Prospects of DNA nanotechnology in stroke repair and regeneration

Abstract

Stroke remains a significant global cause of death and long-term disability, with limited effective treatments for repairing and regenerating damaged brain tissue. Conventional therapy primarily focuses on acute care and preventing recurrence, but it falls short in restoring lost neural function. Recent advances in DNA nanotechnology, enabling precise molecular engineering, targeted delivery, and dynamic bio-functional platforms, offer promising avenues to address this gap. DNA nanostructures have been explored for various stroke therapies, including neuroprotective drug delivery, promoting angiogenesis, reducing inflammation, and guiding stem cells, thanks to their programmability, biocompatibility, and structural adaptability. Additionally, DNA nanodevices paired with imaging agents allow real-time monitoring of cerebral repair processes. Recent studies suggest that DNA nanoparticles could enhance neuronal survival, support functional regeneration, and modify the post-stroke environment. Despite these promising developments, significant challenges remain in vivo stability, immunogenicity, large-scale manufacturing, and safety for translation. This review outlines the current applications of DNA nanotechnology in stroke repair and regeneration, offering mechanistic insights into their therapeutic roles and prospects for clinical translation of DNA-based nanotherapeutics in neuroregenerative medicine.

This review highlights the potential of DNA nanotechnology in stroke therapy and neuroregeneration, focusing on targeted drug delivery, neuroprotection, angiogenesis, and brain repair while addressing challenges in stability and clinical translation.

Introduction

A stroke is clinically defined as a sudden onset of focal neurological dysfunction in the central nervous system¹. It is broadly classified as hemorrhagic (bleeding into the brain by the rupture of a blood vessel) and ischemic (thrombotic or embolic event that causes an impairment of blood flow to an area of the brain)². Stroke is the second leading cause of mortality in middle- to high-income countries and a major cause of disability¹. The incidence of stroke has risen to 85–94 per 1,00,000 but is significantly higher in individuals above the age of 75 years. Furthermore, according to the TOAST (The Trial of Org 10172 in Acute Stroke Treatment), the most widely used mechanistic subclassification system, defines five types of ischemia: (1) large artery atherosclerosis, (2) cardioembolic, (3) small vessel occlusion, (4) stroke of other determined etiology, and (5) stroke of undetermined etiology¹. Similarly, hemorrhagic stroke is further classified into intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH)² (refer Fig. 1). Stroke was considered cardiovascular disease since it affects blood vessels³. In 2018, with the release of ICD-11, it rightly classified stroke as a neurological disorder, prompting increased research into the field⁴. The standard treatments for ischemic stroke include aspirin regimens, recombinant tissue activator (rtPA), and arterial recanalization technology. Surgery intervention is not preferred due to potential risk of disability^5,6. The only FDA-approved drug for acute ischemic stroke, rtPA, cannot be used by a lot of patients and has a narrow time window. Using this causes safety concerns for surgical patients^7,8. Hence, other etiological treatments like aspirin, alteplase, rosuvastatin, and warfarin, and a combination of rtPA with other substances is explored to improve efficacy, increase neuroprotective effects, decreased side effects⁹. Furthermore, the blood-brain barrier (BBB) also restricts the entry of more than 98% of small-molecule drugs and all macromolecular therapeutics from accessing the brain¹⁰. Increasing the lipophilicity of drugs helps them transcend the BBB. However, this is not a universal method since it may inhibit the activity of drugs of interest and cause considerable side effects to peripheral non-target organs. Several novel drug treatments are under research and clinical trials¹¹.

Fig. 1. Types of Strokes, their subsequent sub-types, and causes.

Strokes are classified as hemorrhagic, caused by hypertension, aneurysm rupture, vascular malformations, or trauma, and ischemic, caused by thrombosis, embolism, or systemic hypoperfusion. Created with BioRender.

DNA nanostructures have gained popularity in this field, since they have a programmable sequence, predictable nanostructures, and design functions that enable efficient self-assembly¹². Moreover, with the available research technologies and chemically synthesizable DNA strands, it is possible to make them ideal for intracellular nanostructure construction and specific to stimulus responses, such as increased pH, proteins, and metal ions, which makes this method more diverse¹³. Several reasons favor the use of DNA in constructing biological mimics, functional structures, and materials by molecular self-assembly. Firstly, the well-defined B-form structure, with a right-handed double helix that is formed by two complementary single strands, is about 2 nm in diameter and has 10.5 bases per helical turn, making it ideal. The well-defined B-form structure, with its precise nanoscale dimensions and predictable base-pairing, enables high programmability and structural control. These features translate into key therapeutic advantages, such as precise drug loading, tunable assembly, targeted delivery, and improved interaction with biological systems¹⁴. Second, the single-stranded DNA follows Watson-Crick base pairing to form a double helix, hence forming very predictable interactions. Thirdly, DNA can be used for several practical applications due to its facile synthesis and its chemical stability. Fourthly, it is possible to modify and use DNA with various nanoscale elements that possess different biological, chemical, magnetic, electrical, or optical properties. Next, there is increased spatial accuracy and resolution at the nanoscale since DNA self-assembly is a highly parallel bottom-up fabrication method. Lastly, the inherent sequence specificity, programmability, and addressability make DNA nanostructures more spatiotemporally responsive to a range of external stimuli¹⁵.

This review details the prospective applications of DNA-based nanostructures in stroke therapy diagnostics, medication administration, neuroprotection, and rehabilitation. The review highlights DNA nanodevices that can penetrate the BBB and deliver neuroprotective drugs, their theranostic applications for early diagnosis and monitoring, and the creation of sensitive DNA nanocarriers that respond to stroke-related environmental cues (such as pH and ROS). Furthermore, the integration with CRISPR-Cas systems, aptamers, or RNA-based tools for neurodegeneration and gene regulation and issues with clinical translation, including possible toxicity, targeted effectiveness, and in vivo stability, along with preclinical research on zebrafish and rodent models is also discussed. DNA nanodevices are potential stroke treatment medicines that bridge the gap between nanotechnology and neurotherapeutics.

Basics of DNA nanotechnology

In the 1980s, Seeman first reported that DNA could be used as a structural material. DNA nanotechnology focuses on combining motifs to form ordered lattices of two or three dimensions¹⁶. These structures are formed spontaneously by bringing structural units together by the mechanism of self-assembly¹⁷. The formation of several complex cellular environments depends on the self-assembly of biological macromolecules, such as nucleic acids, lipids, and proteins. The methods of DNA self-assembly have been divided into complementary base pairing, non-canonical base pairing (the formation of G-quadruplexes, the formation of i-motif structures), and the specific recognition of DNA aptamer¹³.

Short single-stranded DNAs are used to build nanostructures like double crossover, triple crossover, 4 × 4, and three-point star structures. Structures, such as nanotubes, 2D lattices, polyhedra, hydrogels, and crystals can be formed by assembling these tiles¹⁸. These nanostructures are broadly created by following rules in these broad categories: 1. Modular self-assembly; 2. Hierarchical assembly of DNA nanostructures; 3. One-pot approach; 4. DNA origami-based strategy¹⁹, Table 1.

Table 1.

