Programmable DNA Nanodevices for Applications in Neuroscience

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Programmable DNA Nanodevices for Applications in Neuroscience Pravin Hivare,§ Chinmaya Panda,§ Sharad Gupta,* and Dhiraj Bhatia* Cite This: https://dx.doi.org/10.1021/acschemneuro.0c00723

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ABSTRACT: The broad area of neuroscience has witnessed an increasing exploitation of a variety of synthetic biomaterials with controlled nanosized features. Different bionanomaterials offer very peculiar physicochemical and biochemcial properties contributing to the development of novel imaging devices toward imaging the brain, or as smartly functionalized scaffolds, or diverse tools contributing toward a better understanding of nervous tissue and its functions. DNA nanotechnology-based devices and scaffolds have emerged as ideal materials for cellular and tissue engineering due to their very biocompatible properties, robust adaptation with diverse biological systems, and biosafety in terms of reduced immune response triggering. Here we present technologies with respect to DNA nanodevices that are designed to better interact with nervous systems like neural cells, advanced molecular imaging technologies for imaging brain, biomaterials in neural regeneration, neuroprotection, and targeted delivery of drugs and small molecules across the blood−brain barrier. Along with comments regarding the progress of DNA nanotechnology in neuroscience, we also present a perspective on challenges and opportunities for applying DNA nanotechnology in applications pertaining to neurosciences. KEYWORDS: Neuroscience, nanotechnology, DNA nanotechnology, protein misfolding, blood-brain barrier applications.11 The field of DNA bionanotechnology has emerged multifold and multidirectional since the pioneering work by Seeman.12 Oligonucleotide-based nanoconstructs act as promising drug delivery agents for in vitro or in vivo applications.13,14 Because of their low immunogenicity and almost no cytotoxicity, DNA-based nanoplatforms are often preferred to metal or polymer-based nanoparticles (NPs).15 The availability of natural nucleases, ligases, and other DNA enzymes help the precise manipulation of DNA. Further, the flexibility and versatility of DNA-based assemblies allow easy integration with other functional entities modifying their surface properties and accessibility.15,16 In this review, we present briefly the concepts currently being explored for interfacing nanodevices and nanomaterials for applications in neuroscience, highlighting some of the main areas of neuroscience and neuronal engineering. We then present how DNA nanotechnology has evolved materials to explore neuronal systems in terms of devices with capacities for crossing the blood-brain barrier (BBB), neuronal cells adhesion and differentiation, imaging and manipulating

1. INTRODUCTION Multiple biomaterials and biofunctionalized nanomaterials have been validated for their interactions and modulations of biological systems at the cellular and molecular levels with very high specificities. We explore these exquisite properties of different bionanomaterials; different nanosystems can sense, stimulate, and interact with different cells and tissues to induce very specific physiological responses like uptake, signaling, migration, differentiation, etc. while minimizing any off-target effects.1−4 While a pallete of nanodevices has been applied to different organs and tissues in the body,5−9 neuroscience remains the area very minimally explored for these applications.10 This is mostly because of the complexities involved in interfacing nanomaterials with neuronal cells and the nervous system. Albeit a bit slow, the recent years have witnessed a significant progress emerging in the contributions of different nanotechnologies to neuroscience research and therapeutics. Functional biological assemblies of proteins and nucleic acids along with other molecular entities perform multiple kinds of complicated, synergistic, and cross-linked tasks required for the efficient performance and survival of cells and living organisms. Inspired by such naturally occurring biological complexes, harnessing the molecular recognition and pattern specificity, scientists have applied a bottom-up approach to construct nanoscaled biodevices that can be used as tiny programmable molecular robots for various © XXXX American Chemical Society

Received: November 10, 2020 Accepted: January 3, 2021

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Figure 1. General approaches of nanodevices to neuroscience. Nanotechnology can be used to develop imaging, delivery, and interacting platforms to understand or abate neurological disorders. (a) Nanodevices and oligonucleotide nanoconstructs are used as specific organelle markers or standards for microscopy techniques. (b) Nanomaterial-based scaffolds for neural differentiation by providing suitable environment and support. (c) Bionanoparticles or peptide conjugates with the capacity to cross the BBB for efficient delivery of small molecules into the CNS (Adapted with modifications from Silva, G. A., Nat. Rev. Neurosci., 2006).

neurons and glial cells with functionalized DNA nanodevices, approaches for differentiating neural regeneration using DNApeptide nanodevices, neuroprotection, etc. Lastly, we provide a perspective of unique underlying challenges when interfacing nanodevices with nervous systems and the possible solutions that might enable future nanotechnologies to overcome the aforesaid hindrances.

applications; the recent conjoining of the fields of neuroscience and nanotechnology has given rise to multiple emergent properties for applications in neuroscience research and therapeutics.21,22 Some of the key applications of nanoneuroscience provide novel routes for cutting-edge research in miniaturization and performance enhancement of synthetic devices, such as nanodevices for neural interfaces (Figure 1). However, because of the complexities of the mammalian nervous system and neural cells and the limited number of nanoneuro studies, the direct applications of nanotechnology in neuroscience have remained largely in the early stages of development. Despite these limitations, multidisciplinary approaches to nanoneuroscience have boosted the research of nanoneuroscience in multiple domains of neurodegenerative and neuroimmunological diseases. In this section, we briefly highlight some of the applications of nanotechnology in different domains of neuroscience. 2.1. Misfolded, Aggregated Pathological Protein Clearance. Misfolding of pathological proteins and the compromised proteostasis quality-control mechanism of the aging cells play crucial roles in exacerbating the neurodegenerative disease pathology.23 Oligomerization and the irreversible formation of soluble or insoluble toxic aggregates of key proteins can be found in major diseases such as Alzheimer’s, Huntington’s, Parkinson’s, amyotrophic lateral sclerosis, frontotemporal dementia, and Creutzfeldt-Jakob disease.24 Misfolded protein deposits such as extracellular βamyloid plaques, intraneuronal tau neurofibrillary tangles, α-

