Advances in DNA nanotechnology for chronic wound management: Innovative functional nucleic acid nanostructures for overcoming key challenges.

Chronic wound management is affected by three primary challenges: bacterial infection, oxidative stress and inflammation, and impaired regenerative capacity. Conventional treatment methods typically fail to deliver optimal outcomes, thus highlighting the urgency to develop innovative materials that can address these issues and improve efficacy. Recent advances in DNA nanotechnology have garnered significant interest, particularly in the field of functional nucleic acid (FNA) nanomaterials, owing to their exceptional biocompatibility, programmability, and therapeutic potential. Among them, FNAs with unique nanostructures have garnered considerable attention. First, they inherit the biological properties of FNAs, including biocompatibility, reactive oxygen species (ROS)-scavenging capabilities, and modulation of cellular functions. Second, based on a precise design, these nanostructures exhibit superior physical properties, stability, and cellular uptake. Third, by leveraging the programmability of DNA strands, FNA nanostructures can be customized to accommodate therapeutic nucleic acids, peptides, and small-molecule drugs, thereby enabling a stable and controlled drug delivery system. These unique characteristics enable the use of FNA nanostructures to effectively address the major challenges in chronic wound management. This review focuses on various FNA nanostructures, including tetrahedral framework nucleic acids (tFNAs), DNA hydrogels, DNA origami, and rolling-circle amplification (RCA) DNA assembly. Additionally, a summary of recent advancements in their design and application for chronic wound management as well as insights for future research in this field are provided.

DNA hydrogels and their derivatives in biomedical engineering applications.

Deoxyribonucleotide (DNA) is uniquely programmable and biocompatible, and exhibits unique appeal as a biomaterial as it can be precisely designed and programmed to construct arbitrary shapes. DNA hydrogels are polymer networks comprising cross-linked DNA strands. As DNA hydrogels present programmability, biocompatibility, and stimulus responsiveness, they are extensively explored in the field of biomedicine. In this study, we provide an overview of recent advancements in DNA hydrogel technology. We outline the different design philosophies and methods of DNA hydrogel preparation, discuss its special physicochemical characteristics, and highlight the various uses of DNA hydrogels in biomedical domains, such as drug delivery, biosensing, tissue engineering, and cell culture. Finally, we discuss the current difficulties facing DNA hydrogels and their potential future development.

Quantitative Characterization of RCA-Based DNA Hydrogels - Towards Rational Materials Design.

DNA hydrogels hold significant promise for biomedical applications and can be synthesized through enzymatic Rolling Circle Amplification (RCA). Due to the exploratory nature of this emerging field, standardized RCA protocols specifying the impact of reaction parameters are currently lacking. This study varied template sequences and reagent concentrations, evaluating RCA synthesis efficiency and hydrogel mechanical properties through quantitative PCR (qPCR) and indentation measurements, respectively. Primer concentration and stabilizing additives showed minimal impact on RCA efficiency, while changes in polymerase and nucleotide concentrations had a stronger effect. Concentration of the circular template exerted the greatest influence on RCA productivity. An exponential correlation between hydrogel viscosity and DNA amplicon concentration was observed, with nucleobase sequence significantly affecting both amplification efficiency and material properties, particularly through secondary structures. This study suggests that combining high-throughput experimental methods with structural folding prediction offers a viable approach for systematically establishing structure-property relationships, aiding the rational design of DNA hydrogel material systems.

Micromechanical Indentation Platform for Rapid Analysis of Viscoelastic Biomolecular Hydrogels.

The advent of biomedical applications of soft bioinspired materials has entailed an increasing demand for streamlined and expedient characterization methods meant for both research and quality control objectives. Here, a novel measurement system for the characterization of biological hydrogels with volumes as low as 75 µL was developed. The system is based on an indentation platform equipped with micrometer drive actuators that allow the determination of both the fracture points and Young's moduli of relatively stiff polymers, including agarose, as well as the measurements of viscosity for exceptionally soft and viscous hydrogels, such as DNA hydrogels. The sensitivity of the method allows differentiation between DNA hydrogels produced by rolling circle amplification based on different template sequences and synthesis protocols. In addition, the polymerization kinetics of the hydrogels can be determined by time-resolved measurements, and the apparent viscosities of even more complex DNA-based nanocomposites can be measured. The platform presented here thus offers the possibility to characterize a broad variety of soft biomaterials in a targeted, fast, and cost-effective manner, holding promises for applications in fundamental materials science and ensuring reproducibility in the handling of complex materials.

