes for eye conditions.

DNA nanostructures, an emerging and rapidly advancing field in drug delivery research, present a next-generation active targeting platform that integrates proteins, aptamers, and peptides [151]. Traditionally, DNA has been considered a carrier of genetic information, but it is now also recognized as a smart material for the construction of nano-architectures in programmable and predictable patterns. Apart from its significance in genetic research and applications across various biological disciplines, such as biomedicine, cancer research, and genetic engineering, DNA’s unique properties—including structural stability, sequence programmability, and predictable self-assembly—have opened up new avenues in the realm of DNA nanostructures. Furthermore, stimulative DNA structures, such as those incorporating hydrogel systems, have shown promise in this field. Following extensive progress in structural design, DNA nanostructures have started to gain widespread application in the biomedical domain, heralding a new wave of disease treatment approaches. 

DNA nanostructures possess a distinctive combination of advantageous properties, including biocompatibility, targeted delivery, design flexibility and precision, triggered release, adaptability, and compatibility with diverse drug types. These characteristics render DNA nanostructures a highly promising and attractive candidate for ocular drug delivery research and development.

The discovery of DNA nanotechnology by Seeman in the early 1980s marked the beginning of the understanding of DNA as a potent material for building logically planned nanostructures outside the realm of biology. They invented DNA nanotechnology that showed the capability of generating nanocarriers of distinct sizes and geometries based on the major complementary base pairs of DNA, GC/AT [158]. Initially, the design of nanostructures was based on sticky binding between DNA strands, but it has progressed to offer several functionalities such as targeting by programming with other substances. Recently, researchers have developed smart DNA-based nanocarriers with dynamic DNA structures that can be switched reversibly [158]. These unique structures can initiate the release of encapsulated drugs into designated areas by reacting to specific stimuli such as strand displacement, pH alterations, and molecular or light-induced reconfigurations. This process effectively utilizes the high specificity and tunability inherent to DNA.

The structural design of DNA-based drug delivery nanocarriers, including 2D grids and 3D objects such as nanotubes, polyhedral, and other complex structures, for the application of DNA nano-systems begins with DNA self-assembly. The idea that DNA can be used as a nanoscale building material stems from the concept that “immovable junctions” can be assembled by rational designs of sequences and coupled to form 2D or 3D structures [166,167,168,169,170]. In fact, Seeman formed a 2D grid using complementary single-strand overhangs called sticky ends and constructed multiple shared-binding closed individual 3D objects such as cubes and tetrahedra based on terrain junctions [167,169,170,171]. Afterward, DNA tile-based self-assembly was proposed as a feasible approach to creating higher-order structures, including nanotubes and nanocages, using modular building blocks with adhesive end interactions [172,173,174].

The DNA origami strategy, first proposed by Rothemund, involves folding a long single-stranded DNA molecule (known as the “scaffold”) into a desired shape with the assistance of many short DNA oligonucleotides (known as “staples”) [161]. This concept has been expanded to design 3D origami structures by arranging DNA helices into different 3D lattices. This technique has progressed to enable the creation of complicated 3D DNA nanostructures with higher productivity and consistency, thereby simplifying the production of various 3D structures. By utilizing the principle of assigning the staple strands of DNA origami as unique pixels, it is possible to precisely fix other molecules such as functional biomolecules, ligands, and nano-sized objects in the desired positions, which offers the possibility of a versatile platform [161].

The development of DNA self-assembly has provided a way to simplify the design of complex DNA nanostructures. Single-stranded tile (SST) self-assembly allows DNA building blocks to form precise shapes without the need for scaffolds by creating loops with connected sticky ends [161]. SSTs interact with each other in a way similar to Lego bricks, through complementary domains. This approach demonstrates unlimited potential in geometric construction, capable of creating not only 2D patterns of complex structures, but also 3D shapes such as alphabets and other forms with complex surfaces, by assembling molecular canvases made up of numerous unique SSTs [163,175,176].

In addition to DNA self-assembly, alternative strategies have been developed for constructing nanostructures, including the use of nanoparticle templates, metal DNA hybrids, and the rolling circle amplification (RCA) approach. Though single DNA duplexes have shown great potential, researchers have focused on enhancing the mechanical properties of DNA nanostructures.