2. CURRENT NANOTECHNOLOGIES IN NEUROSCIENCE RESEARCH Neurons are the basic units of the nervous system. The coordinated activities of neurons result in functional circuits in the brain and central nervous system (CNS).17 Like other tissues of the body, the nervous system is as well and equally prone to damage and diseases like brain injury, neurodegenerative diseases, brain cancer, etc.18 Routine strategies such as drugs, surgery, radiotherapy, and chemotherapy are only partially effective and lead to higher death rates or permanent damage to nervous system, resulting in paralysis or a trauma-like situation for the body.19 These low success rates in neuronal diseases are partially due to the lack of effective and optimal therapy but equally because of a lack of understanding of neuroglia, their structures, their dynamics in combination with other neurons, and their synchronous firing.17,20 Nanoneuroscience is an emerging cocktail of joining forces from the fields of nanotechnology and neuroscience. Nanotechnology mostly involving biomaterials and devices has emerged as an ideal solution for multiple biomedical B

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Figure 2. Applications of nanotechnology in misfolded protein disorders. NP or peptide-NP conjugates can be used as β-sheet linkage breakers to disaggregate and reduce the amyloid load; the nanochaperones by modulating electrostatic interactions with the misfolded proteins can assist in protein refolding, resolubilization, and release.

aggregation inhibitors.34,45 Liposome-mediated efficient delivery of such peptide-functionalized systems through the in vitro BBB and in vivo models has also been depicted.46 Proteincapped iron and cadmium NPs have recently been described to inhibit and disaggregate tau PHFs and were rendered noncytotoxic.47 Because of their small size, surface charge, and excellent surface properties, NPs might lengthen the nucleation phase thereby delaying the aggregation process.45 However, depending on the peptide and particle concentration ratio, NPs can even promote fibrilization.48 It is necessary to mention here that morphological characteristics of ex vivo and in vitro amyloid fibrils are quite distinct as reported by cryoEM, 49,50 and hence their aggregated structures could also be strikingly different. Because of the heterogeneity of aggregation, dynamic self-folding, surface properties, and toxicity, it is often difficult to universally validate the nanotechnology-based therapeutic candidates in animal models. The disordered Aβ peptide has been shown to be toxic to cells with the ability to form membrane pores.51,52 Failure of the protein qualitycontrol mechanism along with the compromised endolysosomal clearance process of the aged cell has been detrimental in Aβ clearance. Toxic Aβ fragments have been reported to be associated with abnormal endosomes and can impair the lysosomal pathway leading to neuronal dysfunction.53,54 A recent reveiw presented by Smith et al. on the endosomal escape of the nanopraticles highlights the current understanding of the different mechanisms of endosome escape such as membrane fusion, osmotic pressure, particle swelling, and membrane destabilization.55 A similar field of relevance is redirecting the misfolded proteins for chaperone-assisted refolding or dragging them for autophagolysosomal degradation. Taking inspiration from the concept of a GroEL/GroES chaperone system as a nanocage,56,57 artificial nanochaperones were developed that can bind the misfolded protein at its exposed hydrophobic segment, and by intramolecular bonding modulation refolding

synuclein, huntingtin, and prion protein are often known to instigate the proteotoxicity in such conformational diseases.24,25 Drug discovery trials against Alzheimer’s initially focused on the inhibitors of β-secretase 1 enzyme (BACE1), which is responsible for a proteolytic cleavage of amyloid precursor protein (APP) to form Aβ.26,27 Despite decreased cerebrospinal fluid (CSF) Aβ levels, cognitive worsening was reported with these drugs.28 Similar conserved proteolytic cleavage events have also been recognized in the case of the prion protein (PrPC).29 Such proteolytic processing is thought to be regulating toxic protein production, degradation, and disease progression. However, because of the varied roles of secretase enzymes in brain function, the shift is now on aggregation inhibitors and chaperone-mediated protein refolding. Protein aggregation kinetics follows a three-step sigmoidal curve,30,31 the first being the nucleation phase, where the protein monomers tend to come together, forming small nucleates or oligomers. When a threshold nucleus size is reached, an exponential phase is observed with the formation of larger protofibrils by the accumulation of oligomers and monomers. The final plateau phase is formed with the formation of relatively stable toxic insoluble mature fibrils (Figure 2).30 Several nanoparticle-mediated methods have been developed to label or disaggregate such proteins for therapeutic benefits. As misfolded proteins consist of ordered cross βsheet-rich structures,32,33 specific β-sheet linkage breakers have also been suggested.34 Gold nanoparticles (AuNPs) have been used to label amyloids in a method called immunogold labeling.35,36 Aβ and tau paired helical filaments (PHFs) were recently tagged with this method for morphological heterogeneity characterization with the help of cryogenic electron microscopy (cryo-EM). 37 Multiple groups have described the roles of small peptides (segments of Aβ itself)38−40 or chemically modified (unnatural amino acid incorporated, Nmethylated) small peptides 41−44 as β-sheet breakers and C

