Functionalizing tetrahedral framework nucleic acids-based nanostructures for tumor in situ imaging and treatment.

Timely in situ imaging and effective treatment are efficient strategies in improving the therapeutic effect and survival rate of tumor patients. In recent years, there has been rapid progress in the development of DNA nanomaterials for tumor in situ imaging and treatment, due to their unsurpassed structural stability, excellent material editability, excellent biocompatibility and individual endocytic pathway. Tetrahedral framework nucleic acids (tFNAs), are a typical example of DNA nanostructures demonstrating superior stability, biocompatibility, cell-entry performance, and flexible drug-loading ability. tFNAs have been shown to be effective in achieving timely tumor in situ imaging and precise treatment. Therefore, the progress in the fabrication, characterization, modification and cellular internalization pathway of tFNAs-based functional systems and their potential in tumor in situ imaging and treatment applications were systematically reviewed in this article. In addition, challenges and future prospects of tFNAs in tumor in situ imaging and treatment as well as potential clinical applications were discussed.

Framework Nucleic Acids-Based VEGF Signaling Activating System for Angiogenesis: A Dual Stimulation Strategy.

Angiogenesis is crucial for tissue engineering, wound healing, and regenerative medicine. Nanomaterials constructed based on specific goals can be employed to activate endogenous growth factor-related signaling. In this study, based on the conventional single-stranded DNA self-assembly into tetrahedral framework nucleic acids (tFNAs), the Apt02 nucleic acid aptamer and dimethyloxallyl glycine (DMOG) small molecule are integrated into a complex via a template-based click chemistry reaction and toehold-mediated strand displacement reaction. Thus, being able to simulate the VEGF (vascular endothelial growth factor) function and stabilize HIF (hypoxia-inducible factor), a functional whole is constructed and applied to angiogenesis. Cellular studies demonstrate that the tFNAs-Apt02 complex (TAC) has a conspicuous affinity to human umbilical vein endothelial cells (HUVECs). Further incubation with DMOG yields the tFNAs-Apt02-DMOG complex (TACD), which promotes VEGF secretion, in vitro blood vessel formation, sprouting, and migration of HUVECs. Additionally, TACD enhances angiogenesis by upregulating the VEGF/VEGFR and HIF signaling pathways. Moreover, in a diabetic mouse skin defect repair process, TACD increases blood vessel formation and collagen deposition, therefore accelerating wound healing. The novel strategy simulating VEGF and stabilizing HIF promotes blood-vessel formation in vivo and in vitro and has the potential for broad applications in the vascularization field.

Tetrahedral framework nucleic acids enhance the chondrogenic potential of human umbilical cord mesenchymal stem cells via the PI3K/AKT axis.

The field of regenerative medicine faces a notable challenge in terms of the regeneration of articular cartilage. Without proper treatment, it can lead to osteoarthritis. Based on the research findings, human umbilical cord mesenchymal stem cells (hUMSCs) are considered an excellent choice for regenerating cartilage. However, there is still a lack of suitable biomaterials to control their ability to self-renew and differentiate. To address this issue, in this study using tetrahedral framework nucleic acids (tFNAs) as a new method in an in vitro culture setting to manage the behaviour of hUMSCs was proposed. Then, the influence of tFNAs on hUMSC proliferation, migration and chondrogenic differentiation was explored by combining bioinformatics methods. In addition, a variety of molecular biology techniques have been used to investigate deep molecular mechanisms. Relevant results demonstrated that tFNAs can affect the transcriptome and multiple signalling pathways of hUMSCs, among which the PI3K/Akt pathway is significantly activated. Furthermore, tFNAs can regulate the expression levels of multiple proteins (GSK3ß, RhoA and mTOR) downstream of the PI3K-Akt axis to further enhance cell proliferation, migration and hUMSC chondrogenic differentiation. tFNAs provide new insight into enhancing the chondrogenic potential of hUMSCs, which exhibits promising potential for future utilization within the domains of AC regeneration and clinical treatment.

Tetrahedral Framework Nucleic Acids Promote Senile Osteoporotic Fracture Repair by Enhancing Osteogenesis and Angiogenesis of Callus.