In the early 1990s, Mirkin et al. achieved a significant breakthrough by creating the first hybrid structure combining nanoparticles and nucleic acids [177]. They employed gold nanoparticles as frameworks for the growth of short DNA strands. Terminal thiol-modified single-stranded DNA oligonucleotides attached to the surface of colloidal gold nanoparticles and duplexes (knowns as linkers) with complementary sticky ends on two transplanted sequences facilitated the self-assembly of gold nanoparticles into clusters. Mirkin observed that the distance between particles in the assembly was proportional to the length of the DNA linker [178,179]. This finding demonstrated that a robust core, combined with multiple double linkers, played a crucial role in maintaining the assembly of DNA-nanoparticle templates. This technique effectively increased the rigidity of the assembly. Subsequently, diverse inorganic nanoparticles, including catalytic noble metals, magnetic oxides, and semiconductors, were integrated with DNA. The DNA–nanoparticle superlattice, which allows for the formation of highly rigid DNA nanostructures through straightforward procedures, holds significant potential for expanding its applications in different types of domains.

The utilization of transition metals to facilitate the coupling and assembly of metal-DNA is another widely employed technique in the construction of DNA nanostructures, as mentioned earlier. This method involves creating DNA-metal hybrid nanostructures, where DNA double helices act as flexible appendages of rigid molecules on the nanoscale, centered around transition metals that can be precisely manipulated and controlled using specific organic molecule pockets [180]. In 2004, Sleiman et al. synthesized DNA complexes consisting of two parallel DNA strands linked to a Ru2-tris(bipyridine) core [181]. Through the design of DNA sequences that allow complementary hybridization, the self-assembly of ring-shaped metal-DNA nanostructures occurred. Subsequent research explored the use of copper ions for nanostructure formation [166]. By incorporating a copper-binding ligand, diphenyl phenanthroline, chemically synthesized between the two DNA strands, a highly stable complex that resisted denaturation during PAGE analysis was formed.

RCA (rolling circle amplification) has proven to be an effective method for efficiently creating DNA nanostructures. RCA involves amplifying multiple copies of a circular DNA template through a nucleic acid amplification technique [182]. These elongated DNA strands produced by RCA have been combined with origami folding techniques to construct a wide range of nanostructures, including nanoribbons, nanotubes, and nanospheres. In 2013, Ouyang et al. successfully developed an RCA-based nanoribbon using only 32 staple strands, demonstrating its easy internalization by cells [183]. Subsequently, with advancements in 3D topological structures based on DNA growth and paper folding, RCA has been employed as a means of merging or inserting therapeutic agents for applications such as drug delivery and targeted therapy [184]. One notable aspect of RCA is its ability to generate nanostructures without a predefined size, setting it apart from other DNA nanostructure formation methods. However, it is important to note that RCA-based nanostructures often have a high payload capacity due to their repetitive units, which should be considered when utilizing them.

The self-assembly property of DNA has enabled the development of DNA nanostructures as highly attractive drug delivery carriers, particularly in terms of biocompatibility. Whereas conventional nanocarriers often face issues with biocompatibility and cell permeation due to their materials and size, DNA nanocarriers generally do not experience these challenges because of DNA’s inherent properties. However, controlling the drug release properties of DNA nanostructures can be challenging when compared to organic and inorganic nanocarriers, as DNA is sensitive to specific pH levels and oxidative environments, causing rapid decay [154]. Additionally, controlling the size and shape of the carrier using only the self-assembly process can pose difficulties. To address these challenges, researchers are attempting to stabilize DNA nanostructures with other materials, such as gold nanoparticles. Even so, this approach may not perfectly control drug release properties. To address this challenge, researchers are investigating the creation of intelligent drug delivery systems that integrate stimuli-responsive materials and DNA nanostructures. One strategy encompasses modifying the surface of DNA nanostructures by incorporating hydrogels, thereby enhancing the responsiveness of these nanocarriers to external stimuli. Smart DNA nanocarriers hold great promise, as they can potentially resolve the biocompatibility issues faced by the mentioned nanocarriers while also improving permeability due to in vivo interactions. Furthermore, these carriers can enable controlled drug release through external stimuli, such as pH changes, molecule interactions, and temperature fluctuations (Table 1). As research in this area continues to progress, the development of smart DNA nanocarriers is anticipated to contribute significantly to overcoming the limitations of current ocular drug delivery systems.