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Figure 3. Nanomaterials for remyelination and biomarker detection. (a) NP-based OPC delivery to the affected area can effectively replenish the degenerated myelin by promoting cell migration and differentiation with increased survival rate. (b) NP-based or DNA-NP conjugates can be used for ultrasensitive, reproducible, and cost-effective disease-specific miRNA detection tools.

can be done.58 Surface-charged gold NPs have been shown to facilitate the crucial resolubilization and refolding steps by employing electrostatic interactions with the denatured cationic proteins.58 A strong adsorption interaction is essential between the NPs and misfolded protein for better folding efficiency. Nanomicelles mimicking heat-shock proteins,59,60 nanogels,61 polymeric NPs,62 liposomes (LPs) along with poly(ethylene glycols) (PEGs), and cyclodextrins as additives can be used for the release of the refolded protein. Additionally, Kameta and co-workers described a nanotube hydrogel-based artificial chaperone system by modulating external cues such as pH and salinity, without adding any specific agents.63 Considering the therapeutic role of autophagy inducers in neurodegenerative diseases,64,65 smart nanosystems for redirecting misfolded proteins for autophagy have also been described. The role of branched polyamines in the clearance of prion protein (PrPSC) from the scrapieinfected neuroblastoma cells through a lysosomal-dependent method has been described.66 The authors further used dendrimers (DDs) to accelerate the prion degradation rate of lysosomal proteases. Quarterization of NPs reduced the toxicity retaining the antiprion properties. Moreover, a chitosan-based NP decorated with the Aβ detection unit GKLVFF and beclin-1-dependent autophagy activation unit TGFQGSHWIHFTANFVNT has been reported to activate autophagy thereby clearing the amyloid beta load in transgenic mice.67 Several other similar smart chaperone-based NPs have been designed to enhance toxic protein clearance in lysosomes or by autophagy activation. 68,69 Undoubtedly autophagy activation is known to have many advantages in protein clearance; however, a more specific and targeted-autophagy activation using such nanoconstructs for tau or huntingtin protein clearance is desirable. 2.2. Disease Biomarker Detection, Neural Regeneration, and Differentiation. Early detection of disease biomarkers provides crucial information about the progress of the disease pathogenicity and could be the best way of effective diagnosis and early therapeutics. Apart from the classical protein-based biomarkers, microRNAs (miRNAs), which negatively regulate gene expression at post-translational levels by binding to the target mRNAs, are often regarded as the new generation of biomarker for neurodegenerative and

neuroimmune diseases. 70 Differential miRNA expression in healthy and diseased individuals has been reported in Alzheimer’s,71 Parkinson’s,72 multiple sclerosis, 73 and other neurological disorders. Because of the low sensitivity, poor reproducibility, high cost, and cross-hybridization issues, current detection methods fall short in low-level, ultrasensitive miRNA detection, paving way for NP-based efficient detection systems. Various optical or electrical readouts providing nanosystems have been described in this regard. 74−76 For example, AuNPs functionalized with a graphene biosensor with quenching capacity have been reported to detect miRNAs in a label-free manner.77 Various ultrasensitive miRNA nanodetection methods capable of detecting up to femto (10−15) or atto (10−18) molar concentrations have been reported using AuNP and tetrahedral DNA conjugates.74,78,79 Several nanotechnology-based therapies focusing on neural regeneration and remyelination have been reported in the recent past. Apart from neurodegenerative diseases, neurodegeneration along with irreversible demyelination are common in neuroimmune diseases such as multiple sclerosis (MS).80 Brain-resident immune glial cells play a critical role in MS progression. A potential neuroconstructive disease-modifying strategy is to direct oligodendrocyte precursor cells (OPCs) to the damaged area, where they can differentiate into mature myelinating oligodendrocytes effectively replenishing the damaged myelin.81 Effective transfer of OPCs or undifferentiated neural stem cells into the CNS can be mediated by NP-based delivery methods (Figure 3a,b). Leukemia inhibitory factor (LIF) has growth factor-like properties and exerts pro-remyelination and anti-inflammatory effects. LIF-coated poly(lactide-co-glycolide) (PLGA) NPs have been shown to promote effective OPC differentiation and have been reported to effectively remyelinate MS lesions in vivo.82 Recently, Osorio-Querejeta et al. utilized extracellular vesicles-based bionanoencapsulation of miR-219a-5p for OPC differentiation and remyelination.83 Moreover, the use of nanocrystalline aqueous gold suspension (CNM-AU8) alone has been shown to repair the demyelinated MS lesions in lysolecithin and cuprizone rodent models improving motor functions.84 NP-coated remyelination and neural regeneration following spinal cord injury (SCI) has also been reported.85,86 PEGylated AuNPs have been employed for mouse models of SCI with reduced inflammatory response, D

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enhance motor function, and increased remyelination.86 Funnel et al. have described a comprehensive review regarding the use of magnetic nanocomposites for neural regeneration.87 Since bionanomaterials can promote implanted cell migration increasing their survival rate, particle-containing cells can prove enormous therapeutic strategies for MS.88,89 2.3. Blood-Brain-Barrier Penetration. The BBB is generally formed by the choroid plexus epithelium at the juncture of the blood-cerebrospinal fluid barrier (BCSFB) and cerebral capillary endothelial cells.90,91 Transmembrane proteins along the cerebral endothelial cells create tight junctions, effectively eliminating passage between the brain extracellular fluid (ECF) and blood.92,93 Such a physical metabolic barrier is essential to create a distinct ECF environment for modulating the solute composition.92 It maintains brain homeostasis and extends CNS protection from neurotoxic xenobiotics and metabolites (Figure 4). The