Senile osteoporotic fracture has aroused increasing attention due to high morbidity and mortality. However, to date, there is no effective therapeutic approach available. Senile osteoporosis is characterized by impaired osteogenesis and angiogenesis, osteoporotic fracture repair could also be promoted by enhancing osteogenesis and angiogenesis. Tetrahedral framework nucleic acids (tFNAs) are a multifunctional nanomaterial that have recently been extensively used in biomedical fields, which could enhance osteogenesis and angiogenesis in vitro. Therefore, we applied tFNAs to intact and femoral fractural senile osteoporotic mice, respectively, to evaluate the effects of tFNAs on senile osteoporosis and osteoporotic fracture repair regarding the osteogenesis and angiogenesis of the callus at the early healing stages and to initially explore the potential mechanism. The outcomes showed that tFNAs had no significant effects on the osteogenesis and angiogenesis of the femur and mandible in intact senile osteoporotic mice within 3 weeks after tFNA treatment, while tFNAs could promote osteogenesis and angiogenesis of callus in osteoporotic fracture repair, which may be regulated by a FoxO1-related SIRT1 pathway. In conclusion, tFNAs could promote senile osteoporotic fracture repair by enhancing osteogenesis and angiogenesis, offering a new strategy for the treatment of senile osteoporotic fracture.

Therapeutic Effects of Self-Assembled Tetrahedral Framework Nucleic Acids on Liver Regeneration in Acute Liver Failure.

Liver failure is a serious disease that is characterized by global hepatocyte necrosis. Hepatocyte proliferation and liver regeneration are critically important for the success of treatments for liver disease. Tetrahedral framework nucleic acids (TFNAs), which are widely used antioxidants and anti-inflammatory nanomaterials, activate multiple proliferation and prosurvival pathways. Therefore, the effects of a TFNA on hepatocyte proliferation and liver regeneration in mouse livers injured by 70% partial hepatectomy (PHx), acetaminophen overdose, and carbon tetrachloride were explored in this study. The TFNA, which was successfully self-assembled from four specifically designed ssDNAs, entered the body quickly and was taken up effectively by hepatocytes in the liver and could eventually be cleared by the kidneys. The TFNA promoted hepatocyte proliferation in vitro by activating the Notch and Wnt signaling pathways. In the three in vivo mouse models of liver injury, the TFNA attenuated the injuries and enhanced liver regeneration by regulating the cell cycle and the P53 signaling pathway. Therefore, by promoting hepatocyte proliferation and enhancing liver regeneration, the TFNA shows potential as an effective therapeutic agent for treating acute liver injury induced by 70% PHx and other factors, thereby preventing the progression to acute liver failure and reducing the associated mortality rate.

A DNA Nanostructure-Based Neuroprotectant against Neuronal Apoptosis via Inhibiting Toll-like Receptor 2 Signaling Pathway in Acute Ischemic Stroke.

Ischemic stroke is a main cause of cognitive neurological deficits and disability worldwide due to a plethora of neuronal apoptosis. Unfortunately, numerous neuroprotectants for neurons have failed because of biological toxicity, severe side effects, and poor efficacy. Tetrahedral framework nucleic acids (tFNAs) possess excellent biocompatibility and various biological functions. Here, we tested the efficacy of a tFNA for providing neuroprotection against neuronal apoptosis in ischemic stroke. The tFNA prevented apoptosis of neurons (SHSY-5Y cells) caused by oxygen-glucose deprivation/reoxygenation through interfering with ischemia cascades (excitotoxicity and oxidative stress) in vitro. It effectively ameliorated the microenvironment of the ischemic hemisphere by upregulating expression of erythropoietin and inhibiting inflammation, which reversed neuronal loss, alleviated cell apoptosis, significantly shrank the infarction volume from 33.9% to 2.7%, and attenuated neurological deficits in transient middle cerebral artery occlusion (tMCAo) rat models in vivo. In addition, blocking the TLR2-MyD88-NF-κB signaling pathway is a potential mechanism of the neuroprotection by tFNA in ischemic stroke. These findings indicate that tFNA is a safe pleiotropic nanoneuroprotectant and a promising therapeutic strategy for ischemic stroke.

Biological Effect of Differently Sized Tetrahedral Framework Nucleic Acids: Endocytosis, Proliferation, Migration, and Biodistribution.

With the advent of nanotechnology, DNA nanostructures have been widely applied in various fields, particularly biology and biomedicine. Tetrahedral framework nucleic acids (TFNAs), a novel type of DNA nanomaterial, have attracted considerable attention due to their simple synthesis, high accessibility, structural stability, and versatility. However, to date, the interaction of differently sized TFNAs with living systems and their ability to be endocytosed and biodistributed in mouse is still not fully understood. To screen for the optimal TFNA size and structures, TFNA endocytosis, proliferation, and migration were tested in adipose stem cells (ASCs). We found that the internalization of differently sized TFNAs in ASCs was remarkably different. Although all TFNAs could enter ASCs, T21 had the best membrane-penetrating ability. After exposure of ASCs to TFNAs of different sizes, the proliferation and migration of cells were enhanced, especially with T21. Importantly, T21 could access the brain and accumulate over time. This study improves our understanding of the influence of TFNA size on the biological behavior of ASCs, which will help in choosing optimal TFNA size for biomedical applications.