Temperature change has been used as a prominent external stimulus to trigger drug release from nanocarriers. The difference between external environment temperature and internal body temperature, as well as temperature changes due to inflammatory responses, enables the temperature to be an essential external stimulus, including the case of eyes [204,205]. The normal surface temperature of the eye is around 35 °C, and it can fluctuate up to 40 °C due to infection or other reasons. Specifically, the external temperature can act as an effective trigger for smart DNA nanocarriers, since double-stranded DNA unwinds into single-stranded DNA when hydrogen bonds between complementary base pairs break as the temperature increases. Researchers have made important advances leveraging temperature change as an external stimulus for DNA nanocarriers. Liu and colleagues developed a self-assembling DNA hydrogel exhibiting sensitivity to both temperature and enzyme reactions [206]. The Y-DNA and linker components formed a thermally sensitive pure DNA hydrogel through the complementary hybridization of sticky ends. However, this strategy does not adequately address temperature responsiveness and self-degradation in the human body, as the gel turns into a solution when temperatures rise from 25 °C to 50 °C due to the base pairing of complementary sticky ends. To overcome these limitations, researchers have incorporated nanomaterials with photothermal properties and nucleic acids to create DNA hydrogels that respond to temperature variations and degrade biologically. These hydrogels can be precisely controlled within the body. Notably, gold nanoparticles exhibit unique photothermal properties, allowing them to convert absorbed light energy into heat [188]. When integrated into a DNA hydrogel, these nanoparticles can act as an activator, initiating transitions between the gel and solution phases. Building on this concept, Song’s team designed a photothermally responsive, self-degradable DNA hydrogel embedding gold nanoparticles for rapid drug release in combined chemo-photothermal treatments [207]. As light illuminates the DOX-AuNP-DNA hydrogel, the resulting heat generation induces DNA hydrogel fragmentation and subsequent DOX release. Similarly, Cui’s group employed gold nanorods conjugated with interference oligonucleotides to develop an HA-DNA hydrogel aimed at gene therapy applications [187]. Here, near-infrared light increases the temperature, breaking the hydrogen bonds within the DNA helix and prompting the hydrogel’s degradation. This process releases the gold nanorods, which modulate pro-inflammatory genes. These instances showcase the potential for regulated drug delivery using DNA hydrogels.

Changes in pH are indeed one of the pathophysiological features that can be used to trigger drug release, especially in cancer therapies. Rapid cancer cell proliferation results in an acidic environment due to hydrolysis or protonation. As pH changes are also observed in the ocular environment in response to eye diseases, it can be considered an effective release control factor for DNA nanocarriers [208,209]. Researchers such as Zhang et al. have showcased pH-responsive metal-organic backbone DNA tetrahedral gates that release drugs in acidic environments due to the formation of quadruplexes through sequence reconstruction [210]. In another example, Song et al. achieved pH responsiveness in PEG-DNA-GNP carriers using an i-motif [190]. At normal physiological pH, drugs are stably incorporated into the M1/MC2 duplex. However, when the environment becomes slightly acidic, M1 forms an i-motif, causing MC2 to dissociate and release the drug. These studies demonstrate that pH-responsive DNA nanocarriers not only provide stable and high drug loading capacity, but they also enable control the release in response to intracellular endosomal/lysosomal acidic environments. This has the potential to improve the efficacy of drug delivery systems, particularly in ocular and cancer therapies, resulting in better clinical outcomes.