The DNA Nanodevices and their Applications

Type of nanodevice	Structure	Therapeutic Application	Advantages	Limitations	References
DNA Origami	Folded 2D/3D DNA structures	Gene silencing, targeted drug delivery	Highly precise, versatile cargo loading. Used in stimuli-responsive or controlled release	Susceptible to nuclease degradation and low stability in physiological conditions; complex and costly synthesis; may trigger immune recognition without protective coatings	15,147
DNA Tiles	Modular lattice-like DNA motifs	Scaffold for a drug or a biomolecule	Easy to synthesize, programmable assembly. Diffusion-based or passive release	Moderate stability and payload limitations due to surface area; potential nuclease susceptibility	23,147
DNA Cages	Hollow polyhedral DNA spheres	Targeted drug encapsulation	BBB penetration, protective encapsulation. Encapsulation-based or stimuli-gated release.	Limited cargo size and risk of enzymatic digestion in circulation
DNA Hydrogels	Crosslinked DNA polymer network	Sustained drug release	Biocompatible, injectable. Sustained release	Weak mechanical strength and unpredictable network properties; costly production; rapid degradation by nucleases	22,148
DNA Walkers	DNA molecular machines	Site-specific controlled drug release	Dynamic motion, programmable targeting. Activity-dependent release.	Sensitive to physiological environments; complex programming and functional design challenges (dynamic systems often less stable in vivo)	149,150
Other NPs in stroke
Liposomes	Phospholipid bilayer vesicles	Transport small drugs & neuroprotectants	Biocompatible, improved circulation, can carry hydrophilic/hydrophobic cargo	Prone to instability (leakage/fusion during storage)	151,152
Polymeric Nanoparticles (e.g., PLGA)	Solid polymer matrix	Sustained drug release, gene delivery	Controlled release, biodegradable, FDA-approved polymer	Initial burst drug release	153,154
Micelles	Amphiphilic self-assembled core–shell.	Deliver small hydrophobic drugs	Enhances solubility, extended half-life	Poor stability in blood	155,156
Dendrimers (e.g., PAMAM)	Branched, tree-like polymers	Gene delivery, anti-inflammatory agents	High loading capacity, defined architecture	Cytotoxicity due to cationic surface groups	157,158
Extracellular Vesicles (EVs) / Exosomes	Cell-derived lipid vesicles	Natural delivery of proteins/miRNA	Innate BBB crossing, low immunogenicity	Difficult large-scale isolation	159,160
Cell Membrane-Derived Nanovesicles	Vesicles coated with specific cell membranes	Targeted delivery to inflammation or clot sites	Homotypic targeting, immune modulation	Complex and variable fabrication process	161
Inorganic Nanoparticles (e.g., CeO₂, MnO₂)	Metal or metal oxide particles	ROS scavenging, oxygen modulation	Enzyme-mimetic activity, supports tissue survival	Long-term toxicity and poor biodegradability	162,163
Nanogels	Crosslinked polymer networks	Controlled release in response to stimuli	High water content, tunable responsiveness	Premature drug leakage	164,165

DNA origami is a bottom-up assembly of the prescribed DNA nano objects, enabling the assembly of more complex and larger structures¹⁵. DNA hydrogels combine desirable features, such as self-healing, biocompatibility, controlled biodegradability, flexible mechanical properties, nutrient permeability, stability against proteases, and stimulus response, making them efficient²⁰. DNA origami is preferred over DNA tiles since it offers higher yield, robustness, and can build complex non-periodic shapes²¹. Two strategies are employed to build DNA origami structures: 1. blunt-end stacking or sticky-end hybridization, and 2. Lengthening the strands by heating or introducing new strands or by PCR²². Use of the DNA origami technique has enabled the construction of DNA cage structures of 60 MDa and of a size range ~10 nm to ~100 nm. Several DNA nanocages with different structures based on the edges and multiple crossover sites have been developed, such as cubes, octahedra, tetrahedra, dodecahedra, trigonal bipyramids, icosahedra, prisms, and buckyballs¹⁹.

Planks assemble hierarchically and are components of DNA tiles. These structures must be rigid and exceed the strength of DNA’s hydrogen bonds for stable self-assembly. It is important to follow a strategic organization of stably bonded single strands to form stable tiles and origami units²³.

DNA can be used as a hydrogel since it can absorb large amounts of water²⁴. DNA acts as a cargo and constitutes the core of the 3D network structure, unlike other nucleic acid-containing hydrogels. This dual property enables a wide range of uses of DNA hydrogels. Hydrogels from DNA strands can be pure or hybridized hydrogels²². DNA walkers are nanostructures that can be programmed to walk on tracks made of DNA, similar to the molecular motors like myosin, dynein, and kinesin, but are relatively slower²⁵.

Pathophysiology of stroke and therapeutic targets

It is believed that ischemic stroke is caused by impaired blood flow, which initiates the ischemic cascade, leading to neuronal injury²⁶ (Fig. 2). Initially, ATP synthesis is blocked due to reduced blood flow, which leads to the failure of high-energy metabolism in neural cells. The blockage of ATP synthesis leads to the inhibition of ion pumps and NMDA receptors. This results in the intracellular accumulation of Ca²⁺, Na⁺, and Cl⁻²⁷. ATP reduction leads to the lysis of mitochondria and lysosomes, promoting calcium influx and hydrolyzing intracellular materials. The increased calcium leads to excitotoxicity due to the release of the excitatory amino acid glutamate²⁸. This leads to the initiation of the apoptotic pathway, since nitric oxide synthase, protein kinase C, and phospholipase A2, which are calcium-dependent enzymes, are activated²⁹. A mitochondrial calcium influx would lead to the activation of downstream apoptotic pathways, inducing apoptosis³⁰ (Fig. 3). Moreover, this also leads to injured neural cells, which release free radicals, reactive oxygen species (ROS), and danger-associated molecular patterns (DAMPs)⁹. These include heme, fibrinogen, high-mobility group box 1 (HMGB1), S100P, heat shock proteins, and matricellular proteins, such as tenascin-C and galectin-3³¹. Upon activation by DAMPs, microglia and astrocytes release inflammatory factors like cytokines and chemokines^32,33, which leads to the subsequent recruitment of leukocytes and their adhesion to endothelial cells. This leads to the alteration of the endothelial cytoskeleton, degenerating pericytes, and the cleavage of endothelial junction proteins due to enzymatic cleavage. This disrupts the BBB³⁴. The BBB, located in the brain, is a structure of microvessels that maintains homeostasis in the central nervous system (CNS). It works as a protective barrier, both physical and chemical, by restricting transduction of potentially toxic and harmful substances^33,35. The BBB can be disrupted by ischemic or hemorrhagic stroke, leading to serious clinical conditions like vasogenic brain edema and hemorrhagic transformation, due to the influx of water molecules and blood components into the brain extracellular space³⁶. The BBB is located at the center of the neurovascular unit (NVU). The neurovascular unit is a dynamic barrier consisting of the cellular components, intercellular connections, and molecular signaling systems between blood and the central nervous system. A rich transport system is facilitated by several cellular components, like endothelial cells, glial cells, vascular cells, and neurons, connected by adherens junctions, gap junctions, tight junctions, and pericyte-endothelial junctions³⁷. Excitatory pathways are key regulators in ischemic stroke. Hence, certain drugs that regulate the excitatory amino acids have been considered as an important aspect of treatment. Presently, the pathway to inhibit glutamate release has been explored in several clinical trials, such as glutamate release inhibitors, glutamate receptor modulators, GABA receptor agonists, and 5-HT1 receptor agonizts. Certain natural ingredients like astragaloside IV and baicalin are used to regulate downstream signaling pathways, such as calcium influx, since they are known to inhibit excitatory toxicity and play a neuroprotective role^30,38.

Fig. 2. Ischemic cascade events in the neurovascular unit during acute stroke.

Deprivation of oxygen and glucose induces the release of glutamate from astrocytes, leading to excitotoxic neuronal injury and activation of microglia. This releases DAMPs, further activating microglia and promoting pro-inflammatory cytokine release, leading to BBB disruption. The infiltration of leukocytes amplifies the neuroinflammation, while neurons emit signals to stimulate astrocytes and microglia to release neuroprotective factors. Figure adapted from ref.³². Copyright: Nature Publishing Group.

Fig. 3. Pathophysiology of Ischemic Stroke.

