Light-Activated Nanodiamond-Based Drug Delivery Systems for Spatiotemporal Release of Antisense Oligonucleotides.

Nanodiamonds (NDs) are considered promising delivery platforms, but inaccurate and uncontrolled release of drugs at target sites is the biggest challenge of NDs in precision medicine. This study presents the development of phototriggerable ND-based drug delivery systems, utilizing ortho-nitrobenzyl (o-NB) molecules as photocleavable linkers between drugs and nanocarriers. UV irradiation specifically cleaved o-NB molecules and then was followed by releasing antisense oligonucleotides from ND-based carriers in both buffer and cellular environments. This ND system carried cell nonpermeable therapeutic agents for bypassing lysosomal trapping and degradation. The presence of fluorescent nitrogen-vacancy centers also allowed NDs to serve as biological probes for tracing in cells. We successfully demonstrated phototriggered release of antisense oligonucleotides from ND-based nanocarriers, reactivating their antisense functions. This highlights the potential of NDs, photocleavable linkers, and light stimuli to create advanced drug delivery systems for controlled drug release in disease therapy, opening possibilities for targeted and personalized treatments.

Interaction of Sp1 and APP promoter elucidates a mechanism for Pb2+ caused neurodegeneration.

A ubiquitously expressed transcription factor, specificity protein 1 (Sp1), interacts with the amyloid precursor protein (APP) promoter and likely mediates APP expression. Promoter-interaction strengths variably regulate the level of APP expression. Here, we examined the interactions of finger 3 of Sp1 (Sp1-f3) with a DNA fragment containing the APP promoter in different ionic solutions using atomic force microscope (AFM) spectroscopy. Sp1-f3 molecules immobilized on an Si substrate were bound to the APP promoter, which was linked to the AFM tips via covalent bonds. The interactions were strongly influenced by Pb2+, considering that substituting Zn2+ with Pb2+ increased the binding affinity of Sp1 for the APP promoter. The results revealed that the enhanced interaction force facilitated APP expression and that APP overexpression could confer a high-risk for disease incidence. An increased interaction force between Sp1-f3 and the APP promoter in Pb2+ solutions was consistent with a lower binding free energy, as determined by computer-assisted analysis. The impact of Pb2+ on cell morphology and related mechanical properties were also detected by AFM. The overexpression of APP caused by the enhanced interaction force triggered actin reorganization and further resulted in an increased Young's modulus and viscosity. The correlation with single-force measurements revealed that altered cellular activities could result from alternation of Sp1-APP promoter interaction. Our AFM findings offer a new approach in understanding Pb2+ associated neurodegeneration.

Stepwise Ligand-induced Self-assembly for Facile Fabrication of Nanodiamond-Gold Nanoparticle Dimers via Noncovalent Biotin-Streptavidin Interactions.

Nanodiamond-gold nanoparticle (ND-AuNP) dimers constitute a potent tool for controlled thermal heating of biological systems on the nanoscale, by combining a local light-induced heat source with a sensitive local thermometer. Unfortunately, previous solution-based strategies to build ND-AuNP conjugates resulted in large nanoclusters or a broad population of multimers with limited separation efficiency. Here, we describe a new strategy to synthesize discrete ND-AuNP dimers via the synthesis of biotin-labeled DNA-AuNPs through thiol chemistry and its immobilization onto the magnetic bead (MB) surface, followed by reacting with streptavidin-labeled NDs. The dimers can be easily released from MB via a strand displacement reaction and separated magnetically. Our method is facile, convenient, and scalable, ensuring high-throughput formation of very stable dimer structures. This ligand-induced self-assembly approach enables the preparation of a wide variety of dimers of designated sizes and compositions, thus opening up the possibility that they can be deployed in many biological actuation and sensing applications.

Photoresponsive Self-Assembled DNA Nanomaterials: Design, Working Principles, and Applications.

Photoresponsive DNA nanomaterials represent a new class of remarkable functional materials. By adjusting the irradiation wavelength, light intensity, and exposure time, various photocontrolled DNA-based systems can be reversibly or irreversibly regulated in respect of their size, shape, conformation, movement, and dissociation/association. This Review introduces the most updated progress in the development of photoresponsive DNA-based system and emphasizes their advantages over other stimuli-responsive systems. Their design and mechanisms to trigger the photoresponses are shown and discussed. The potential application of these photon-responsive DNA nanomaterials in biology, biomedicine, materials science, nanophotonic and nanoelectronic are also covered and described. The challenges faced and further directions of the development of photocontrolled DNA-based systems are also highlighted.