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vances have paved the way for strategies to overcome the BBB for efficient drug delivery without inviting peripheral immune cells. Various NP delivery trends emphasized the size, shape, stiffness, charge, surface modifications, efficient drug loading capacity, programmability, lipophilicity, reduced off-target effects, and biocompatibility as well as biodistribution.97,98 NP-engineered targeted drug-loading systems have demonstrated a wide design variety and surface decorations owing to their extraordinary surface-to-mass ratio and physicochemical properties severely improving particle pharmacokinetics. Another strategy is to attach a brain endothelium receptortargeting ligand or antibody to the outer NP surface for efficient uptake.99,100 However, such strategies have been shown to be trapped in the endothelium or being transported to lysosomes if NP-ligand binding strength is high, delaying the release.101 PEGylated and peptide/ligand/antibody-coated cationic LP-based delivery methods are often preferred because of their specificity and charge providing easy uptake.102 Recently, Song et al. developed a high-density lipoprotein nanoconstruct containing apolipoprotein E (ApoE), which can target and degrade Aβ in an animal model.103 Along with LPs, nanovesicles and brain endothelial cell-derived exosomes have been described to cross the BBB in zebrafish for siRNA delivery. 104 Recently, AuNPs with exosome modifications were shown to cross BBB in vivo.105 DDs are novel branched cascade molecules with the potential of conjugating with PEG or transferrin effectively crossing the BBB. Poly(amido amine) (PAMAM) is the most widely used DD, which has been implicated in stroke-related injury,106 and it can protect against Aβ-induced memory loss in AD.107 It is often difficult to compare different brain-targeting strategies because of different analytical and experimental setups, varied animal or cell lines, and a diverse array of NPs being used.

3. OVERVIEW OF DNA NANOTECHNOLOGY AND ITS BIOLOGICAL APPLICATIONS Since its inception in the early 1980s,12 the concept of nanotechnology-based constructs made entirely of DNA has gained momentum. After the demonstration of a DNA-based cube in the early 1990s,108 three-dimensional (3D) DNA nanostructures have been widely used to deliver cargo inside cells or tissues.15 Self-assembly and DNA origami are the two famous methods to have varieties of 3D structures with drug molecules trapped inside. 15 These carefully engineered, welltailored 3D DNA nanocapsules can be made to release the entrapped drug with certain chemical or environmental stimuli.109−111 Controlled drug release and the biodegradability of the delivery system are often concerns in the nanotechnology field. DNA cages provide excellent control over the reversible modulation of the pore size of the nanodevice, release of the trapped payload, and ratiometric analysis. Ligands, particular concentrations of ions, other specific small molecules like cyclic di GMP (cdGMP), the pH of the buffer, and other nucleotide sequences can also act as a stimulus.109,111,112 Reversible openings of DNA polyhedron and tetrahedron structures depending on the pH of the buffer have been reported.113 An origami-based cuboid box was designed with six DNA sheets with a manageable DNA lid prepared using a toe-hold extension that can open in the presence of external nucleotide sequences.114 Recently, a multilock substrate control vault was also reported.115,116 Selective encapsulation, environment sensing, followed by controlled cargo release are ensured by these carefully

Figure 4. Overview of the BBB. The BBB formed by the cerebral endothelial cells creating tight junctions effectively eliminates the passage between blood and brain ECF thereby creating brain homeostasis. The endothelial cells contain specific receptors for essential solutes and metabolites for neuro maintenance.

neurovascular unit (NVU) cells (viz., neurons, pericytes, endothelium, astroglial cells, and vascular smooth muscle cells) are responsible for maintaining BBB permeability by forming a membrane around blood vessels.93 Compromised BBB and neurovascular pathways often lead to the passage of peripheral immune components into the CNS, instigating a cascade of immune chain reactions. Neuroinflammation is recognized as a common underlying cause of many neurodegenerative and neuroimmune diseases.94 Being the primary gateway to the brain, the BBB often has regulated transport pumps for specific polar solutes, whereas small lipophilic molecules can pass through the membrane by direct penetration. Generally, solutes take two different pathways: paracellular transport (passage between endothelial cells) and transcellular transport (passage through the endothelial cells crossing the cytoplasm).95 The transport of ions, nutrients such as essential amino acids, glucose, and hormones is facilitated by highly specific ion pumps, nutrient transporters, ABC transporters, etc. The transport of a few macromolecules and circulating peptides follows adsorption, receptor-mediated transcytosis, and pinocytosis.93,96 Recent technological adE

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engineered structures. Temperature-sensing biodegradable smooth drug release platforms that can release the payload upon sensing a suitable temperature have been described.111,112 Logic gate-based smart aptamer-DNA nanoconjugates hold enormous potential in specificity and delivery.14,116 For a detailed review of such systems, readers are encouraged to go through ref 15. With the interaction properties of cell-specific aptamer-antigen binding, targeted delivery is possible (Figure 5). Apart from drug delivery