Arterial blockage that lowers cerebral blood flow and oxygen delivery is the cause of an ischemic stroke. ATP-dependent ion pumps are hampered by the ensuing energy failure, which causes depolarization, glutamate excitotoxicity, and Ca2+ overload. These occurrences cause oxidative stress, mitochondrial malfunction, and the initiation of cell-death pathways. Tissue damage is exacerbated concurrently by leukocyte infiltration, breakdown of the blood–brain barrier, and the release of inflammatory mediators. A crucial treatment target is the surrounding penumbra, which is still recoverable while the ischemic core has permanent necrosis. Created with BioRender.

The brain is more sensitive to oxidative stress due to its higher oxidative metabolism. Reactive oxidative species (ROS), mainly produced by mitochondrial succinate dehydrogenase and NADPH oxidase, are a result of the impaired homeostasis between the body’s oxidation and antioxidant systems³⁹. The dynamic balance of ROS is destroyed in the case of ischemia due to the expression and activity of enzymes like NOS, cyclooxygenase (COX), xanthine dehydrogenase/xanthine oxidase, coenzyme II oxidase, monoamine oxidase (MAO), and ROS inhibitors like superoxide dismutase (SOD), catalase (CAT), peroxidase, and glutathione peroxidase (GSH-Px). The activity of these enzymes maintains a balance in normal physiological conditions⁴⁰.

Apoptosis is induced in three main ways in brain ischemia: the mitochondrial pathway, the death receptor pathway, and the endoplasmic reticulum stress pathway. The Bcl2 protein family, p53 gene, caspase family, Fas gene, and the JAK/STAT pathway are key members of the apoptosis pathway. Bcl2 is prominent in brain ischemia reperfusion injury, since it has an endogenous neuroprotective effect. The p53 accumulation in the plasma membrane directly activates Bax to trigger cell apoptosis. It also increases the permeability of the outer mitochondrial membrane, leading to the release of cytochrome C after binding with Bcl2⁴¹. Once caspase 3 is induced, cell apoptosis is initiated and executed. The activity of caspase 3 leads to the lysis of several intracellular components in the mitochondria and the death receptor-mediated apoptosis pathway. The activation of the caspase family is caused by the Fas receptors and the Fas/FasL system³⁰, Table 2.

Table 2.

Summary of stroke pathophysiological targets

Target	Role in Stroke	Therapeutic Strategy	Potential DNA Nanodevice use	References
BBB disruption	Barrier breakdown hinders drug delivery, increases leakage	BBB-penetrating nanocarriers	DNA cages, aptamer-modified nanocarriers	166,167
Oxidative stress	Causes damage to lipids, proteins, and neurons	Antioxidant delivery, ROS scavenging systems	ROS-responsive nanostructures	30,168
Neuroinflammation	Worsens ischemic injury due to cytokine release	Anti-inflammatory drug delivery, cytokine inhibition	DNA hydrogels	169
Apoptosis	Programmed neuronal death, tissue loss	siRNA, gene silencing, neuroprotective drugs	DNA origami for siRNA delivery, apoptosis sensing	30,170

Therapeutics for stroke

Current therapeutics for stroke

Some other alternatives for treatments for ischemic stroke apart from thrombolysis via r-tPA are demonstrated in Fig. 4. Mechanical thrombectomy, an endovascular process to restore the blood flow by removing the thrombus of an intracranial occlusion⁴². The role of neuroprotective agents like NMDAR and GABA agonists, calcium channel blockers, immune modulators, and hypoglycaemic therapies is to counteract the harmful molecular and biochemical events causing irreversible ischemic damage⁴³. Vagus Nerve Stimulation (VNS) has shown a reduction in infarct volumes and improvement in neurological deficits in a stroke model^44,45. Antithrombotic agents are divided into two classes: antiplatelet agents and anticoagulant agents. The former is used to prevent non-cardioembolic stroke, while the latter is used in the efficient prevention of cardioembolic stroke⁴⁶. Transplantation of neural stem cells (NSC) in the brain can promote neural recovery post stroke; however, the mechanism of NSC is still unknown⁴⁷. Human urinary kallidinogenase (HUK), an upcoming drug, reduces cerebral infarction volume, mortality, and disability rate⁴⁸.

Fig. 4. Different therapeutics used for treating ischemic stroke.

The goals of ischemic stroke treatment are to prevent neuronal damage, encourage recovery, and restore cerebral blood flow. Thrombolytic therapy: when administered intravenously within the therapeutic window, recombinant tissue plasminogen activator (rt-PA) destroys the clot and restores perfusion. Endovascular thrombectomy: in cases of major vessel obstruction, fast reperfusion is achieved by mechanically removing the obstructing clot using stent retrievers or aspiration catheters. Heparin, clopidogrel, and aspirin are antiplatelet and anticoagulant medications that lower the risk of recurrent thromboembolic episodes. Neuroprotective agents: To maintain the ischemic penumbra, experimental treatments target oxidative stress, apoptosis, and excitotoxicity. Anti-inflammatory and BBB stabilizers: subsequent harm is decreased by substances that alter immune responses or preserve vascular integrity. Rehabilitation and regenerative therapies: neurorehabilitation, growth factor administration, and stem cell transplantation improve functional recovery and plasticity. When taken as a whole, these therapeutic approaches emphasize both short-term measures to restore blood flow and long-term plans to enhance neurological results. Created with BioRender.