Tetrahedral Framework Nucleic Acid Delivered RNA Therapeutics Significantly Attenuate Pancreatic Cancer Progression via Inhibition of CTR1-Dependent Copper Absorption.

Copper is vital for various life processes, whereas severely toxic at excess level. Intracellular copper homeostasis is strictly controlled by a set of transporters and chaperones encoded by the copper homeostasis genes. Increasing evidence has shown that copper is usually overloaded in multiple malignancies, including pancreatic cancer, which has an extremely poor prognosis. Recently, silencing the SLC31A1 gene, which encodes a major transmembrane copper transporter (CTR1), has been demonstrated to be an effective means for reducing the malignant degree of pancreatic cancer by downregulating the cellular copper levels. Herein, we utilized tetrahedral framework nucleic acids (tFNAs) as vehicles to overcome the biological barriers for delivering small molecular RNAs and efficiently transferred two kinds of CTR1 mRNA-targeted RNA therapeutics, siCTR1 or miR-124, into PANC-1 cells. Both therapeutic tFNAs, termed t-siCTR1 and t-miR-124, prevented copper intake more effective than the free RNA therapeutics via efficiently suppressing the expression of CTR1, thereby significantly attenuating the progression of PANC-1 cells. In this study, therapeutic tFNAs are constructed to target metal ion transporters for the first time, which may provide an effective strategy for future treatment of other metal metabolism disorders.

Improved Antiviral Activity of Classical Swine Fever Virus-Targeted siRNA by Tetrahedral Framework Nucleic Acid-Enhanced Delivery.

DNA self-assembled nanostructures have been considered as effective vehicles for biomolecule delivery because of their excellent biocompatibility, cellular permeability, noncytotoxicity, and small size. Here, we report an efficient antiviral strategy with self-assembled tetrahedral framework nucleic acids (tFNAs) delivering small interfering RNA (t-siRNA) to silence classical swine fever virus (CSFV) gene in porcine host cells. In this study, two previously reported siRNAs, C3 and C6, specifically targeting the CSFV genome were selected and modified on tFNAs, respectively, and termed t-C3 and t-C6. Results indicate that t-C3 and t-C6 can inhibit the viral proliferation of CSFV in kidney derived porcine cells, PK-15, effectively and that inhibition was markedly stronger than free siRNA-C3 or siRNA-C6 only. In addition, the DNA nanostructure also has high cargo-carrying capacity, allowing to deliver multiple functional groups. To improve the antiviral ability of tFNAs, a dual-targeting DNA nanostructure t-C3-C6 was constructed and used to silence the CSFV gene in porcine host cells. This study found that t-C3-C6 can inhibit the viral release and replication, exhibiting outstanding anti-CSFV capabilities. Therefore, these dual-targeting tFNAs have great potential in virus therapy. This strategy not only provides a novel method to inhibit CSFV replication in porcine cells but also verifies that tFNAs are effective tools for delivery of antiviral elements, which have great application potential.

Nanomaterials and Aging.

As the proportion of the elderly population increases, more and more people suffer from aging-related diseases. Even if aging is inevitable, prolonging the time of healthy aging, delaying the progression of aging-related diseases, and the incidence of morbidity can greatly alleviate the pressure on individuals and society. Current research and exploration in the field of materials related to aging are expanding tremendously. Here, we present a summary of recent research in the field of nanomaterials relevant to aging. Some nanomaterials, such as silica nanomaterials (NMs) and carbon nanotubes, cause damage to the cells similar to aging processes. Other nanomaterials such as fullerenes and metalbased nanomaterials can protect the body from endogenous and exogenous harmful substances such as ROS by virtue of their excellent reducing properties. Another new type of nucleic acid nanomaterial, tetrahedral framework nucleic acids, works effectively against cell damage. This material selectively clears existing senescent cells in the tissue and thus prevents the development of the chronic inflammatory environment caused by senescent cells secreting senescence-associated secretory phenotype to the surroundings. We believe that nanomaterials have tremendous potential to advance the understanding and treatment of aging-related disorders, and today's research only represents the beginning stages.

The protective effect of tetrahedral framework nucleic acids on periodontium under inflammatory conditions.