A DNA/Poly-(L-lysine) Hydrogel with Long Shelf-Time for 3D Cell Culture.

Deoxyribonucleic acid (DNA)-based hydrogels are emerging as promising functional materials for biomedical applications. However, the shelf-time of DNA hydrogels in biological media is severely shortened by nucleases, which limit the application of DNA hydrogels. Herein, a DNA hydrogel with long shelf-time is reported for 3D cell culture. Poly-(L-lysine) (PLL) is introduced as both a cross-linker and a protectant. The electrostatic interaction between PLL and DNA drove the formation of hydrogel. PLL coating on DNA increased the steric hindrance between DNA and nucleases, thus weakening the digestion of nucleases toward phosphodiester bond. As a result, the shelf-time of DNA/PLL hydrogel for 3D cell culture is extended from generally 1 day to longer than 15 days, which has not been achieved previously. Notably, poly-AS1411-aptamers are integrated to DNA/PLL hydrogels for anchoring U87 cells, and the cell encapsulation efficiency of the DNA/PLL hydrogels with aptamer is 4-time higher than that of the hydrogels without aptamer. DNA/PLL hydrogel provided a favorable microenvironment to support the proliferation of cells, which formed cell spheroid in 15 days. This protective coating strategy solves the long-standing problem on the shelf-time of DNA hydrogel, and is envisioned to promote the development of DNA hydrogel in more biomedical applications.

Rational Design of DNA Hydrogels Based on Molecular Dynamics of Polymers.

In recent years, DNA has emerged as a fascinating building material to engineer hydrogel due to its excellent programmability, which has gained considerable attention in biomedical applications. Understanding the structure-property relationship and underlying molecular determinants of DNA hydrogel is essential to precisely tailor its macroscopic properties at molecular level. In this review, the rational design principles of DNA molecular networks based on molecular dynamics of polymers on the temporal scale, which can be engineered via the backbone rigidity and crosslinking kinetics, are highlighted. By elucidating the underlying molecular mechanisms and theories, it is aimed to provide a comprehensive overview of how the tunable DNA backbone rigidity and the crosslinking kinetics lead to desirable macroscopic properties of DNA hydrogels, including mechanical properties, diffusive permeability, swelling behaviors, and dynamic features. Furthermore, it is also discussed how the tunable macroscopic properties make DNA hydrogels promising candidates for biomedical applications, such as cell culture, tissue engineering, bio-sensing, and drug delivery.

Preparation Strategies, Functional Regulation, and Applications of Multifunctional Nanomaterials-Based DNA Hydrogels.

With the extensive attention of DNA hydrogels in biomedicine, biomaterial, and other research fields, more and more functional DNA hydrogels have emerged to match the various needs. Incorporating nanomaterials into the hydrogel network is an emerging strategy for functional DNA hydrogel construction. Surprisingly, nanomaterials-based DNA hydrogels can be engineered to possess favorable properties, such as dynamic mechanical properties, excellent optical properties, particular electrical properties, perfect encapsulation properties, improved magnetic properties, and enhanced antibacterial properties. Herein, the preparation strategies of nanomaterials-based DNA hydrogels are first highlighted and then different nanomaterial designs are used to demonstrate the functional regulation of DNA hydrogels to achieve specific properties. Subsequently, representative applications in biosensing, drug delivery, cell culture, and environmental protection are introduced with some selected examples. Finally, the current challenges and prospects are elaborated. The study envisions that this review will provide an insightful perspective for the further development of functional DNA hydrogels.

Double-Network DNA Macroporous Hydrogel Enables Aptamer-Directed Cell Recruitment to Accelerate Bone Healing.