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technology has started to be used and explored, other areas are devices for neuronal differentiation and engineering and tools for crossing the BBB and smart devices for imaging various metabolites in the brain and nervous system in living organisms. 4.1. Designer, Biofunctionalized DNA Nanodevices as Scaffolds for Neuronal Differentiation and Engineering. Ma et al. studied the effect of the tetrahedral DNA nanostructures (TDNs) on neuroectodermal (NE-4C) stem cells that have been used as an in vitro model system of the nervous system and can further can proliferate and differentiate into the neuronal lineage. The treatment of TDNs promoted a self-renewal of the neuroectodermal (NE-4C) stem cells via activating the Wnt/β-catenin pathway, which is crucial for the regulation of the cell cycle and neuronal differentiation by inhibiting the Notch signaling pathway. The addition of TDNs resulted in the proliferation and differentiation of NE-4C stem cells and has great potential in nerve tissue regeneration.128 Zhou et al. showed that the TDNs can be delivered into dental pulp stem cells (DPSCs) derived from the human dental pulp tissue without the assistance of any transfection reagents. Furthermore, TDNs promoted the osteo/odontogenic differentiation and the proliferation of DPSCs by an activation of the classical Notch signaling pathway.129 Ma et al. demonstrated the quick internalization of TDNs in NE-4C stem cells and the effect of TDNs on cell proliferation, migration, and differentiation. The cell migration was induced by TDNs by activating the RHOA/ROCK2 signaling pathway. On the one hand, TDNs can further be used for a dual purpose, where TDNs help in the neural stem cells proliferation, migration, and differentiation; on the other hand, TDNs can act as drug carriers that could help in an increase in neurogenesis such as neurotrophic growth factors.130 The human neural stem/ progenitor cells (hNSCs) are very difficult to culture, as they require the extracellular matrix molecules. The extracellular matrix molecules, such as laminin or collagen type IV, derive from the human and animal origin, which supports attachment and regulates cell survival and proliferation. Li et al. developed a 12 amino acid novel peptide sequence similar to that of tissue-derived full laminin molecules based on the Ile-Lys-ValAla-Val, or IKVAV, sequence: Ac-Cys-Cys-Arg-Arg-Ile-LysVal-Ala-Val-Trp-Leu-Cys. This peptide supported the hNSCs attachment, proliferation, and differentiation into neurons. The poly(ethylene glycol) hydrogels conjugated with this peptide, showed the hNSCs attachment and proliferation on the surface of hydrogels, and also promoted the cell migration inside the hydrogels (Figure 6a).131 Ronit Freeman et al. showed the immobilization of the peptide-DNA (P-DNA) molecules on a surface through complementary DNA tethers, which direct the cells to adhere and spread reversibly over multiple cycles. Further they have used two different signals, which can act as ON and OFF signals for the migration of the neural stem cells.132 The DNA molecule and the synthetic peptide sequence are the covalent constructs that link to each other and are called as peptide−oligonucleotide conjugates (POCs). POC-derived materials incorporate the programmable selfassembly of different oligonucleotides with the bioactivity and chemical diversity of polypeptides. MacCulloch et al. have explained the different strategies to synthesize POCs (Figure 6b) from synthetic monomers such as phosphoramidites and functionalized amino acids. These POCs have applications in the diverse fields of the controlled behavior of the cell, hybrid self-assembling systems, and the enhanced coupling of POCs

Figure 5. Schematic diagram showing the various applications of DNA nanotechnology. Drug-loaded DNA-NPs for controlled delivery inside the cell, use of siRNA in gene therapy, and aptamer for targeted delivery to specific organ and NPs-DNA hydrogels.

systems, DNA nanostructure-based disease-specific microRNA detection systems have also been popularized. Detection of microRNAs in body fluids is the key to identify complex diseases before the appearance of pathological symptoms, as already described available methods often fall short to faithfully detect miRNAs. Engineered DNA nanoswitches have been reported to detect miRNA in an RNA extract.117,118 The nanoswitches can switch from off (linear) to on (looped) states when bound to the miRNA, thereby facilitating gel electrophoresis-based easy detection. With DNA systems with pH sensitivity and the capability to detect ions, several Clensors for chloride ion transport quantification,119 pH detectors,120 and antibody/protein detectors121,122 have been described. Further, DNA nanotechnology has also found its application in bioimaging. Multifunctionalized quantum dots or AuNPs encapsulated in a DNA icosahedron can mark specific endocytic pathways.123 DNA tiles formed by the origami method have been standardized for super-resolution imaging techniques like SIM, STED, or PALM.124−127 DNA origami efficiently calibrates the brightness, quantum efficiency, yield, and distance with multiple fluorophores. DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT) has been described for 3D ultrahigh resolution imaging for fixed samples.126

4. APPLICATIONS OF DNA NANOTECHNOLOGY IN NEUROSCIENCE AND NEUROENGINEERING The applications of many DNA nanodevices in biological systems have only recently been started to be realized. Some of the areas of application are related to neuroscience and neuroengineering or neuromodulation, where DNA nanoF

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Figure 6. (a) DNA hydrogel scaffolds for neuronal differentiation. Reprinted with permission from ref 131. Copyright 2014, John Wiley and Sons. (b) Different chemical approaches for the POCs. Reprinted with permission from ref 133. Copyright 2003, Royal Society of Chemistry. (c) The effect of DNA nanodevices on stem cell modulation, proliferation, and differentiation.