DNA nanodevices in stroke

As mentioned before, stroke can be caused by intracranial ischemia or spontaneous hemorrhage. The use of TdNs (tetrahedral DNA nanostructures) has been explored to have a therapeutic effect by reducing the damage caused by ischemic stroke or intracerebral hemorrhage. These materials can modulate neuroinflammation and cellular survival pathways. This has been proven in in vitro models by creating acute ischemic stroke due to hypoxia (deprivation of oxygen and glucose). Reoxygenation in neuronal cell lines, such as SH-SY5Y cells, using TdNs has shown a reversal in neuronal loss and has improved the ischemic microenvironment by reducing apoptosis⁴⁹. It was reported in a study by Zhou et al. that tetrahedral framework nucleic acids (TdNs) act via multiple targets to exhibit significant neuroprotective effects in ischemic stroke. They have the ability to cross an intact as well as a compromised BBB via internalization into brain capillary endothelial cells by endocytosis or penetration. It was observed that TdNs protected neuronal cells, such as SH-SY5Y, by interfering with the ischemic cascade, reducing excitotoxicity and oxidative stress, and inhibiting apoptosis in ischemia-reperfusion-mimicked conditions by oxygen-glucose deprivation. The upregulation of Bcl2 and the downregulation of Bax and Caspase-3. In a transient middle cerebral artery occlusion (tMCAO) rat model, TdNs demonstrated significant neuroprotective effects in vivo. These benefits included a substantial reduction in infarct volume, decreasing from approximately 34to 2.7% of the total brain volume, a reversal of neuronal loss, and an attenuation of neurological deficits. Mechanistically, TdNs improved the ischemic microenvironment by simultaneously upregulating erythropoietin expression and inhibiting inflammation. The core molecular pathway that leads to these protective actions is the inhibition of the TLR2–MyD88–NF-κB signaling cascade, which effectively suppresses inflammatory and apoptotic signaling within the ischemic brain tissue⁴⁹. Moreover, TdNs are known for regulating astrocytic polarization by suppressing the A1 phenotype (reducing A1 markers and inflammatory cytokines (C3, IL-1β, TNF-α, IL-6)), which is pro-inflammatory. It also promotes the A2 phenotype (characterized by increased expression of A2 markers and neurotrophic factors, such as S100A10, NGF, and GDNF), which is neuroprotective in nature for ischemic stroke. This helps in improving the ischemic microenvironment by reducing neuroinflammation and contributes to neuronal protection due to the A1 to A2 phenotypic shift⁵⁰. DNA sensing innate immune pathways strongly influence post-stroke neuroinflammation. Post-stroke, the cGAS-STING signaling pathway is activated due to the mitochondrial DNA released from damaged cells, which leads to pro-inflammatory microglial activation and exacerbation of neuronal damage. It has been seen in experimental stroke models that there is suppression of inflammatory signaling and reduction of neuroinflammation by targeting the STING pathway using C-176, a small molecule inhibitor. This provides future opportunities to use engineered DNA nanostructures to control such signaling pathways and induce immune responses that follow stroke, and minimize excessive inflammatory activation. The application of direct engineering of microglial polarization using DNA nanodevices is an emerging area⁵¹. Furthermore, when we look at the ICH model, there has been use of tFNAs that carry siRNA that target chemokine receptor 2 (CCR2 (tFNA-siCCR2)), and downregulate the expression of CCR2. This results in the recruitment of pro-inflammatory and immune cells. This improved neurological outcomes by clearing the hematoma and by the mitigation of neuroinflammation due to balanced expression of inflammatory mediators⁵². There have been several studies pertaining to nanomaterials being used as a neuroprotective agent; however, it has been difficult to take these therapies and techniques up to the clinical level due to the neurotoxic potential of many nanomaterials and also the complex nervous system. However, TdNs are a DNA nanomaterial and hence have several biological advantages. It has higher stability and biocompatibility and can be easily taken up by mammalian cells, making them a highly popular and effective therapeutic for neuromedications⁵³. Studies have indicated that they are capable of neuronal differentiation of neural stem cells (NSCs) and can promote the proliferation and migration of cells. Therapies that involve NSCs and tFNAs have been shown to improve spinal cord injury. Moreover, it is shown that tFNAs can protect PC12 cells from apoptosis. This property can be useful in therapeutic solutions for neurodegenerative diseases like Parkinson’s and Alzheimer’s⁵⁴. The tFNAs are known to target the Toll-like receptor (TLR-2) pathway in acute ischemic stroke recovery. The TLR-2 pathway is responsible for the inflammation and oxidative stress in the ischemic tissue. It has been shown that if the TLR-2 pathway is blocked, there is a reduction in neuronal death, limiting infarct volume, and it improves functional recovery, making it a crucial target for intervention⁴⁹. Moreover, it has also been shown that TdNs can interfere in the ischemic cascade that involves excitotoxicity, apoptosis, and oxidative stress, and confer neuroprotection to the neuronal SH-SY5Y cells. It has been shown that the neurological deficits that were a result of acute ischemic stroke, such as impaired cognition, motor skills, and high disability, were greatly alleviated by the effect of TdNs⁵⁵. They improved survival and behavioral outcomes and also reduced the infarct size. Furthermore, microscopic analysis of the brain tissue has shown that the animals treated with TdNs had less neuronal loss than the saline controls. The qualitative labeling of the NeuN/DAPI confirms the reversal of ischemia-induced neuronal depletion in both the cortex and the penumbral regions. tFNA-treated models, compared to untreated AIS models, have been shown to have preserved neurons and, to some extent, enhanced neurogenesis, which shows a promising ability for neuroprotection and regeneration potential in the tFNAs⁴⁹. Furthermore, the potential of tFNAs was also seen in tMCAO models, where their administration was seen to significantly reduce the infarct volume size and reduce mortality rates. Further, there was modulation of excitotoxicity, oxidative stress, and apoptosis in the in vivo as well as in vitro studies. The most significant result is that the tFNAs have shown a particular molecular mechanism underlying their efficacy, which is mediated by the inhibition of TLR2 signaling⁴⁹. Furthermore, TdNs have also been promising therapeutic agents in non-neurological conditions. They can act as anti-inflammatory agents in periodontitis and also as antioxidants in acute kidney injury. They have demonstrated consistent results in both cellular and animal models, by reducing the infarct size and restoring the complex neurological functions of motor coordination and sensory processing. They also have a pleiotropic action, which is advantageous in conditions like AIS, where several pathways, such as calcium overload, oxidative stress, neuroinflammation, and other pathological mechanisms, are involved. They have also been effective in nanomolar concentrations, highlighting their biocompatibility and biodegradability, which distinguish them from other nanomaterials⁵³. There has been no reporting of significant adverse effects when compared with saline controls. They are superior in terms of biosafety to polymeric, metallic, and liposomal nanoparticles, since there are no cytotoxic by-products. The spectrum of TdNs is broad since they have very versatile structures. They can be used as neuroprotective agents as well as nanocarriers for therapeutic payloads. TdNs are better drug delivery agents in AIS, compared to other nanoparticle platforms, since they are structurally robust, ideal for synthesizing for large yields, and are not toxic. These TdNs are a promising neuroprotective agent and drug delivery system for stroke and could open a therapeutic window for existing interventions, such as thrombolysis⁴⁹.

Along with TdNs, DNA origami is used to integrate DNA sheets with tPA binding sites and thrombin-responsive DNA fasteners; these can rapidly target the site of the thrombus and begin the mechanism of action by exposing active tPA when thrombin concentration exceeds threshold⁵⁶. Moreover, it is possible to harness organ-specific delivery of the GOI by using DNA origami structures encapsulated in LNPs⁵⁷. Banik et al. have reported that brain cell-derived exosomes were used to encapsulate an ATP-responsive DNA aptamer sensor to allow for targeted delivery across the BBB⁵⁸.

Several other kinds of polymers can be used as therapeutic agents. Certain polymers can change their hydrophilicity or hydrophobicity, degradability, and ionizability to respond to certain internal stimuli like ROS, GSH, pH, enzyme activity, and some external stimuli like laser and ultrasound, known as stimuli-responsive polymers. These stimuli-responsive polymers can be integrated with DNA nanostructures since they provide a programmable and biocompatible scaffold, in turn adding a property of environment-sensitive behavior in the nanocarrier. These devices help in allowing targeted cargo release, additional structural stability, and enhanced responsiveness to pathological conditions. For instance, it is possible to graft polymers responsive to ROS, GSH, or pH variations to DNA nanostructures in order to enable site-specific drug release at ischemic stroke sites^59,60. Some hydrogels are used as a stimulus-responsive nanodevice to help with diabetes. Diabetic wounds have a very adverse environment, characterized by oxygen deficiency, reduced angiogenesis, and increased oxidative stress. Hydrogels have properties that promote angiogenesis and exert antioxidant stress, and they can be used as clinically efficient modes of healing such wounds. The antioxidant activity of the gel is governed by polydopamine and a metal–organic framework-derived catalytic enzyme mimic (ε-polylysine-coated mesoporous manganese cobalt oxide), which is present in the gel. The hydrogel network is created using Schiff’s base reaction between hydrazide-modified hyaluronic acid and aldehyde-modified pectin. Moreover, these can catalytically generate oxygen by ROS, and they exhibit a good hemostatic performance along with biocompatibility. This shields skin cells from ROS and also reduces death by hypoxia. It has sped up the healing of wounds in diabetic rats. Several different strategies are employed to create these stimulus-responsive nanomaterials. To make them pH-responsive, the backbone of the nanoparticle consists of functional groups that can be protonated in acidic conditions⁶¹. As a result, the nanostructure is destroyed due to the disturbance of the equilibrium in the particle. This destruction leads to the release of the drug from the structure by rearrangement, disintegration, or expansion. Moreover, the use of acid-responsive bonds can also be used, which are unstable at lower pH and break; as a result, the nanostructure is disrupted, and the drug is released⁶². For making them enzyme-responsive, specifically for cancer treatment, structures that get destroyed under overexpression of tumor-associated enzyme matrix metalloproteinase (MMP2, MMP9, MMP13) and cathepsin B are preferred⁶³. These enable the release of the cargo at specific sites where the tumor is present due to enzyme-specific cleavage. Similarly, since cancer cells have a higher amount of ROS, thioketal nanoparticles, thioether phosphatidylcholines, and boronic esters are used for drug release at tumor-specific sites⁶⁴. To deal with the GSH response, materials that are degraded by high GSH levels, like disulfide, manganese dioxide, and diselenide bonds, are used. Their degradation would lead to drug release at the specific site. The employment of such site-specific techniques enables the accumulation of the drug in the tumor and increases therapeutic effects⁶⁵.