Recruiting endogenous bone marrow mesenchymal stem cells (BMSCs) in vivo to bone defect sites shows great promise in cell therapies for bone tissue engineering, which tackles the shortcomings of delivering exogenous stem cells, including limited sources, low retention, stemness loss, and immunogenicity. However, it remains challenging to efficiently recruit stem cells while simultaneously directing cell differentiation in the dynamic microenvironment and promoting neo-regenerated tissue ingrowth to achieve augmented bone regeneration. Herein, a synthetic macroporous double-network hydrogel presenting nucleic acid aptamer and nano-inducer enhances BMSCs recruitment, and osteogenic differentiation is demonstrated. An air-in-water template enables the rapid construction of highly interconnective macroporous structures, and the physical self-assembly of DNA strands and chemical cross-linking of gelatin chains synergistically generate a resilient double network. The aptamer Apt19S and black phosphorus nanosheets-specific macroporous hydrogel demonstrate highly efficient endogenous BMSCs recruitment, cell differentiation, and extracellular matrix mineralization. Notably, the enhanced calvarial bone healing with promising matrix mineralization and new bone formation is accompanied by adapting this engineered hydrogel to the bone defects. The findings suggest an appealing material approach overcoming the traditional limitations of cell-delivery therapy that can inspire the future design of next-generation hydrogel for enhanced bone tissue regeneration.

Programming Injectable DNA Hydrogels Yields Tumor Microenvironment-Activatable and Immune-Instructive Depots for Augmented Chemo-Immunotherapy.

Injectable hydrogels have attracted increasing attention for promoting systemic antitumor immune response through the co-delivery of chemotherapeutics and immunomodulators. However, the biosafety and bioactivity of conventional hydrogel depots are often impaired by insufficient possibilities for post-gelling injection and means for biofunction integration. Here, an unprecedented injectable stimuli-responsive immunomodulatory depot through programming a super-soft DNA hydrogel adjuvant is reported. This hydrogel system encoded with adenosine triphosphate aptamers can be intratumorally injected in a gel formulation and then undergoes significant molecular conformation change to stimulate the distinct release kinetics of co-encapsulated therapeutics. In this scenario, doxorubicin is first released to induce immunogenic cell death that intimately works together with the polymerized cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) in gel scaffold for effectively recruiting and activating dendritic cells. The polymerized CpG ODN not only enhances tumor immunogenicity but minimizes free CpG-induced splenomegaly. Furthermore, the subsequently released anti-programmed cell death protein ligand 1 (aPDL1) blocks the corresponding immune inhibitory checkpoint molecule on tumor cells to sensitize antitumor T-cell immunity. This work thus contributes to the first proof-of-concept demonstration of a programmable super-soft DNA hydrogel system that perfectly matches the synergistic therapeutic modalities based on chemotherapeutic toxicity, in situ vaccination, and immune checkpoint blockade.

Synthesis and Biomedical Applications of DNA Hydrogel.

Deoxyribonucleic acid (DNA), as a natural polymer material, carries almost all the genetic information and is recognized as one of the most intelligent natural polymers. In the past 20 years, there have been many exciting advances in the synthesis of hydrogels using DNA as the main backbone or cross-linking agent. Different methods, such as physical entanglement and chemical cross-linking, have been developed to perform the gelation of DNA hydrogels. The good designability, biocompatibility, designable responsiveness, biodegradability and mechanical strength provided by DNA building blocks facilitate the application of DNA hydrogels in cytoscaffolds, drug delivery systems, immunotherapeutic carriers, biosensors and nanozyme-protected scaffolds. This review provides an overview of the main classification and synthesis methods of DNA hydrogels and highlights the application of DNA hydrogel in biomedical fields. It aims to give readers a better understanding of DNA hydrogels and development trends.

Tumor Microenvironment-Responsive Hydrogel for Direct Extracellular ATP Imaging-Guided Surgical Resection with Clear Boundaries.