into larger molecules.133 Scheideler et al. developed a highthroughput lithography technique that directs the simultaneous assembly of various solid-phase ligands across length scales within minutes. Such DNA-directed techniques enable the mechanistic insight into how tissues encode regulatory information through the spatial presentation of heterogeneous signals on cell surfaces. 134 4.2. DNA-Based Devices with Enhanced Blood-Brain Barrier Capacities. Physical or chemical perturbation of the BBB leading to the opening of tight junctions through increased permeability by paracellular diffusion can also lead to toxicity and neuronal dysfunction. Physical methods such as focused ultrasound,135,136 hyperosmotic agent-based disruption,137,138 and photochemical and photodynamic internalization methods139,140 can be employed for weakening the BBB thereby facilitating the transport and delivery of entities into brain. These available targeted delivery methods, however, often involve rupturing the barrier thereby increasing the chances of infection.90 Despite their theranostics success at laboratory-scale experiments, NP-triggered neurotoxicity has become a concern over the years. Metallic NPs can be neurotoxic and have been reported to impair the autophagy flux and induce DNA damage.141 Most nanodelivery methods suffer from neuroglial toxicity, poor elimination, undesired interactions, intrabrain accumulation, cell cycle arrest, reactive oxidative stress, and necrosis over certain concentrations.142 Moreover, drug modification strategies such as increasing lipophilicity come at a cost of reduced specificity, and such

uptakes can also have off-target issues.90,143 A high degree of programmability and tailoring often renders the delivery method unsuitable for real-world delivery applications. Intranasal administration has also been demonstrated to bypass the BBB;144 however, its safety, efficacy, and permeability are still under consideration. Because of their size and charge, many functionalized DNA conjugates often find it difficult to cross the BBB and therefore fail to reach their desired target decreasing the drug loading and ultimate efficacy. Recently, a BBB targeting a spherical nucleic acid (SNA)-based fluorophore was reported.145,146 SNAs can bind to the scavenger receptors (SR) of a cell membrane enabling efficient transcytosis compared to free DNAs.145 Polystyrene-b-DNA (PS-b-DNA) micelles were created using the polystyrene-b hydrophobic polymer matrix hybridized with DNA, which has the capability to cross the in vitro or in vivo BBB.146 The authors used the micelles loaded with the NIR-II near-infrared dye for enhanced bioimaging applications. Organic SNAs are often preferred over their inorganic counterparts for delivery applications of NIR-II dye because of their accommodating core.146 Further, a 3D DNA nanocage-based delivery system with or without a brain-targeting peptide was reported with the ability to cross the in vitro and in vivo BBB models (Figure 7).147 The nanocages were found noncytotoxic and were more efficient than nanotubes. These studies, even though they appear promising, are still in the preliminary stages, and the integrity of the barrier modulated by the DNA devices still remains to be explored. G

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Figure 7. 3D DNA nanocages or peptide-conjugated nanocages with the potential to cross the BBB. DNA nanomaterials, shuttle peptide nanoconjugates, and specific receptor (LDLR and transferrin receptor) targeting peptide-conjugated bioanoparticles can penetrate the BBB to deliver drugs to the CNS. DNA−Protein conjugates can also be used for delivery applications.13

Targeting BBB receptors via functionalized NPs is often preferred for selective targeting. Combining DNA nanoconstructs with specific ligands against the receptors is the most favorable method for crossing the BBB. Low-density lipoprotein receptors (LDLRs)148,149 and transferrin receptors (TfRs)100,150,151 are extensively used for receptor-mediated transcytosis, whereas other receptors like leptin152 and insulin receptors153 have also been considered because of their considerable level of expression, LDLR-targeting protein ligands, antibody, shuttle peptides such as ApoE,149,154 Angiopep-2,154−156 or Peptide-22.157 Angiopep-2 is a shuttle peptide and has been shown to transport drugs such as doxorubicin158 and paclitaxel.155 Angiopep-2 in conjugation with paclitaxel (ANG1005) can enter the brain by targeting LRP1 and is currently set to enter a phase III clinical trial for breast cancer and glioma.159 Similarly, TfR1 is also a promising target. DNA-coated AuNPs have been described to cross the BBB with a focused ultrasound opening.160 However, the passage of DNA nanoconjugates through the BBB without applying a physical trigger is extremely limited. Further, various small peptides have been reported to cross the BBB acting as informational molecules. BBB shuttle peptides are shown to enter the brain parenchyma via transcytosis or endocytosis without affecting the integrity of the BBB.161 Regional permeability across the BBB and the specific uptake of peptides are still under intense investigation. Over the last years, various synthetic peptide molecules have also emerged, overcoming the pitfalls of the natural peptides.161 For a more detailed review on BBB penetrating proteins and DNA−

protein nanoconjugates, readers are encouraged to go through comprehensive reviews on this topic.13,162 4.3. DNA Nanotechnology for Biosensing in Brain. Neuroinflammation triggered by brain-resident microglia and astrocytes significantly affect the course of many neurodegenerative diseases.94 Molecular pattern recognition receptors (PRRs) on glial cells, when they come across dangerassociated molecular patterns (DAMPs) or specific misfolded proteins,163−165 can activate the cellular degradation machinery to trap and degrade the pathogens. PRRs, in particular, toll-like receptors (TLRs), can activate the inducible nitric oxide synthase (iNOS), which fills the membrane-bound phagosome with nitric oxide (NO), which is toxic to the pathogen.166 Though there are various small molecular systems dedicated for NO detection in live cells, many fail to provide exact spatial and quantitative information about small ionic or molecular signatures in cellular physiology and diseases. A novel DNAbased detection system was recently described in zebrafish brain (Figure 8).167 The NOckout probe consists of a NOsensing dye (diaminorhodamine (DAR)) and a normalizing fluorophore (A647) attached via PEG linkers to complementary ends of a 24-base pair DNA duplex strand (Figure 8a). As nonself DNA, the carefully designed DNA system is recognized by the TLR receptors and induces nitric oxide synthase (NOS2) activity in microglia. This system can localize itself in the phagosomes of microglial cells after being phagocytose reporting NOS2 activity (Figure 8b). The sensing dye diaminorhodamine (DAR) in the presence of NO can form a triazole compound (DAR-T), which is highly fluorescent and is pH-insensitive. The relative fluorescence of H

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Figure 8. DNA nanotechnology for applications in biosensing. (a) DNA-based NOckout probe designed by Veetil et al.167 formed through the assembly of 24-mer DNA strands, NO-sensing fluorophore, and normalizing fluorophore can be recognized by the TLR receptors on microglia and can localize in phagosomes. (b) Time-lapse images of NOckout probes uptake by the zebrafish microglia, colocalization with apoptotic body marker before phagocytosis, and subsequent localization in the phagosomes. (a, b) Reproduced with permission from ref 167. Copyright 2020, PNAS. (c) Schematic of how DNA nanodevices act as a biosensor, where the structural arrangement or conformation of the nanodevices changes in the presence of an external trigger, the output of which can be either colorimetric, fluorescence signal, biochemical change, or biological response.