Furthermore, DNA technology can now be integrated with next-generation therapeutic platforms, because of the advancement in genomic engineering in recent years. Today, nucleic acids, proteins, and genome editing systems like CRISPR-Cas9 complexes and guide RNAs can be delivered by programmable DNA nanocages that act as protective carriers⁶⁶. Furthermore, it is possible to make precise nucleotide modifications by using genome editing techniques like base editors and prime editors, which can induce modifications without introducing double-strand DNA breaks^67,68. It is possible to correct mutations and modulate gene expression pathways of oxidative stress, inflammation, and neuronal survival post-ischemia using such tools. Moreover, DNA nanostructures’ versatile nature helps in integrating them with optogenetics modules, which help in temporally and spatially precise delivery of the cargo by enabling light-controlled activation^69,70. In disorders like stroke, DNA nanotechnology helps in advanced genome editing for achieving precise and controllable therapeutic delivery.

DNA nanodevices in stroke diagnosis and imaging

MRI and CT are the commonly used diagnostic techniques for analyzing the severity and location of stroke by providing accurate brain images; however, both of the methods are expensive and not easily accessible⁷¹. Some of the common techniques used for diagnosing stroke biomarkers in a clinical setting include quantitative real-time PCR, enzyme-linked immunosorbent assay, immunoturbidimetry, single-molecule array, liquid chromatography-tandem mass spectrometry, next-generation sequencing, and biosensing technologies⁷². Stroke diagnosis and imaging using circulating miRNAs, exosome-derived miRNAs, and intracellular pathway analysis, each with its own advantages and limitations, have been reported. DNA nanodevices help overcome these challenges through their programmability, sensitivity, and ability to function in complex biological environments.

A type of RNA with 18–25 nucleotides that is expressed endogenously is known as miRNA (microRNAs), and it plays a crucial role in various regulatory events, such as cell proliferation, differentiation, and apoptosis. According to recent studies, abnormal miRNA expression is linked with the occurrence, development, and metastasis of ischemic stroke and tumors, making miRNAs a potent biomarker for early ischemic stroke diagnosis and prognostic monitoring. However, miRNA analysis in a clinical setting is limited due to low concentration, similar sequences, and a complex intracellular environment. Hence, there is a need for developing approaches that are sensitive, specific, and stable for miRNA detection for clinically relevant conditions. Conventional techniques for miRNA detection in vitro conditions include reverse transcription-quantitative polymerase chain reaction (RT-PCR), northern blotting, and microarray. These methods require further RNA extraction from samples via complex, time-consuming processes, which can cause further degradation of the RNA, resulting in poor-quality RNA and reduced reproducibility of outcomes. Catalyzed hairpin assembly [CHA] and HCR are enzyme-free amplification methods that have been used more often because of their high amplification efficiency, minimal background signal, and isothermal reaction conditions. Nevertheless, the limitations of these methods are the biostability of DNA probes, which can degrade in the presence of nucleases, leading to false-positive results. In order to mitigate this limitation, several inorganic nanomaterials are also used as carriers for DNA probes; however, these external nanoparticles could induce toxicity, even though they provide an excellent sensing capability. DNA tetrahedral nanostructures (DTN) have attracted a lot of attention in the field of bio-analysis owing to their extraordinary structural stability and biocompatibility. In a study done by the researchers, they introduced another method for miRNA detection in tumors by using DTN-mediated HCR (DTN-HCR). Functionalised DTN nanoprobes are formed by conjugating HCR to one of the vertices of DTN via complementary base pairing. Further, by conjugating DNA hairpin structures at the vertex of DTN, the overall bio-stability of the probe improves. The increased bio-stability comes from the fact that the DTN can structurally protect the hairpin loop by creating steric hindrance. This extra layer of protection further reduces the false-positive results and improves miRNA detection⁷³.

An alternative to circulative miRNA in the bloodstream, miRNA found in exosomes [exo-miRs] are known to have higher abundance and stability because of the protection from the lipid bilayer membrane⁷⁴. In disease diagnostics, exo-miRs have gained considerable attention because of their enhanced preservation in liquid biopsy, owing to their stability when exposed to enzymes present in bodily fluids, and thereby acting as a potential biomarker for liquid biopsy. Synaptic communication and neuroglial interaction are influenced by the exosomes released from cells in the CNS, such as neurons, astrocytes, and microglia⁷⁵. A promising advantage in disease diagnosis via liquid biopsy is shown by exo-miRs because of their presence in urine, blood, plasma, and saliva. An optimized and instantaneous monitoring of exo-miRs ensures a convenient disease diagnosis in patients. miR-126, miR-223, miR-124, and miR-9 showcase a promising ischemic stroke diagnosis, because of higher specificity and sensitivity in comparison with free circulation miRNA. A study conducted showed that miR-223 was able to have an AUC of 0.859 with78.8% specificity and 84.0% sensitivity in diagnosing stroke⁷⁶. However, there are limitations in detecting exo-miRs in situ when using molecular sensors. These limitations arise because of the inherent properties of exo-miRs, including their complex composition, small size, low concentration, and presence of membrane obstruction. In order to overcome the aforementioned limitations, the newly designed molecular biosensors should be able to enter a confined space to identify the target and should have a uniform and constant signal output when present in a nanoscale environment in exosomes. Nucleic acid-incorporated biosensors are known to have unique qualities that allow them to overcome the limitations mentioned above because of their small size, natural biocompatibility, exceptional programmability, and enhanced ability to recognize molecules. Currently, in much research, several DNA nanomaterials like tetrahedral DNA, hairpin DNA, branched DNA, and spherical DNA are used as biosensors for the detection of exo-miRs⁷⁷.

DNA assemblies are considered primary materials used in biosensing imaging owing to their low immunogenicity; along with this, they are able to enhance the sensitivity and specificity of imaging by carrying multifunctional units, such as fluorophores, signal amplification elements, and targeting ligands. These multifunctional units are also important in precision imaging and personalized medicine²². It is possible to create high-purity DNA nanocages via the self-assembly technique. This structure may enter the brain as soon as 15 min after intrathecal injection and remain there for at least 12 h, according to PET imaging using Zr-89-tagged DNA nanocages. The biodistribution of the same was confirmed by fluorescent imaging with a DNA nanocage labeled with 800CW. MRI and TTC staining of brain tissues ex vivo showed a significant reduction in the brain infarct volume when administered to rat models of ischemic stroke (Fig. 5). Extensive mechanistic investigations showed that DNA nanocages may reduce the expression of Caspase-3 in rats suffering from ischemic stroke and efficiently scavenge ROS in vitro⁷⁸.

Fig. 5. PET and MRI scans showing DNA nanocage uptake in rat brain.

Figure adapted from⁷⁸. Copyright: Nature Publishing Group.