Most solid tumors are clinically treated using surgical resection, and the presence of residual tumor tissues at the surgical margins often determines tumor survival and recurrence. Herein, a hydrogel (Apt-HEX/Cp-BHQ1 Gel, termed AHB Gel) is developed for fluorescence-guided surgical resection. AHB Gel is constructed by tethering a polyacrylamide hydrogel and ATP-responsive aptamers together. It exhibits strong fluorescence under high ATP concentrations corresponding to the TME (100-500 µm) but shows little fluorescence at low ATP concentrations (10-100 nm) such as those in normal tissues. AHB Gel can rapidly (within 3 min) emit fluorescence after exposure to ATP, and the fluorescence signal only occurs at sites exposed to high ATP, resulting in a clear boundary between the ATP-high and ATP-low regions. In vivo, AHB Gel exhibits specific tumor-targeting capacity with no fluorescence response in normal tissue, providing clear tumor boundaries. In addition, AHB Gel has good storage stability, which is conducive to its future clinical application. In summary, AHB Gel is a novel tumor microenvironment-targeted DNA-hybrid hydrogel for ATP-based fluorescence imaging. It can enable the precise imaging of tumor tissues, showing promising application in fluorescence-guided surgeries in the future.

DNA hydrogels for bone regeneration.

DNA-based biomaterials have been proposed for tissue engineering approaches due to their predictable assembly into complex morphologies and ease of functionalization. For bone tissue regeneration, the ability to bind Ca2+ and promote hydroxyapatite (HAP) growth along the DNA backbone combined with their degradation and release of extracellular phosphate, a known promoter of osteogenic differentiation, make DNA-based biomaterials unlike other currently used materials. However, their use as biodegradable scaffolds for bone repair remains scarce. Here, we describe the design and synthesis of DNA hydrogels, gels composed of DNA that swell in water, their interactions in vitro with the osteogenic cell lines MC3T3-E1 and mouse calvarial osteoblast, and their promotion of new bone formation in rat calvarial wounds. We found that DNA hydrogels can be readily synthesized at room temperature, and they promote HAP growth in vitro, as characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, atomic force microscopy, and transmission electron microscopy. Osteogenic cells remain viable when seeded on DNA hydrogels in vitro, as characterized by fluorescence microscopy. In vivo, DNA hydrogels promote the formation of new bone in rat calvarial critical size defects, as characterized by micro-computed tomography and histology. This study uses DNA hydrogels as a potential therapeutic biomaterial for regenerating lost bone.

pH-Responsive and Reversible A-Motif-Based DNA Hydrogel: Synthesis and Biosensing Application.

Functional DNA hydrogels with various motifs and functional groups require perfect sequence design to avoid cross-bonding interference with themselves or other structural sequences. This work reports an A-motif functional DNA hydrogel that does not require any sequence design. A-motif DNA is a noncanonical parallel DNA duplex structure containing homopolymeric deoxyadenosines (poly-dA) strands that undergo conformation changes from single strands at neutral pH to a parallel duplex DNA helix at acidic pH. Despite this and other advantages over other DNA motifs like no cross-bonding interference with other structural sequences, the A-motif has not been explored much. We successfully synthesized a DNA hydrogel by using an A-motif as a reversible handle to polymerize a DNA three-way junction. The A-motif hydrogel was initially characterized by electrophoretic mobility shift assay, and dynamic light scattering, which showed the formation of higher-order structures. Further, we used imaging techniques like atomic force microscopy and scanning electron microscope to validating its hydrogel like highly branched morphology. pH-induced conformation transformation from monomers to gel is quick and reversible, and was analysed for multiple acid-base cycles. The sol-to-gel transitions and gelation properties were further examined in rheological studies. The use of the A-motif hydrogel in the visual detection of pathogenic target nucleic acid sequence was demonstrated for the first time in a capillary assay. Moreover, pH-induced hydrogel formation was observed in situ as a layer over the mammalian cells. The proposed A-motif DNA scaffold has enormous potential in designing stimuli-responsive nanostructures that can be used for many biological applications.

Stimuli-Responsive Self-Degradable DNA Hydrogels: Design, Synthesis, and Applications.

DNA hydrogels play an increasingly important role in biomedicine and bioanalysis applications. Due to their high programmability, multifunctionality and biocompatibility, they are often used as effective carriers for packing drugs, cells, or other bioactive cargoes in vitro and in vivo. However, the stability of the DNA hydrogels prevents their in-demand rapid release of cargoes to achieve a full therapeutic effect in time. For bioanalysis, the generation of signals sometimes needs the DNA hydrogel to be rapidly degraded when sensing target molecules. To meet these requirements, stimulus-responsive DNA hydrogels are designed. By responding to different stimuli, self-degradable DNA hydrogels can switch from gel to solution for quantitative bioanalysis and precision cargo delivery. This review summarizes the recently developed innovative methods for designing stimuli-responsive self-degradable DNA hydrogels and showed their applications in the bioanalysis and biomedicines fields. Challenges, as well as prospects, are also discussed.