DAR-T to the normalizing fluorophore is regarded as an output signal of the sensor. Veetil et al. developed a DNA icosahedron containing the neurosteroid dehydroepiandrosterone (DHEA), which can release the payload upon photoirradiation.168 The icosahedron was coated with lipid motifs for easy anchoring and attachment to the cell membrane. The neurosteroid DHEA cargo was conjugated with dextran through a photocleavable covalent linkage. Upon irradiation at a specific wavelength, the linkage breaks, releasing the cargo. The small-molecule release can also be quantified in real-time by evaluating the fluorescence of Alexa 647 attached to the DNA icosahedron. The authors further went on to demonstrate a light-triggered controlled cargo release in Caenorhabditis elegans coelomocytes. This nanodevice could be extremely helpful in a spatiotemporal understanding of neurogenesis and the kinetics of neuronal activation. Such nanodevices have pioneered the field of DNA nanotechnologymediated live-cell imaging and can be a great imaging tool to study cell activation and growth at the molecular level.

systems and neuroscience. Some of the examples would include the use of 3D DNA cages to trap and release drugs, small molecules, neurotransmitters, and neuronal developmental factors in nervous systems. Similarly, using DNA nanodevices the receptors on neuronal cells like bone-morphometric-protein, cadherins, and laminins could be programmed in a controlled manner to offer specific neuronal differentiations. The biggest advantages that DNA nanodevices offer is their spatial and functional specificities for incorporating these ligands to have highly targeted cellular effects. One of the key advantages that DNA offers is its robustness to be coupled to almost any chemical entity, and these hybrid devices are definitely poised to overcome three main challenges in neuronal targeting and activitygreater specificity, modulation of neuronal physiology, and minimal side effects. Being modular in nature, DNA nanodevices can also be made for multitasking, that is, carry out a diverse set of specific cellular functions in nervous systems. The biggest success of DNA nanodevices in neuroscience in coming times will be our detailed understanding of neuronal systems in terms of quantitative chemical signaling and spatial organization in the molecular details. To achieve this, the design and engineering aspects of DNA nanotechnology will have to join hands with experts from neurosciences and clinicians. While these challenges are addressed, still the daunting challenge that lies before DNA nanodevices is crossing the blood-brain barrier. The cells and tissues in the CNS are extremely well-protected from physical and mechanical injury as well as being immunologically safe within the BBB and blood-retina barrier. One of the biggest challenges for DNA nanodevices is to be

5. CHALLENGES AND PERSPECTIVE Despite tremendous progress in integrating multiple domains of scientific areas for applying nanotechnology to neuroscience, a large number of challenges remain to be addressed. However, the impact that might arise from addressing such challenges in terms of our understanding of nervous systems, what changes in it during diseases, and how we can modulate the nervous systems at the molecular scaleswill be phenomenal. DNA-based nanotechnologies are now well-poised to be tailored for direct applications in challenging areas of nervous I