Alternative approaches to achieving pH responsiveness in DNA nanocarriers involve utilizing Watson–Crick- and Hoogsteen-type triplex motifs. These motifs form duplexes at neutral pH and they transition to TAT and CGC DNA triplets at lower pH values [211,212]. Yuwei et al. designed acid-resistant DNA hydrogels for stability in acidic environments by copolymerizing acrylamide monomers with adenine (A)- and cytosine (C)-rich oligonucleotides through free radical polymerization reactions [213]. Changes in Hoogsteen interactions and electrostatic forces, depending on the pH, induce binding or dissociation of the DNA hydrogel. In this study, pH-responsive DNA hydrogels were further developed for oral drug delivery against hostile acidic environments such as the stomach (pH 1.2), duodenum (pH 5.0), and small intestine (pH 7.2). Successful drug administration was confirmed through in vitro and in vivo studies. Moreover, Fu et al. sought to design a pH-responsive DNA motif that was not limited to a specific sequence [214]. By incorporating five pH-sensitive adenine/cytosine (A/C) mismatches evenly throughout the stem region, they successfully destabilized the hairpin structure, enabling it to lose its structure and hybridize with a 20-nucleotide-long DNA strand, forming a DNA duplex. However, when the pH changed during oxidation, the protonated adenine formed A/C base pairs with cytosine, causing the DNA duplex to dissociate. This dynamic process was shown to be reversible when the solution pH alternated between 5.0 and 8.0, highlighting its potential applicability in dynamic DNA nanotechnology.

Small molecules found in the body, such as ATP and GSH, have the potential to act as endogenous stimuli that trigger conformational changes in DNA nanostructures. These small molecule-responsive DNA nanocarriers offer innovative perspectives for disease diagnosis and the development of therapeutic strategies. ATP concentrations vary in different intracellular and extracellular environments and among several organs in the body. Notably, intracellular and extracellular ATP levels are higher than normal in tumor cells or in the presence of inflammation [215]. This mechanism presents an appealing opportunity for designing DNA nanostructures that respond to ATP stimulation. Since DNA itself lacks ATP reactivity, most hybrid nano-assemblies are constructed using ATP aptamers [193,216,217]. Ran et al. introduced a strategy for drug delivery via nano-assemblies composed of graphene oxide, two single-stranded DNAs, and ATP aptamers [218]. Supramolecular π–π stacking interactions between graphene oxide and the drug resulted in high loading efficiency. It was confirmed that the formation of ATP/ATP aptamer complexes in the presence of ATP triggers the dissociation of nano-assemblies, promoting drug release in high ATP concentration environments, such as the cytosol, compared to ATP-deficient extracellular fluid. This approach paves the way for targeted drug delivery systems based on endogenous stimuli. In addition, Xu recently developed ATP-responsive DNA-polyacrylamide nanohydrogels using ATP aptamer as a cross-linker [219]. Following the gel–sol transition induced by ATP, DOX incorporated in the G-C bilayer structure was released, demonstrating anti-cancer cytotoxic effects. In the presence of high levels of intracellular ATP, the ATP aptamer competitively binds to ATP, preventing DNA strand hybridization and causing the disassembly of nanogel by grafting polyacrylamide backbone chains.

Glutathione (GSH), the most prevalent antioxidant molecule in organisms, serves as an excellent material for controlling the morphology of nanocarriers. Similar to ATP, GSH exists at levels four times higher in tumor tissue compared to normal cells and has a higher concentration within cells than in extracellular fluid. This makes GSH a viable candidate for efficient drug delivery in vivo [220]. In contrast to ATP-responsive nano-assemblies, which require ATP aptamers, GSH-responsive designs can employ disulfide cross-linked DNA, offering a significant advantage. Chen et al. reported the formation of DNA nanogels using cross-linking disulfide bonds that released drugs in response to elevated GSH levels [196]. Additionally, some researchers have constructed GSH-responsive nanocarriers through electrostatic interactions between materials and DNA. Specifically, Wang and colleagues developed polyplexes based on polymers containing p-(2,4-dinitrophenyloxybenzyl)-ammonium cationic moieties [221]. GSH specifically cleaves p-2,4-dinitrophenyl ether, converting the ammonium cation to a carboxylic acid anion. This charge-reversal mechanism allows for stable, GSH-responsive drug release while addressing the issue of conventional cationic polymers impeding intracellular release due to strong electrostatic binding to negatively charged DNA. Furthermore, research is broadening to include trigger-sensing drug delivery studies using different types of molecules such as specific enzymes, oligonucleotides, and metal ions [199,202,222,223,224]. It is anticipated that efforts will continue to develop methods that accurately release drugs within the complex environment of the human body.