To study specific endocytic pathways, researchers can now encapsulate gold nanoparticles (AuNPs) and multi-functionalized quantum dots within a DNA icosahedron. DNA tiles, formed using the DNA origami method, serve as standard tools for high-resolution microscopy techniques, such as SIM, STED, and PALM. DNA origami can easily calibrate quantum efficiency, brightness, yield, and distance with multiple fluorophores. For fixed samples, DNA-PAINT (DNA-point accumulation for imaging in nanoscale topography) has been used to achieve 3D ultrahigh resolution⁷⁹. DNA-PAINT acted as an alternative to SMLM-based imaging methods. In this method, we can see reversible binding and unbinding of fluorescently tagged oligonucleotides and DNA origami to get randomized photo-switching. Through this imaging method, one can also get images of the DNA nanostructures with a resolution of sub-10 nm^80–82. For achieving super high resolutions, researchers have combined DNA nanotechnology with advanced hardware technique⁸³. SMLM-based super-resolution imaging shows limited performance with thick samples; to overcome this limitation, Schueder et al. used 3D DNA -PAINT in combination with spinning disk confocal hardware; this combination was able to give resolution up to 20 nm in the x–y plane and 80 nm in the z-axis with depths up to 10 mm, allowing whole-cell imaging⁸⁴. It is possible to modify tetrahedral DNA nanostructures with various fluorophores, making them useful as platforms for biosensing and bioimaging⁸⁵. In an experiment by Fan and coworkers in 2016, tetrahedral DNA nanostructures were labeled with different probes, such as near-infrared (NIR) dye Dylight-755 (Dy) and isotope 99mTc, allowing for both NIR fluorescence and single-photon emission computed tomography (SPECT). They observed that when they used folic acid as their targeting ligand, resulting in FA-Dy-99mTc-TDN, which can simultaneously use both NIR and SPECT for in vivo tumor imaging⁸⁶. In another study done by Liu and coworkers to show that targeted drug delivery and cancer cell imaging can be done hand in hand, they used a modified TDN with two different aptamers - MUC1 and AS1411. In this modified version, the MUC1 aptamer had a quencher molecule, Cy5, adjacent to it. The fluorescence signals are only seen when MUC1-positive cells are present, which causes the MUC1 aptamer to bind to the MUC1 protein⁸⁷.

In the field of stimulus-responsive DNA nanostructures, one of the main research fields includes biosensing. DNA sequences respond to a variety of external stimuli apart from their nucleotide composition, including changes in ion concentration, pH levels, and peptides. The above-mentioned response mechanisms act as a basis for the development of biosensors. DNA nanostructures have unique abilities that can be used in biosensing applications, such as great controllability, which allows control of the distance between sensors and hence increases detection specificity; inherent biocompatibility and penetration power; integrating with hybridized chain reactions to accomplish enhanced signal amplification; improved detection specificity by being conjugated with other materials; combining with multiple detectable beacons like fluorophores, metal ions, or chemicals. DNA nanostructure biosensing applications focus on the detection of low concentrations of substances in a fast manner⁸⁸. Zhang et al., in their study, showed that a DNA hairpin-loaded DNA tetrahedron was used for readily detecting intracellular RNA and cell membrane proteins. Interestingly, what caught the eyes of many researchers is the downstream reactions occurring after the detection. Combining biosensors with different downstream reaction designs can aid applications from detecting up to cellular capture⁸⁹. The potential molecular mechanisms by which DNA nanocages act in ischemic stroke are shown in Figs. 6–8.

Fig. 7. Nanoparticles (self-fueled nano-penetrators) used in the alleviation of ischemic stroke.

Figure adapted from⁹⁰. Copyright: Nature Publishing Group.

Fig. 6. Possible mechanism of action of DNA nanocages in ischemic stroke.

Created with BioRender.

Fig. 8.

Pipeline for evaluating DNA nanocages in stroke medicine.

Gene regulation and regenerative therapies

The recent advances in nanotechnology for therapeutic applications focus on carrier-free nanoassemblies, DNA-based drug delivery, and their potential in treating ischemic stroke, cancer, and neurodegenerative diseases. DNA nanostructures, like tetrahedral DNA conjugates (TDN) and gold nanoparticles (AuNP), are used for ultrasensitive microRNA detection and efficient gene silencing. This plays a potent role in drug resistance and targeted drug delivery, especially for cancer. These nanostructures exhibit neurotherapeutic potential, which promotes nerve regeneration, protects against oxidative stress, and supports neuroprotection in diseases like retinal damage and neurodegeneration.

The nanoassembly was co-assembled using a photothermal photosensitizer, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), and a photothermal-activatable nitric oxide donor, N, N′-di-sec-butyl-N, N′-dinitroso-1,4-phenylenediamine (BNN6), producing stable structures over a broad range of molar ratios. It was identified that a 1:3 ratio of DiR: BNN6 was optimal for stability and NO production; it was also efficient for laser-driven autonomous motion through gas bubble generation, allowing deep clot penetration. These self-fueled nano penetrators have an extremely high fuel loading capacity, outperforming conventional PLGA nanoparticles and the clinical thrombolytic drug lumbrokinase in animal models. Ischemic stroke was also alleviated by modulating platelet aggregation, cyclic GMP, and microvascular repair because of NO release⁹⁰.