Stimuli-Responsive Self-Assembled DNA Nanomaterials for Biomedical Applications.

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

Mitochondrial Delivery of Therapeutic Agents by Amphiphilic DNA Nanocarriers.

The first example of mitochondrial delivery of the anticancer drug doxorubicin (Dox) is presented by lipid-functionalized DNA nanocages (LNCs). Dox localized in mitochondria induces significant cytotoxicity and cellular apoptosis in MCF-7 compared with Dox localized in lysosomes. These results suggest that LNC has the potential to be an outstanding tool in the treatment of specific organelle-related diseases such as cancers.

A Reversible DNA Logic Gate Platform Operated by One- and Two-Photon Excitations.

We demonstrate the use of two different wavelength ranges of excitation light as inputs to remotely trigger the responses of the self-assembled DNA devices (D-OR). As an important feature of this device, the dependence of the readout fluorescent signals on the two external inputs, UV excitation for 1 min and/or near infrared irradiation (NIR) at 800 nm fs laser pulses, can mimic function of signal communication in OR logic gates. Their operations could be reset easily to its initial state. Furthermore, these DNA devices exhibit efficient cellular uptake, low cytotoxicity, and high bio-stability in different cell lines. They are considered as the first example of a photo-responsive DNA logic gate system, as well as a biocompatible, multi-wavelength excited system in response to UV and NIR. This is an important step to explore the concept of photo-responsive DNA-based systems as versatile tools in DNA computing, display devices, optical communication, and biology.

Conformational Change of Self-Assembled DNA Nanotubes Induced by Two-Photon Excitation.

Two-photon-regulated, shape-changing DNA nanostructures are demonstrated by integrating a DNA nanotube with a two-photon photocleavable module that enables the opening of the cavities of tube, and becomes partially single-stranded in response to two-photon excitation under 800 nm fs laser pulses.

Nanoneedle-assisted delivery of site-selective peptide-functionalized DNA nanocages for targeting mitochondria and nuclei.

Peptide-functionalized DNA nano-objects selectively target mitochondria and the nucleus by means of nanoneedle-assisted delivery. This technology preserves the cell viability and structural integrity of nanostructures and assists the nano-objects in escaping degradation by endocytosis. This method opens up a new avenue for further in vitro studies of intracellular behaviors of DNA assemblies and their interactions in specific organelles.

TNA-Mediated Antisense Strategy to Knockdown Akt Genes for Triple-Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) remains a significant challenge in terms of treatment, with limited efficacy of chemotherapy due to side effects and acquired drug resistance. In this study, a threose nucleic acid (TNA)-mediated antisense approach is employed to target therapeutic Akt genes for TNBC therapy. Specifically, two new TNA strands (anti-Akt2 and anti-Akt3) are designed and synthesized that specifically target Akt2 and Akt3 mRNAs. These TNAs exhibit exceptional enzymatic resistance, high specificity, enhance binding affinity with their target RNA molecules, and improve cellular uptake efficiency compared to natural nucleic acids. In both 2D and 3D TNBC cell models, the TNAs effectively inhibit the expression of their target mRNA and protein, surpassing the effects of scrambled TNAs. Moreover, when administered to TNBC-bearing animals in combination with lipid nanoparticles, the targeted anti-Akt TNAs lead to reduced tumor sizes and decreased target protein expression compared to control groups. Silencing the corresponding Akt genes also promotes apoptotic responses in TNBC and suppresses tumor cell proliferation in vivo. This study introduces a novel approach to TNBC therapy utilizing TNA polymers as antisense materials. Compared to conventional miRNA- and siRNA-based treatments, the TNA system holds promise as a cost-effective and scalable platform for TNBC treatment, owing to its remarkable enzymatic resistance, inexpensive synthetic reagents, and simple production procedures. It is anticipated that this TNA-based polymeric system, which targets anti-apoptotic proteins involved in breast tumor development and progression, can represent a significant advancement in the clinical development of effective antisense materials for TNBC, a cancer type that lacks effective targeted therapy.

Ultra-stable threose nucleic acid-based biosensors for rapid and sensitive nucleic acid detection and in vivo imaging.