The ongoing research and development of DNA-based nanomaterials with varied structures and shapes for use in drug delivery systems highlight the promising potential of this field. Numerous studies have demonstrated that DNA nanocages can efficiently enter cells with the assistance of a transfection agent, such as lipofectamine, and can accumulate in the cytoplasm while maintaining their intact structure for up to 48 h [225]. Moreover, DNA nanotubes have exhibited no toxicity to living cells, even when combined with other molecules such as ligands or fluorescent dyes [226,227]. Despite continued efforts to utilize proteins or similar peptides found in the body to create drug delivery carriers with excellent biocompatibility, it has been reported that intraocular administration of nanoparticles modified with these materials can cause eye inflammation [228]. This underscores the importance of good biocompatibility when using DNA-based materials for ocular drug delivery [229]. Though most studies on DNA nanocarriers have focused on their preparation and application for cancer treatment, recent research has begun exploring their potential in ocular drug delivery systems (Table 2).

Researchers such as Kim et al. used a single plasmid DNA molecule compressed with polyethylene glycol-substituted polylysine (CK30PEG) to deliver cystic fibrosis transmembrane modulators to patients with cystic fibrosis [240]. These DNA nanoparticles were introduced into retinal tissue using subretinal injections and did not cause any local toxicity or inflammation. Moreover, recent studies have shown that DNA nanoparticles are effective in eye drop delivery as well. Willem et al. developed drug delivery carriers by hybridizing oligonucleotides to DNA nanoparticles that surround a lipid core [231]. The experiment confirmed improved residence time in porcine and human corneal tissue compared to the original drug, highlighting the potential to increase the in vivo efficiency of these drug delivery systems. Subsequent studies conducted by other research groups have supported the potential of DNA nanocarriers for intraocular drug delivery [232,233,235]. In an in vivo experiment reported by one research group, nanoparticles were injected into the eye tissue of rats and it was found that although the drug appeared diffused when compared to injections into the vitreous cavity, adhesion to retinal tissue was still achieved up to 5 days [232]. As demonstrated in another study, the nanoparticles effectively lowered intraocular pressure compared to the native drug due to the increased adhesion of NPs to the corneal surface for up to 4 h in vitro and up to 1 h in vivo (in pig eyes and rats) [233]. In a separate investigation, Trav-NP was shown to maintain the drug effect for up to 4 h following eye drop instillation and outstanding biocompatibility was confirmed without any indications of apoptosis [235].

In a recently published study on ocular drug delivery involving DNA nanocarriers, researchers developed a DNA nanocomposite by using microRNA instead of organic or inorganic materials [237]. In this study, they selected tetrahedral frame nucleic acid (tFNA), which has biological functions, as the carrier. They attached microRNA-22-3p (miR-22) to the tFNA to treat progressive retinal ganglion cell loss and axonal damage caused by glaucoma. The study demonstrated that tFNA could facilitate miR-22 uptake in retinal neurons, as evident from the results in NMDA-treated RGC-5 cells. NMDA-treated RGC-5 cells were divided into time-based groups and exposed to Cy5-miR-22 and Cy5-tFNA-miR22, respectively. Notably, the Cy5 fluorescence of tFNA-miR22 started increasing significantly at 3 h and peaked at 6 h, whereas the Cy5 fluorescence of miR-22 remained faint at this time point. Although miR-22 gradually entered cells over 24 h, its entry efficiency at 24 h was still lower than that of tFNA-miR22 (33.5% vs. 40.4%). In subsequent experiments aimed at assessing the ability of tFNA-miR22 to modulate the TrkB-BDNF signaling pathway in retinal neurons, tFNA-miR22 demonstrated greater efficacy compared to other groups. Consistent with expectations, both mRNA and protein levels of BDNF were significantly reduced after NMDA induction in the study. However, after treatment with tFNAs-miR22, both TrkB and BDNF expression levels were significantly higher compared to other groups. These findings indicate that tFNAs-miR22 selectively activates TrkB and restores BDNF expression in damaged retinal neurons. These findings not only demonstrate the potential of DNA nanostructures in the treatment of eye diseases, but also highlight the successful establishment of a straightforward, yet effective, delivery system for relevant microRNAs.