Alternatively, tetrahedral DNA conjugates and AuNPs are capable of detecting ultrasensitive miRNA in femto- or attomolar concentrations^91–93. MicroRNA detection in body fluids can be useful to identify the disease before the onset of pathological symptoms⁷⁹. miRNA detection has been done by engineered DNA nano-switches from an RNA extract. These switches, when linear (off), can be turned to loops (on) upon binding with miRNA, which can be easily detected in gel-electrophoresis^79,94,95. In a study done by Shen et al., they wanted to see if a DNA origami template could improve folding and enhance fluorescence intensity. Hence, they used an aptamer - Broccoli and DFHBI-1T flurogen for characterizing FLAPs scaffolded on DNA nanostructure in an in-vitro system. The aptamer Broccoli was conjugated with rectangular DNA origami at different valencies and positions, forming single- and multi-broccoli nanostructures. In the presence of DFHBI-1T, the single Broccoli aptamer conjugated with DNA origami showed 10 times higher fluorescence. Similar enhancement was observed when Broccoli aptamers were conjugated to 2D DNA brick nanostructures or scaffolded with double-helix templates. They also studied different lengths of DNA nanostructure and confirmed that fluorescence enhancement was observed in any double-stranded DNA template⁹⁶. An increase in the development of gene therapy has made researchers look into the potential of functional nucleic acids, like aptamers, miRNAs, siRNAs, and mRNAs, for tissue regeneration. Nucleic acid-based devices provide a very promising approach for nucleic acid delivery, owing to their phenomenal biocompatibility, easy synthetic process, high transfection efficiency, and minimal off-target effects⁸⁸. DNA nanocages based on the required type of therapy are tailored in a way that they can carry various types of drug cargoes⁹⁷. One such example is DNA nanocarriers used in immunotherapy, where they can carry unmethylated CpG sequences, which enhance immune response, or monoclonal antibodies and other drugs as immune checkpoint inhibitors. The inhibitors will help the immune system to recognize and attack cancerous cells⁹⁸. DNA nanocarriers can also seamlessly integrate nucleic acid therapeutics because of their similar chemical structures. Modern macromolecular drugs like miRNA, siRNA, and antisense oligonucleotides can be delivered using these carriers, aiding in targeting and altering gene expression or protein production⁹⁷. DNA nanocarriers allow specific targeted chemotherapeutic drug delivery when they are intercalated into the DNA duplex, and also overcome drug resistance. A widely used cancer therapy is suppressing gene expression, because it is precise and does not have any adverse effect⁸⁵. Lee et al. conjugated TDN with folate and anti-luciferase siRNA to target the luciferase gene in tumors. The modified TDN was injected into nude mice via the tail vein, and was accumulated highest in the kidney and tumor, with low levels in the heart, spleen, liver, and lung. In KB cells overexpressing folate receptors, luciferase activity showed a 60% decrease in bioluminescence, whereas there was no decrease in bioluminescence; folate conjugated with anti-luciferase siRNA alone showed no effect⁹⁹. Another study on suppression of gene expression by Ding and his co-workers used a double-bundle TDN to deliver an antisense oligonucleotide (ASO) targeting the c-raf proto-oncogene. In a reducing environment, the disulfide bond linking the ASO to the TDN is cleaved, releasing the ASO. Once released, the ASO binds to c-raf mRNA¹⁰⁰. DNAzymes or deoxyribozymes are considered therapeutic agents themselves for various diseases because they have the ability to recognize and cleave their substrates^101,102. In an experiment by Meng et al, on gene regulation, they designed TDN for DNAzyme D13 delivery. It was found that this DNAzyme was able to cleave mRNA involved in cell proliferation - c-Jun. It was observed that the conjugated TDN with DNAzyme showed high cellular penetration, and as a result, a decrease in cell proliferation was seen due to c-Jun mRNA silencing activity. The above-mentioned studies showcase TDN advantages in gene therapy¹⁰³. DNA tetrahedron (TD), along with its phenomenal drug delivery ability, also has a wide range of neurotherapeutic efficiencies, some of which include neuroprotective and neurotherapeutic effects, neural stem cell proliferation, and the ability of neuronal differentiation¹⁰⁴. As per the recent data, it is observed that TDN is able to regulate cellular behavior, having different effects on a variety of cell types¹⁰⁵. A study done by Shi and coworkers observed an increase in autophagy in chondrocytes after tFNA exposure at 250 × 10–9 M concentration¹⁰⁶. Zhou and colleagues observed that tFNA promoted proliferation and osteogenic differentiation when exposed to human periodontal ligament stem cells¹⁰⁷. tFNA could also enhance proliferation and differentiation of neural stem cells, as shown by Ma and colleagues, in neuroectodermal stem cells¹⁰⁸. A study done by Yao on Schwann cells shows that incubating tFNA at a concentration of 125 × 10–9 M for 24 h showed enhanced cell proliferation, migration, and also activated NGF/PI3K/AKT neuroprotective signaling pathway, leading to secretion of functional proteins¹⁰⁹. TDNs' therapeutic ability was also seen in facial nerve crush models, where they could promote the recovery of nerve conduction, muscle movement, and repair injured myelin sheaths and axons. TDNs also affected the expression of marker proteins like neurotrophic factor, myelin basic protein (MBP), nerve growth factor, and peripheral myelin protein, which promote injured neuronal repair and form mature myelin sheaths during the remyelination process^105,109. In injuries caused by ischemia-reperfusion, it is accompanied by high ROS levels leading to retinal ganglion cell damage, causing apoptosis, and may lead to irreversible visual damage¹¹⁰. RGCs, being terminally differentiated, are not able to regenerate themselves, and there are fewer efficient drugs for treating vision loss due to oxidative stress and apoptosis in RGCs^111–113, in order to overcome this Qin and his colleagues used tert-butyl peroxide to develop RGC models of oxidative stress. In their results, they observed that tFNA was able to lower the cellular ROS and shield cells against oxidative stress by regulation of intracellular oxidation-associated enzymes via activation of akt/nrf2 signaling pathway¹¹⁴. In another experiment, Li formulated a novel DNA nanocomposite by conjugating tFNA to microRNA-22-3p (tFNA-miR22). They observed that tFNA can deliver mirr-22 to damaged RGCs successfully and also has an additional neuroprotective effect on glaucoma. Synergistic effects between tFNA and miR-22 were also observed; the conjugate can specifically activate tyrosine kinase receptor B to have a neuroprotective effect on RGCs by increasing the expression level of brain-derived growth factor (BDNF)¹¹⁵. As per the previous literature, tFNA shows high compatibility with aptamers¹⁰⁵. In a study by Tian, they conjugated AS1411 to one of the four strands of tFNA, formulating a modified aptamer-tFNA. Under hypoxic conditions, the aptamer-modified tFNA is efficient in tumor-targeted drug delivery in comparison to unmodified tFNA; along with this, the modified tFNA is able to suppress tumor cell proliferation¹¹⁶. Various targeting molecules like folic acid, cell-penetrating peptides, and affibody molecules are conjugated with tFNA to increase the specificity^117,118.

Preclinical studies and model systems

Zebrafish, a teleost fish, has gained popularity amongst the research community because of its low maintenance, high fecundity, transparent embryos, and captivating visualization of nanomaterial uptake and growth^119,120. The circulatory system of zebrafish is a singular circuit comprising of heart-gills-body-heart, enabling easy uptake of chemicals directly to the circulatory system¹²¹. In comparison to the rodent model, zebrafish allows larger chemical molecule screening at reasonable costs. The genome of zebrafish is 70% similar to that of humans, making it a prominent in vivo model¹²². There are several models for inducing ischemic stroke in zebrafish; one of the most common models is generating ischemia by creating hypoxia in both larvae and adults by submerging them in a low-oxygen tank to create hypoxia or chemically induced hypoxia, commonly by using CoCl₂¹²³.

In another study, they observed the global effects of hypoxia on developing embryos; these effects include pericardial edema, deformation in the spine, and abnormalities in head¹²⁴. An ischemic event is characterized by locomotor impairment and neuronal loss. However, pathology does not mimic a thrombosis or embolism that would be observed clinically. Global hypoxic models are useful for cross-checking the findings from a rodent model to another species; however, they may showcase limitations regarding the specificity for drug discovery. A focal ischemic event model can be developed by using photosensitive dye - Rose Bengal¹²³. Ponatinib is a withdrawn leukemia drug and can cause thrombosis. It was observed that 2dpf zebrafish larvae treated with 1ug/ml ponatinib for 24 h duration showcased cerebral thrombosis, which was confirmed with histology and cerebral angiography. Due to the ongoing development of cerebrovasculature, this time stamp is perfect to treat zebrafish larvae in order to see the effects of ponatinib on cerebrovasculature¹²⁵.

Another model for cerebral ischemia is cell ablation via bacterial nitroreductase catalysis of toxic metronidazole, under the control of a neuroendothelial cell-specific promoter, which can damage brain tissues. Chen et al. created a model of cerebral ischemia linked with vascular regeneration which is driven by lymphatic invasion by the method of cell ablation¹²⁶. The induction of vessel damage causes brain-specific apoptosis and inflammation, indicating that this model can positively replicate ischemic stroke pathology¹²³.

A study done by Kansara et al. used zebrafish larvae to understand the time- and geometry-dependent uptake of DNA nanocages. For this study, the time-dependent uptake was studied from 4 to 96 h after treatment, while the geometry-dependent uptake included DNA nanocages of different shapes like: tetrahedron (TdN), icosahedron (ID), cube, and buckyball (BB), which were studied on 72 h post-fertilized (hpf) larvae for 12 h. In addition to this, the exposed zebrafish larvae were also monitored for developmental changes by assessing at the molecular levels and genetic expressions. Zebrafish larvae and embryos exposed to DNA TdNs showed phenomenal internalization as well as tissue-specific uptake. In order to see any transcriptional alterations being induced after TdNs exposure, the study demonstrated that gene expression of genes for cardiovascular development, dorsoventral axis development, tail formation, and floorplate development was not significantly altered under DNA TdN exposure. Hence, we can conclude that TdNs does not induce any alteration in the developing genes of zebrafish¹²².