A portable colorimetric point-of-care testing platform for MicroRNA detection based on programmable entropy-driven dynamic DNA network modulated DNA-gold nanoparticle hybrid hydrogel film.

Point-of-care testing (POCT) platforms for microRNA (miRNA) detection have attracted considerable attention in recent years, due to the increasingly important role of miRNA as biomarkers for the diagnosis of many diseases, such as cancers. However, several limitations such as the requirement of enzyme-related amplification system, expensive preservation cost, sophisticated analysis instruments and tedious operations of conventional miRNA biosensing devices severely hinder their widespread applications. In this work, a portable and smart colorimetric analysis platform was developed by employing the ultrathin DNA-gold nanoparticle (AuNP) hybrid hydrogel film as the signaling unit and the enzyme-free entropy-driven dynamic DNA network (EDN) as the signal converter and amplification unit. By programming the DNA sequences of the EDN, the EDN could respond to a specific miRNA, with miRNA-155 or miRNA-21 as the model target, and release a converter DNA with amplified concentration to further trigger the release of AuNPs from the hydrogel film as a colorimetric signal output. To avoid the use of sophisticated spectral instruments, digital analysis based on primary three-color channel (R/G/B) was further introduced by using user-friendly camera and image processing software, and a detection limit at pM level was achieved. Moreover, by introducing H2O2-mediated AuNPs enlargement procedure in the colorimetric analysis platform, the detection limit for miRNA target could further be enhanced to fM level. The POCT platform is also portable and storable with a good storage stability for at least 45 days, suggesting its great potential in practical diagnosis applications.

Black phosphorus nanosheets-enabled DNA hydrogel integrating 3D-printed scaffold for promoting vascularized bone regeneration.

The classical 3D-printed scaffolds have attracted enormous interests in bone regeneration due to the customized structural and mechanical adaptability to bone defects. However, the pristine scaffolds still suffer from the absence of dynamic and bioactive microenvironment that is analogous to natural extracellular matrix (ECM) to regulate cell behaviour and promote tissue regeneration. To address this challenge, we develop a black phosphorus nanosheets-enabled dynamic DNA hydrogel to integrate with 3D-printed scaffold to build a bioactive gel-scaffold construct to achieve enhanced angiogenesis and bone regeneration. The black phosphorus nanosheets reinforce the mechanical strength of dynamic self-healable hydrogel and endow the gel-scaffold construct with preserved protein binding to achieve sustainable delivery of growth factor. We further explore the effects of this activated construct on both human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) as well as in a critical-sized rat cranial defect model. The results confirm that the gel-scaffold construct is able to promote the growth of mature blood vessels as well as induce osteogenesis to promote new bone formation, indicating that the strategy of nano-enabled dynamic hydrogel integrated with 3D-printed scaffold holds great promise for bone tissue engineering.

Microneedle Array Encapsulated with Programmed DNA Hydrogels for Rapidly Sampling and Sensitively Sensing of Specific MicroRNA in Dermal Interstitial Fluid.

Author: Please verify that the changes made to improve the English still retain your original meaning.Detection of microRNA (miRNA) in dermal interstitial fluid (ISF) has emerged as clinically useful in health status monitoring. However, it remains a great challenge owing to the difficult sampling and low abundance. Here, we report a DNA hydrogel microneedles (MNs) array to realize rapid enrichment and sensitive detection of miRNA in ISF. The MNs' patch consists of methacrylate hyaluronic acid (MeHA) equipped with a smart DNA circuit hydrogels' system (MeHA/DNA), in which an appropriate miRNA input enables triggering a cascading toehold-mediated DNA displacement reaction to catalytically cleave cross-linking points to generate amplified fluorescence (FL) for miRNA detection. The MeHA/DNA-MNs patch with high mechanical strength can extract adequate ISF in a short time (0.97 ± 0.2 mg in 5 min) in vivo because of its supreme water affinity. Additionally, the cascading toehold-mediated DNA displacement signal amplification reaction allows for sensitive detection of the low-abundant miRNAs down to 241.56 pM. The DNA hydrogels' MNs present potential for minimally invasive personalized diagnosis and real-time health monitoring in clinical applications.