The human genome's nucleotide sequence variation, such as single nucleotide mutations, can cause numerous genetic diseases. However, detecting nucleic acids accurately and rapidly in complex biological samples remains a major challenge. While natural deoxyribonucleic acid (DNA) has been used as biorecognition probes, it has limitations like poor specificity, reproducibility, nuclease-induced enzymatic degradation, and reduced bioactivity on solid surfaces. To address these issues, we introduce a stable and reliable biosensor called graphene oxide (GO)- threose nucleic acid (TNA). It comprises chemically modified TNA capture probes on GO for detecting and imaging target nucleic acids in vitro and in vivo, distinguishing single nucleobase mismatches, and monitoring dynamic changes in target microRNA (miRNA). By loading TNA capture probes onto the GO substrate, the GO-TNA sensing platform for nucleic acid detection demonstrates a significant 88-fold improvement in the detection limit compared to TNA probes alone. This platform offers a straightforward preparation method without the need for costly and labor-intensive isolation procedures or complex chemical reactions, enabling real-time analysis. The stable TNA-based GO sensing nanoplatform holds promise for disease diagnosis, enabling rapid and accurate detection and imaging of various disease-related nucleic acid molecules at the in vivo level. STATEMENT OF SIGNIFICANCE: The study's significance lies in the development of the GO-TNA biosensor, which addresses limitations in nucleic acid detection. By utilizing chemically modified nucleic acid analogues, the biosensor offers improved reliability and specificity, distinguishing single nucleobase mismatches and avoiding false signals. Additionally, its ability to detect and image target nucleic acids in vivo facilitates studying disease mechanisms. The simplified preparation process enhances practicality and accessibility, enabling real-time analysis. The biosensor's potential applications extend beyond healthcare, contributing to environmental analysis and food safety. Overall, this study's findings have substantial implications for disease diagnosis, biomedical research, and diverse applications, advancing nucleic acid detection and its impact on various fields.

Nanometre-Scale Biosensors Revolutionizing Applications in Biomedical and Environmental Research.

Driven by the convergence of nanotechnology, biotechnology, and materials science, the field of biosensors has witnessed remarkable advancements in recent years [...].

Versatile nanodiamond-based tools for therapeutics and bioimaging.

Nanodiamonds (NDs) are a remarkable class of carbon-based nanoparticles in nanomedicine which have recently become a hot topic of research due to their unique features including functionalization versatility, tunable opto-magnetic properties, chemical stability, minimal cytotoxicity, high affinity to biomolecules and biocompatibility. These attractive features make NDs versatile tools for a wide range of biologically relevant applications. In this feature article, we discuss the opto-magnetic properties of negatively charged nitrogen vacancy (NV-) centres in NDs as fluorescence probes. We further discuss the frequently used chemical methods for surface chemistry modification of NDs which are relevant for biomedical applications. The in vitro and in vivo biocompatibility of modified NDs is also highlighted. Subsequently, we give an overview of recent state-of-the-art biomedical applications of NDs as versatile tools for bioimaging and detection, and as targeting nanocarriers for chemotherapy, photodynamic therapy, gene therapy, antimicrobial and antiviral therapy, and bone tissue engineering. Finally, we pinpoint the main challenges for NDs in biomedical applications which lie ahead and discuss perspectives on future directions in advancing the field for practical applications and clinical translations.

C-176 loaded Ce DNase nanoparticles synergistically inhibit the cGAS-STING pathway for ischemic stroke treatment.

The neuroinflammatory responses following ischemic stroke cause irreversible nerve cell death. Cell free-double strand DNA (dsDNA) segments from ischemic tissue debris are engulfed by microglia and sensed by their cyclic GMP-AMP synthase (cGAS), which triggers robust activation of the innate immune stimulator of interferon genes (STING) pathway and initiate the chronic inflammatory cascade. The decomposition of immunogenic dsDNA and inhibition of the innate immune STING are synergistic immunologic targets for ameliorating neuroinflammation. To combine the anti-inflammatory strategies of STING inhibition and dsDNA elimination, we constructed a DNase-mimetic artificial enzyme loaded with C-176. Nanoparticles are self-assembled by amphiphilic copolymers (P[CL35-b-(OEGMA20.7-co-NTAMA14.3)]), C-176, and Ce4+ which is coordinated with nitrilotriacetic acid (NTA) group to form corresponding catalytic structures. Our work developed a new nano-drug that balances the cGAS-STING axis to enhance the therapeutic impact of stroke by combining the DNase-memetic Ce4+ enzyme and STING inhibitor synergistically. In conclusion, it is a novel approach to modulating central nervus system (CNS) inflammatory signaling pathways and improving stroke prognosis.