The closest model to human ischemic stroke is occlusion of the middle cerebral artery [MCA] and its branches. This occlusion accounts for almost 70% of the infarcts¹²⁷. One of the common methods to induce MCA stroke models in rodents is by intra-arterial suture occlusion of MCA (MCAo). This method has been used more than 40% of the time in approximately 2600 studies on neuroprotection after ischemic stroke, owing to its less invasive nature, and it also does not require craniectomy; hence cranial structure damage can easily be avoided¹²⁸. The time frame to induce MCAo using suture varies from 60 min, 90 min, 120 min or permanent occlusion in rats. These can successfully induce infarction 88%-100% of the time, whereas subarachnoid hemorrhage 12% of the time¹²⁹. The advantage of the MCAo model includes its close mimicry to the human ischemic stroke, including penumbra formation, and has high reproducibility and large infarct volumes, allowing precise control of ischemia and reperfusion. This method is known to cause reliable BBB damage, neuronal death, inflammation, and measurable behavioral deficits. Other stroke models include: intraluminal suture, craniotomy model, photothrombosis model, endothelin-1 model, embolic stroke model¹³⁰.

DNA nanostructures have demonstrated promising potential in translational medicine, specifically in novel applications like targeted drug delivery, biosensing, molecular imaging, and nucleic acid therapeutics¹³¹. Nevertheless, many challenges need to be addressed before these systems progress from lab-based research to preclinical and clinical trials. The first limitation being the stability of DNA nanostructures in a physiological environment, because nuclease-mediated degradation may potentially affect circulation time and therapeutic performance. Additionally, there is a need for extensive analysis on biodistribution, pharmacokinetics, immunogenicity, and long-term toxicity during development of preclinical studies¹³². From a translational standpoint, moving forward in clinical application requires stringent regulatory adherence. some of them includes standardizing the characterization of physicochemical properties, large-scale reproducibility, and adherence to Good Manufacturing Practices (GMP) for production of clinical-grade materials. To properly evaluate the safety and efficacy of DNA-based nanotherapeutics, well-defined regulatory pathways and clinical trials are necessary¹³³. Tackling these scientific and regulatory challenges is crucial to bridge the gap between lab-based discoveries and clinical applications, Table 3.

Table 3.

Preclinical Studies Using DNA Nanodevices in Neurological Disorders

Model System	Disease/Condition	DNA nanodevice Used	Outcome	Reference
Zebrafish	Stroke-like hypoxia model	DNA hydrogel with antioxidant cargo	Reduced neuronal damage, improved survival	64,123
Rodent (MCAO)	Ischemic stroke model	tFNAs	Neurological recovery, improved infarct volume	52
Rat	BBB permeability assay	Aptamer-tagged DNA cage	Effective brain delivery, increased stability	49,171

Challenges and future perspectives

Even though the DNA nanocages showcase a variety of advantages, there is still limited use of them in in vivo explorations because the investigation of targeting and intracellular fate of these cages in in vivo models is limited. The limitations of the DNA nanocages arise due to certain molecular barriers, such as: 1. Inefficient delivery: In cases of passive targeting, there is rapid clearance by the liver and kidneys, which could cause potential toxicity or reduce serum concentration, along with limited cell membrane penetration and lysosomal entrapment^22,88; 2. Stability: Exogenous DNA is vulnerable to host scavenging and DNases, further leading to structural damage of the nanocarrier. Drug delivery of DNA origamis is limited due to denaturation, dissociation in the tumor microenvironment, and nuclease degradation, further limiting its industrialization^22,88; 3. Limited toxicity evaluation: even though the degraded DNA is considered to be biocompatible, its biosafety is yet to be evaluated. Though degraded DNA nanostructures are generally considered biocompatible, long-term biosafety is unknown. Further studies are required to assess the safety and the potential harm, immune response, inflammation, and genotoxicity from origami by-products. TdNs offer a chance to explore these limits^22,88,122; and 4. Production difficulties: It is complex to manufacture DNA nanomaterials due to variations in raw material purity and reaction conditions. It is essential to focus on cost reduction and efficacy improvement for precise, large-scale production due to high cost and low yields, which restrict practical application²².

Chan W.C.W et al, found only 0.7% of NPs are able to reach tumor sites, showcasing the inefficiency of EPR (enhanced permeability and retention) based drug delivery^134,135. The EPR effect is influenced by size, type, and TME of the tumor. NP accumulation is restricted in cases of larger tumors because of their heterogeneous vasculature. Warburg effect fuels growth in tumors and can weaken the EPR effect in advanced cancers, ultimately hindering effective drug delivery and challenging the clinical translation of nanomedicines^22,136. To overcome these limitations, several new strategies are being explored for cytoplasmic delivery and lysosomal escape^137,138. Stability can be enhanced through coating and chemical modifications^139–141. Advancing this field into clinical applications requires scaling up, because the current experimental procedures require microgram quantities. Recent technological advancements have been reported to improve the scalability in the production of DNA nanostructure^142,143. Another approach eliminated the need for staple strands by allowing the construction of single-molecule DNA origami using a long, custom-designed ssDNA scaffold synthesized via PCR or in vivo methods¹⁴⁴. This ss-DNA origami strategy has also shown strong compatibility with RNA-based origami structures¹⁴⁵. Utilizing DNA nanostructures as vaccine display scaffolds owing to their capability to present antigenic proteins or molecules in a defined 3-dimensional structure has been receiving a lot of attention in research areas. The modified vaccine vectors are said to help give immunity to previously challenging epitome which are not able to be exposed to the immune system in a defined immunogenicity configuration¹⁴⁶.

Conclusion

Stroke is considered a leading global health challenge, since it causes severe mortality and is associated with long-term disability, owing to the narrow therapeutic window due to the presence of the BBB. The emergence of DNA nanotechnology in recent years has offered a wide range of therapeutic potential, especially for neurological disorders. These nanodevices are multifunctional, programmable, and biocompatible, and act as a bridge for critical therapeutic gaps in diseases. DNA devices are capable of being programmed, which enables precise control over the size, shape, and cargo loading for drug delivery. There are diverse types of therapies, such as neuroprotective peptides, small molecules, siRNA, miRNA, and CRISPR, for which nanodevices can be employed. Through these mechanisms, it is possible to direct site-specific therapies in a variety of diseases. Imaging of disease biomarkers using DNA-based nanodevices, using fluorescence and AFM, has been made possible, which helps in knowing the real-time disease progression and designing specific treatment regimes. Furthermore, nanodevices can be devised for microenvironment-specific treatment, such as acidosis, oxidative stress, and enzyme-specific release, ensuring controlled release of the drug in the site of injury. This is known to ensure neuroprotection and increase recovery. Moreover, by ensuring the stable delivery of siRNA, miRNA, and CRISPR tools, DNA nanocarriers help in modulating key signaling pathways by suppressing apoptosis and reducing neuroinflammation. DNA nanostructures have been known to target TLR2 and other pro-inflammatory cascades, which help in mitigating neuronal death and promote angiogenesis and neurogenesis. DNA nanodevices have been used for successful BBB penetration, and have proved that there is a lower infarct volume, improved behavioral recovery, and sustained release. However, certain challenges need to be addressed, such as stability in circulation, large-scale reproducibility, and long-term safety. Hybrid DNA nanostructures could be used to enhance their functions. DNA nanodevices are a promising therapeutic solution, especially for neurological disorders; further research to translate this into clinical trials is needed in order to understand the precise regulatory and signaling pathways.

Author contributions

K.K. conceived the original idea and wrote the original manuscript. S.C. and K.J. provided the first draft. A.K. and D.B. conceived the idea and required funding. All the authors discussed and helped in writing and critically reading the manuscript.

Peer review

Peer review information

Communications Biology thanks Tanveer A. Tabish, Julien Rossignol, Sarah Youssef and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Dr Marjan Gharagozloo and Dr Ophelia Bu.

Competing interests

Dhiraj Bhatia is an Editorial Board Member for Communications Biology, but was not involved in the editorial review of, nor the decision to publish this article. All other authors declare no competing interests