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(3) Ali, M. R. K., Wu, Y., Ghosh, D., Do, B. H., Chen, K., Dawson, M. R., Fang, N., Sulchek, T. A., and El-Sayed, M. A. (2017) Nuclear Membrane-Targeted Gold Nanoparticles Inhibit Cancer Cell Migration and Invasion. ACS Nano 11 (4), 3716−3726. (4) Rauch, J., Kolch, W., Laurent, S., and Mahmoudi, M. (2013) Big Signals from Small Particles: Regulation of Cell Signaling Pathways by Nanoparticles. Chem. Rev. 113 (5), 3391−3406. (5) Flores, A. M., Hosseini-Nassab, N., Jarr, K.-U., Ye, J., Zhu, X., Wirka, R., Koh, A. L., Tsantilas, P., Wang, Y., Nanda, V., Kojima, Y., Zeng, Y., Lotfi, M., Sinclair, R., Weissman, I. L., Ingelsson, E., Smith, B. R., and Leeper, N. J. (2020) Pro-efferocytic nanoparticles are specifically taken up by lesional macrophages and prevent atherosclerosis. Nat. Nanotechnol. 15 (2), 154−161. (6) Fries, C. N., Curvino, E. J., Chen, J.-L., Permar, S. R., Fouda, G. G., and Collier, J. H. (2020) Advances in nanomaterial vaccine strategies to address infectious diseases impacting global health. Nat. Nanotechnol., 1−14. (7) Weiss, C., Carriere, M., Fusco, L., Capua, I., Regla-Nava, J. A., Pasquali, M., Scott, J. A., Vitale, F., Unal, M. A., Mattevi, C., Bedognetti, D., Merkoçi, A., Tasciotti, E., Yilmazer, A., Gogotsi, Y., Stellacci, F., and Delogu, L. G. (2020) Toward NanotechnologyEnabled Approaches against the COVID-19 Pandemic. ACS Nano 14 (6), 6383−6406. (8) Kamaly, N., He, J. C., Ausiello, D. A., and Farokhzad, O. C. (2016) Nanomedicines for renal disease: current status and future applications. Nat. Rev. Nephrol. 12 (12), 738−753. (9) Esteban-Fernández de Á vila, B., Angsantikul, P., RamírezHerrera, D. E., Soto, F., Teymourian, H., Dehaini, D., Chen, Y., Zhang, L., and Wang, J. (2018) Hybrid biomembrane−functionalized nanorobots for concurrent removal of pathogenic bacteria and toxins. Sci. Robot. 3 (18), No. eaat0485. (10) Silva, G. A. (2006) Neuroscience nanotechnology: progress, opportunities and challenges. Nat. Rev. Neurosci. 7 (1), 65−74. (11) Niemeyer, C. M. (2002) The developments of semisynthetic DNA-protein conjugates. Trends Biotechnol. 20 (9), 395−401. (12) Seeman, N. C. (1982) Nucleic acid junctions and lattices. J. Theor. Biol. 99 (2), 237−247. (13) Stephanopoulos, N. (2020) Hybrid Nanostructures from the Self-Assembly of Proteins and DNA. Chem. 6 (2), 364−405. (14) Douglas, S. M., Bachelet, I., and Church, G. M. (2012) A LogicGated Nanorobot for Targeted Transport of Molecular Payloads. Science (Washington, DC, U. S.) 335 (6070), 831−834. (15) Bhatia, D. D., Wunder, C., and Johannes, L. Self-assembled, programmable DNA nanodevices for biological and biomedical applications. ChemBioChem. 2020. DOI: 10.1002/cbic.202000372. “in press”. (16) Chen, Y.-J., Groves, B., Muscat, R. A., and Seelig, G. (2015) DNA nanotechnology from the test tube to the cell. Nat. Nanotechnol. 10 (9), 748−760. (17) Südhof, T. C. (2018) Towards an Understanding of Synapse Formation. Neuron 100 (2), 276−293. (18) Silberberg, D., Anand, N. P., Michels, K., and Kalaria, R. N. (2015) Brain and other nervous system disorders across the lifespan  global challenges and opportunities. Nature 527 (7578), S151− S154. (19) Aldape, K., Brindle, K. M., Chesler, L., Chopra, R., Gajjar, A., Gilbert, M. R., Gottardo, N., Gutmann, D. H., Hargrave, D., Holland, E. C., Jones, D. T. W., Joyce, J. A., Kearns, P., Kieran, M. W., Mellinghoff, I. K., Merchant, M., Pfister, S. M., Pollard, S. M., Ramaswamy, V., Rich, J. N., Robinson, G. W., Rowitch, D. H., Sampson, J. H., Taylor, M. D., Workman, P., and Gilbertson, R. J. (2019) Challenges to curing primary brain tumours. Nat. Rev. Clin. Oncol. 16 (8), 509−520. (20) Vainchtein, I. D., and Molofsky, A. V. (2020) Astrocytes and Microglia: In Sickness and in Health. Trends Neurosci. 43 (3), 144− 154. (21) Acarón Ledesma, H., Li, X., Carvalho-De-Souza, J. L., Wei, W., Bezanilla, F., and Tian, B. (2019) An atlas of nano-enabled neural interfaces. Nat. Nanotechnol. 14 (7), 645−657.

efficiently delivered to the CNS without a disruption of protecting cells and sheets. Systemic and local side effects with delivery and primary functioning of DNA nanodevices still pose a major challenge for applying DNA nanodevices in vivo. However, despite the challenges, the prospective applications of DNA nanodevices in neuroscience offer tremendous opportunities to understand and explore neuronal physiology and to develop effective therapies in the same for clinical applications.

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AUTHOR INFORMATION

Corresponding Authors

Sharad Gupta − Biological Engineering discipline and Center for Biomedical Engineering, Indian Institute of Technology Gandhinagar, Palaj 382355, Gandhinagar, India; Email: [email protected] Dhiraj Bhatia − Biological Engineering discipline and Center for Biomedical Engineering, Indian Institute of Technology Gandhinagar, Palaj 382355, Gandhinagar, India; orcid.org/0000-0002-1478-6417; Email: dhiraj.bhatia@ iitgn.ac.in

Authors

Pravin Hivare − Biological Engineering discipline, Indian Institute of Technology Gandhinagar, Palaj 382355, Gandhinagar, India; orcid.org/0000-0001-7746-1081 Chinmaya Panda − Biological Engineering discipline, Indian Institute of Technology Gandhinagar, Palaj 382355, Gandhinagar, India; orcid.org/0000-0002-9575-6913 Complete contact information is available at: https://pubs.acs.org/10.1021/acschemneuro.0c00723 Author Contributions

P.H., C.P., S.G., and D.B. wrote the manuscript. P.H. created all the figures. All authors discussed the paper. Author Contributions

§

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We sincerely thank all the members of the D.B. and S.G. groups for critically reading the manuscript and their valuable feedback. C.P. and P.H. thank IITGN-MHRD, Government of India, for fellowship. D.B. thanks SERB, Government of India, for Ramanujan Fellowship and BRNS-BARC for research funding. P.H. acknowledges D. Barbhaya and H. Sham for assistance with figure creation. S.G. thanks SERB for Core research grant. The work in host laboratories is funded by MHRD and DST-SERB and GSBTM, Government of India. The figures were created with the help of CorelDRAW 2019, BioRender.com, and Blender 2.90 software.

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