Wavelength-Dependent, Orthogonal Photoregulation of DNA Liberation for Logic Operations.

In this study, we synthesized two phosphoramidites based on 2,7-bis-{4-nitro-8-[3-(2-propyl)-styryl]}-9,9-bis-[1-(3,6-dioxaheptyl)]-fluorene (BNSF) and 4,4'-bis-{8-[4-nitro-3-(2-propyl)-styryl]}-3,3'-di-methoxybiphenyl (BNSMB) structures as visible light-cleavable linkers for oligonucleotide conjugation. In addition to the commercial ultraviolet (UV) photocleavable (PC) linker, the BNSMB linker was further applied as a building component to construct photoregulated DNA devices as duplex structures, which are functionalized with fluorophores and quenchers. Selective cleavage of PC and BNSMB is achieved in response to ultraviolet (UV) and visible light irradiations as two inputs, respectively. This leads to controllable dissociation of pieces of DNA fragments, which is followed by changes of fluorescence emission as signal outputs of the system. By tuning the number and position of the photocleavable molecules, fluorophores, and quenchers, various DNA devices were developed, which mimic the functions of Boolean logic gates and achieve logic operations in AND, OR, NOR, and NAND gates in response to two different wavelengths of light inputs. By sequence design, the photolysis products can be precisely programmed in DNA devices and triggered to release in a selective and/or sequential manner. Thus, this photoregulated DNA device shows potential as a wavelength-dependent drug delivery system for selective control over the release of multiple individual therapeutic oligonucleotide-based drugs. We believe that our work not only enriches the library of photocleavable phosphoramidites available for bioconjugation but also paves the way for developing spatiotemporal-controlled, orthogonal-regulated DNA-based logic devices for a range of applications in materials science, polymers, chemistry, and biology.

Reinforced self-assembly of donor-acceptor π-conjugated molecules to DNA templates by dipole-dipole interactions together with complementary hydrogen bonding interactions for biomimetics.

One of the most important criteria for the successful DNA-templated polymerization to generate fully synthetic biomimetic polymers is to design the complementary structural monomers, which assemble to the templates strongly and precisely before carrying polymerization. In this study, water-soluble, laterally thymine-substituted donor-acceptor π-conjugated molecules were designed and synthesized to self-assemble with complementary oligoadenines templates, dA(20) and dA(40), into stable and tubular assemblies through noncovalent interactions including π-π stacking, dipole-dipole interactions, and the complementary adenine-thymine (A-T) hydrogen-bonding. UV-vis, fluorescence, circular dichroism (CD), atomic force microscopy (AFM), and transmission electron microscopy (TEM) techniques were used to investigate the formation of highly robust nanofibrous structures. Our results have demonstrated for the first time that the dipole-dipole interactions are stronger and useful to reinforce the assembly of donor-acceptor π-conjugated molecules to DNA templates and the formation of the stable and robust supramolecular nanofibrous complexes together with the complementary hydrogen bonding interactions. This provides an initial step toward DNA-templated polymerization to create fully synthetic DNA-mimetic polymers for biotechnological applications. This study also presents an opportunity to precisely position donor-acceptor type molecules in a controlled manner and tailor-make advanced materials for various biotechnological applications.

Nucleic Acids and Their Analogues for Biomedical Applications.

Nucleic acids are emerging as powerful and functional biomaterials due to their molecular recognition ability, programmability, and ease of synthesis and chemical modification. Various types of nucleic acids have been used as gene regulation tools or therapeutic agents for the treatment of human diseases with genetic disorders. Nucleic acids can also be used to develop sensing platforms for detecting ions, small molecules, proteins, and cells. Their performance can be improved through integration with other organic or inorganic nanomaterials. To further enhance their biological properties, various chemically modified nucleic acid analogues can be generated by modifying their phosphodiester backbone, sugar moiety, nucleobase, or combined sites. Alternatively, using nucleic acids as building blocks for self-assembly of highly ordered nanostructures would enhance their biological stability and cellular uptake efficiency. In this review, we will focus on the development and biomedical applications of structural and functional natural nucleic acids, as well as the chemically modified nucleic acid analogues over the past ten years. The recent progress in the development of functional nanomaterials based on self-assembled DNA-based platforms for gene regulation, biosensing, drug delivery, and therapy will also be presented. We will then summarize with a discussion on the advanced development of nucleic acid research, highlight some of the challenges faced and propose suggestions for further improvement.