Thermal-Responsive MXene-DNA Hydrogel for Near-Infrared Light Triggered Localized Photothermal-Chemo Synergistic Cancer Therapy.

Stimuli-responsive DNA hydrogels are promising candidates for cancer treatment, as they not only possess biocompatible and biodegradable 3D network structures as highly efficient carriers for therapeutic agents but also are capable of undergoing programmable gel-to-solution transition upon external stimuli to achieve controlled delivery. Herein, a promising platform for highly efficient photothermal-chemo synergistic cancer therapy is established by integrating DNA hydrogels with Ti3 C2 TX -based MXene as a photothermal agent and doxorubicin (DOX) as a loaded chemotherapeutic agent. Upon the irradiation of near-infrared light (NIR), temperature rise caused by photothermal MXene nanosheets triggers the reversible gel-to-solution transition of the DOX-loaded MXene-DNA hydrogel, during which the DNA duplex crosslinking structures unwind to release therapeutic agents for efficient localized cancer therapy. Removal of the NIR irradiation results in the re-formation of DNA duplex structures and the hydrogel matrix, and the recombination of free DOX and adaptive hydrogel transformations can also be achieved. As demonstrated by both in vitro and in vivo models, the MXene-DNA hydrogel system, with excellent biocompatibility and injectability, dynamically NIR-triggered drug delivery, and enhanced drug uptake under mild hyperthermia conditions, exhibits efficient localized cancer treatment with fewer side effects to the organisms.

Biodegradable and Antioxidant DNA Hydrogel as a Cytokine Delivery System for Diabetic Wound Healing.

Impaired diabetic wound healing is associated with the persistence of chronic inflammation and excessive oxidative stress, which has become one of the most serious clinical challenges. Wound dressings with anti-inflammatory and reactive oxygen species (ROS)-scavenging properties are desirable for diabetic wound treatment. In this study, a shape-adaptable, biodegradable, biocompatible, antioxidant, and immunomodulatory interleukin-33 (IL-33)-cytogel is developed by encapsulating IL-33 into physically cross-linked DNA hydrogels and used as wound dressings to promote diabetic wound healing. The porous microstructures and biodegradable properties of the IL-33-cytogel ensure the local sustained-release of IL-33 in the wound area, where the sustained-release of IL-33 is maintained for at least 7 days. IL-33-cytogel can induce local accumulation of group 2 innate lymphoid cells (ILC2s) and regulatory T cells (Tregs), as well as M1-to-M2 transition at the wound sites. Additionally, the antioxidant and biocompatible characteristics of DNA hydrogels promote the scavenging of intracellular ROS without affecting cell viability. As a result, local inflammation in the diabetic wound area is resolved upon IL-33-cytogel treatment, which is accompanied by improved granulation tissue regeneration and accelerated wound closure. This study demonstrates a promising strategy in tissue engineering and regenerative medicine by incorporating DNA hydrogels and cytokine immunotherapy for promoting diabetic wound healing.

Applications of Functional DNA Materials in Immunomodulatory Therapy.

In recent years, nanoscale or microscale functional materials derived from DNA have shown great potential for immunotherapy as superior delivery carriers. DNA nanostructures with excellent programmability and addressability enable the precise assembly of molecules or nanoparticles. DNA hydrogels have predictable structures and adjustable mechanical strength, thus being advantageous in controllable release of cargos. In addition, utilizing systematic evolution of ligands by exponential enrichment technology, a variety of DNA aptamers have been screened for specific recognition of ions, molecules, and even cells. Moreover, a wide variety of chemical modifications can further enrich the function of DNA. The unique advantages of functional DNA materials make them extremely attractive in immunomodulation. Recently, functional DNA materials-based immunotherapy has shown great potential in fighting against many diseases like cancer, viral infection, and inflammation. Therefore, in this review, we focus on discussing the progress of the applications of functional DNA materials in immunotherapy; before that, we also summarize the characteristics of the functional DNA materials descried above. Finally, we discuss the challenges and future opportunities of functional DNA materials in immunomodulatory therapy.