Periodontitis is a common disease that causes periodontium defects and tooth loss. Controlling inflammation and tissue regeneration are two key strategies in the treatment of periodontitis. Tetrahedral framework nucleic acids can modulate multiple biological behaviors, and thus, their biological applications have been widely explored. In this study, we investigated the effect of tFNAs on periodontium under inflammatory conditions. Lipopolysaccharide and silk ligature were used to induce inflammation in vivo and in vitro. The results displayed that tFNAs decreased the release of pro-inflammatory cytokines and levels of cellular reactive oxygen species in periodontal ligament stem cells, which promoted osteogenic differentiation. Furthermore, animal experiments showed that tFNAs ameliorated the inflammation of the periodontium and protect periodontal tissue, especially reducing alveolar bone absorption by decreasing inflammatory infiltration and inhibiting osteoclast formation. These findings suggest that tFNAs can significantly improve the therapeutic effect of periodontitis and have the great potential significance in the field of periodontal tissue regeneration.

Tetrahedral Framework Nucleic Acids Deliver Antimicrobial Peptides with Improved Effects and Less Susceptibility to Bacterial Degradation.

Antimicrobial peptides (AMPs) have been an attractive alternative to traditional antibiotics. However, considerable efforts are needed to further enhance their antimicrobial effects and stability against bacterial degradation. Tetrahedral framework nucleic acids (tFNAs), a new class of three-dimensional nanostructures, have been utilized as a delivery vehicle. In this study, tFNAs were combined for the first time with an antimicrobial peptide GL13K, and the effects of the resultant complexes against Escherichia coli (sensitive to GL13K) and Porphyromonas gingivalis (capable of degrading GL13K) were investigated. tFNA-based delivery enhanced the effects of GL13K against E. coli. The tFNA vehicle both increased bacterial uptake and promoted membrane destabilization. Moreover, it enhanced the effects of GL13K against P. gingivalis by protecting the peptide against degradation in the protease-rich extracellular environment. Therefore, tFNA provides a delivery vehicle for AMPs targeting a broad range of disease.

PEGylated Protamine-Based Adsorbing Improves the Biological Properties and Stability of Tetrahedral Framework Nucleic Acids.

Recently, many researchers have reported that DNA nanostructures, such as tetrahedral framework nucleic acids (tFNAs), have great potential to be useful tools in clinical and laboratory applications due to their programmable shapes, functional sites, and biological responses. However, finite endocytosis and stability in cells and body fluids compromise the functions of DNA nanostructures as a result of various adverse factors. In this study, we successfully synthesized PEGylated protamine, and tFNAs were adsorbed to it in a proper ratio of nitrogen in protamine to phosphorus in tFNAs (N/P ratio) as the functional complex. Furthermore, we demonstrated that PEGylated protamine-adsorbed tFNAs show a more prominent positive effect on cell viability and proliferation than naked tFNAs do. An increase in endocytosis can be observed in three different tissue-derived cells with the PEG-protamine-tFNA (PPT) complex. The increased endocytic ability is mediated by multiple pathways; moreover, the stimulatory effect of the PPT complex on the endocytic ability is dramatically blocked by the inhibition of the caveola-dependent pathway. Consistently, when tFNAs are stabilized by PEGylated protamine, they often tend to escape from lysosomes and survive for a longer period in biological fluids rather than being rapidly eliminated from the kidneys. The in vitro and in vivo results of our study demonstrate that the PPT complex method is a feasible, potent, and low-cost strategy that improves tFNA biocompatibility, stability, and internalization. This study provides evidence supporting the possibility of implementing PPTs for use in drug delivery, bioimaging, and gene transfection in the future.

Tetrahedral Framework Nucleic Acids Promote Corneal Epithelial Wound Healing in Vitro and in Vivo.

Poor post-traumatic wound healing can affect the normal function of damaged tissues and organs. For example, poor healing of corneal epithelial injuries may lead to permanent visual impairment. It is of great importance to find a therapeutic way to promote wound closure. Tetrahedral framework nucleic acids (tFNAs) are new promising nanomaterials, which can affect the biological behavior of cells. In the experiment, corneal wound healing is used as an example to explore the effect of tFNAs on wound healing. Results show that the proliferation and migration of human corneal epithelial cells are enhanced by exposure to tFNAs in vitro, possibly relevant to the activation of P38 and ERK1/2 signaling pathway. An animal model of corneal alkali burn is established to further identify the facilitation effect of tFNAs on corneal wound healing in vivo. Clinical evaluations and histological analyses show that tFNAs can improve the corneal transparency and accelerate the re-epithelialization of wounds. Both in vitro and in vivo experiments show that tFNAs can play a positive role in corneal epithelial wound healing.