Biologically stable threose nucleic acid-based probes for real-time microRNA detection and imaging in living cells.

We successfully fabricated threose nucleic acid (TNA)-based probes for real-time monitoring of target miRNA levels in cells. Our TNA probe is comprised of a fluorophore-labeled TNA reporter strand by partially hybridizing to a quencher-labeled TNA that is designed to be antisense to a target RNA transcript; this results in effective quenching of its fluorescence. In the presence of RNA targets, the antisense capture sequence of the TNA binds to targeted transcripts to form longer, thermodynamic stable duplexes. This binding event displaces the reporter strand from the quencher resulting in a discrete "turning-on" of the fluorescence. Our TNA probe is highly specific and selective toward target miRNA and is able to distinguish one to two base mismatches in the target RNA. Compared with DNA probes, our TNA probes exhibited favorable nuclease stability, thermal stability, and exceptional storage ability for long-term cellular studies. Our TNA probes are efficiently taken up by cells with negligible cytotoxicity for dynamic detection of target miRNAs and can also differentiate the distinct target miRNA expression levels in different cell lines. This work illuminates for using TNA as a building component to construct a biocompatible probe for miRNA detection that offers alternative molecular reagents for miRNA-related diagnostics.

Cellular uptake, tissue penetration, biodistribution, and biosafety of threose nucleic acids: Assessing in vitro and in vivo delivery.

Compared with siRNAs or other antisense oligonucleotides (ASOs), the chemical simplicity, DNA/RNA binding capability, folding ability of tertiary structure, and excellent physiological stability of threose nucleic acid (TNA) motivate scientists to explore it as a novel molecular tool in biomedical applications. Although ASOs reach the target cells/tumors, insufficient tissue penetration and distribution of ASOs result in poor therapeutic efficacy. Therefore, the study of the time course of drug absorption, biodistribution, metabolism, and excretion is of significantly importance. In this work, the pharmacokinetics and biosafety of TNAs in living organisms are investigated. We found that synthetic TNAs exhibited excellent biological stability, low cytotoxicity, and substantial uptake in living cells without transfection. Using U87 three-dimensional (3D) multicellular spheroids to mimic the in vivo tumor microenvironment, TNAs showed their ability to penetrate efficiently throughout the whole multicellular spheroid as a function of incubation time and concentration when the size of the spheroid is relatively small. Additionally, TNAs could be safely administrated into Balb/c mice and most of them distributed in the kidneys where they supposed to excrete from the body through the renal filtration system. We found that accumulation of TNAs in kidneys induced no pathological changes, and no acute structural and functional damage in renal systems. The favourable biocompatibility of TNA makes it attractive as a safe and effective nucleic acid-based therapeutic agent for practical biological applications.

Recent Developments in Aptasensors for Diagnostic Applications.

Aptamers are exciting smart molecular probes for specific recognition of disease biomarkers. A number of strategies have been developed to convert target-aptamer binding into physically detectable signals. Since the aptamer sequence was first discovered, a large variety of aptamer-based biosensors have been developed, with considerable attention paid to their potential applications in clinical diagnostics. So far, a variety of techniques in combination with a wide range of functional nanomaterials have been used for the design of aptasensors to further improve the sensitivity and detection limit of target determination. In this paper, the advantages of aptamers over traditional antibodies as the molecular recognition components in biosensors for high-throughput screening target molecules are highlighted. Aptamer-target pairing configurations are predominantly single- or dual-site binding; the design of recognition modes of each aptamer-target pairing configuration is described. Furthermore, signal transduction strategies including optical, electrical, mechanical, and mass-sensitive modes are clearly explained together with examples. Finally, we summarize the recent progress in the development of aptamer-based biosensors for clinical diagnosis, including detection of cancer and disease biomarkers and in vivo molecular imaging. We then conclude with a discussion on the advanced development and challenges of aptasensors.