Ongoing research in the drug delivery field aims to treat diseases by directly delivering microRNA, akin to the use of aptamers as drug molecules. In particular, since the emergence of COVID-19, interest in nucleic acid therapeutics has surged, with diverse applications being explored, including the treatment of eye diseases. In neovascular eye disorders such as diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity, miRNA expression levels are known to be disrupted [241]. As such, delivering miRNA not only holds promise for treating ganglion cell damage caused by glaucoma, but also suggests the potential for addressing neovascularization induced by a variety of diseases. Given that mRNA is a type of nucleic acid, it is highly unstable, susceptible to rapid degradation due to environmental changes, specific proteins, and being recognized as an endogenous molecule, potentially triggering immune responses. Consequently, a carrier is necessary. Lipid nanoparticles (LNPs) have been widely employed as carriers for mRNA. However, when utilizing DNA nanostructures, it is possible to control interactions with mRNA by diversifying the structure and properties of the materials, thus underscoring the significance of DNA nanostructures in mRNA delivery in future developments. Moreover, previous studies have demonstrated the efficacy of combining DNA nanostructures and mRNA in treating ocular conditions. As a result, research in this area is expected to continue gaining attention and contribute to advancements in targeted therapies for eye diseases.

Indeed, the progression of research on ocular drug delivery carriers has frequently seen carriers that demonstrate effectiveness in systemic diseases such as cancer extend their applicability to the realm of ocular treatment. For instance, Viral S. Kansara et al.’s study examined the therapeutic effect of DNA nanoparticles (DNPs) consisting of single DNA molecules compacted with 10 kDa polyethylene glycol (PEG)-substituted lysine 30-mers (CK30PEG) in retinal diseases [238]. Prior to investigating the treatment impact on retinal disorders, the researchers conducted a study on cystic fibrosis. It is clear that ocular drug delivery utilizing several DNA nanocarriers, such as the previously described hydrogel-based smart DNA nanocarrier, has not yet been extensively explored. However, considering that hydrogel-based ocular drug delivery systems are evaluated as promising and extensively used in ocular drug delivery and that carriers proven effective in other fields have extended to the realm of ocular treatment, hydrogel-based smart DNA nanocarriers are indispensable for the future of ocular drug delivery. Moreover, due to the success of integrating DNA nanocarriers with mRNA in treating ocular diseases and the increasing interest and progress in the field of nucleic acid therapeutics, there is substantial potential for further development and innovation in ocular drug delivery using DNA nanocarriers.

DNA nanostructures hold promise in the field of ocular drug delivery; however, they currently face a myriad of challenges and opportunities. Practical application difficulties stem from variations in drug circulation, distribution, metabolism, potency, and degradation, which depend on the interactions between DNA and cellular behavior. The potential risks associated with DNA nanostructures for ocular drug delivery parallel those observed in nanostructures designed to address toxicity, side effects, and drug resistance challenges in anti-tumor therapies [183,242]. As a deoxynucleotide polymer, DNA is prone to degradation in blood circulation. Furthermore, due to its small size and biocompatibility, DNA can readily bind to single-stranded RNA, such as genes in the cell nucleus, which may result in dysregulation of gene expression. Despite these risks, the high biocompatibility of DNA, a naturally occurring biological molecule, underscores its potential for further development into complex and advanced forms to achieve efficient medical applications. Presently, this field remains in its early stages and practical research focused on biomedical applications is limited. Predominantly, studies are conducted on in vitro cell cultures, tissues, or animal models, such as mice and rabbits. To expand the use of DNA nanostructure-based drug delivery systems in human medicine, researchers must consider immune and circulatory functions, as well as address the challenges of high-purity manufacturing and mass production of DNA nanostructures, which are factors that significantly impact commercial applications. Rigorous preclinical and clinical trials, combined with innovative manufacturing techniques, will be instrumental in overcoming the current limitations and bringing these novel drug delivery systems closer to clinical application. Ultimately, these efforts may lead to groundbreaking therapies for ocular diseases, improving eye care and patients’ quality of life.