Tetrahedral framework nucleic acids: Nanokeys unlocking a new era of precision biomedicine

Graphical abstract

Highlights

• •The review integrates structural, mechanistic, and translational insights of tFNAs across multiple diseases.
• •It highlights pathway-specific regulation linking tFNAs to regenerative outcomes.
• •The review emphasizes tFNAs’ dual role as bioactive molecules and programmable delivery vectors for precision medicine.
• •By comparing cross-disciplinary evidence, this work defines tFNAs as next-generation DNA nanotherapeutics with clinical potential.

Abstract

Tetrahedral framework nucleic acids (tFNAs), a crucial component of DNA nanotechnology, have emerged as versatile tools in biomedical research, largely owing to their distinctive structural features and multifaceted capabilities. Recent studies have highlighted the ability of tFNAs to specifically target biological pathways, facilitate cellular uptake, and enhance therapeutic efficacy. tFNAs also show considerable potential in addressing challenges such as drug resistance and poor bioavailability. However, several challenges, including limited in vivo stability, low drug-loading capacity, and potential long-term toxicity, need to be addressed for successful clinical translation. This review comprehensively explores the latest advancements in tFNAs, focusing particularly on their applications and mechanisms in treating a wide array of diseases. From bone diseases to ophthalmic disorders, tFNAs not only promote bone regeneration but also enhance therapeutic efficacy through targeted drug delivery. They also play a role in combating infectious diseases, skin and soft tissue repair, neurological and mental disorders, hepatorenal diseases, and cancer. In addition, tFNAs have demonstrated significant potential in regenerative medicine, immunomodulation, and gene delivery. This work highlights the future potential of DNA nanotechnology in precision medicine, paving the way for next-generation therapeutic strategies.

Introduction

Tetrahedral Framework Nucleic Acids (tFNAs), also known as tetrahedral DNA nanostructures (TDNs), are a key innovation in DNA nanotechnology and have attracted increasing attention for their unique structures and broad biomedical applications. tFNAs are three-dimensional structures self-assembled from four carefully designed single-stranded DNAs (ssDNAs) based on the complementary base pairing rules, enabling precise molecular targeting, efficient gene delivery, and diverse therapeutic interventions[1]. tFNAs exhibit numerous advantageous properties, including precise molecular targeting, high biocompatibility, low immunogenicity, non-toxicity, ease of assembly, resistance to nuclease degradation, and remarkable mechanical strength[[2], [3], [4], [5], [6]]. tFNAs can be rapidly internalized by cells via caveolin-mediated endocytosis and, upon functionalization with a nuclear localization signal (NLS), can escape lysosomal degradation, enabling efficient membrane penetration and making them highly effective vectors for drug and gene delivery[4,7,8]. Their stability and favorable biological profile position them as promising candidates for precision medicine, offering potential solutions to longstanding challenges in drug delivery, tissue repair, and disease treatment.

Although the biological functions of DNA and RNA have been extensively studied, the advent of tFNAs represents a remarkable breakthrough that offers distinct advantages over conventional DNA nanostructures. This review systematically summarizes the topic from the perspective of the diverse disease applications of tFNAs, focusing on their structural design and modification strategies, uptake and targeting mechanisms, and regulation of genes and signaling pathways. In addition, it highlights the unique biological advantages and clinical translational potential of tFNAs, while providing an in-depth discussion of their current challenges and future directions. The review not only providesresearchers with a comprehensive understanding of the unique role of tFNAs in nanomedicine but also offers valuable theoretical insights and practical guidance for advancing their clinical translation.

Nucleic acids

In 1869, Swiss scientist Friedrich Miescher first discovered nucleic acids in pus cells and named them “nuclein” due to their presence in the cell nucleus. In 1889, Richard Altmann renamed “nuclein” to “nucleic acid”, although DNA and RNA had not yet been distinguished. In 1929, Phoebus Levene identified the components of DNA, including the four bases A, C, G, and T. In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty demonstrated through experiments that DNA could be the carrier of genetic information. In 1953, James Watson and Francis Crick proposed the double-helix model of DNA, where A pairs with T, and C always pairs with G. Between 1961 and 1966, Robert W. Holley et al. deciphered the genetic code. In 1990, sequencing of the human genome began, and in 2001, the complete human genome sequence was published. Since the mid-20th century, nucleic acid research has rapidly advanced, becoming a core area of modern biological and medical research. Today, nucleic acid research is widely applied in fields such as disease diagnosis, gene therapy, and personalized medicine[9].

Nucleic acids can be classified into DNA, RNA, and synthetic nucleic acids, also known as nucleic acid analogs. These compounds are structurally similar to naturally occurring RNA and DNA and include peptide nucleic acid (PNA), morpholino oligonucleotide (MO), locked nucleic acid (LNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA)[[10], [11], [12], [13], [14]]. Each type of nucleic acid has modifications in its molecular backbone that distinguish it from natural DNA or RNA, providing unique advantages for research and applications. The human genome contains approximately 3 billion DNA base pairs, whereas small interfering RNA (siRNA) consists of only 21 nucleotides[15,16]. DNA is generally larger than RNA, as it primarily functions to store genetic information, while RNA plays a more prominent role in information transfer and functional execution[17]. In most cases, naturally occurring DNA molecules are double-stranded, whereas RNA molecules are single-stranded. However, some viruses have double-stranded RNA genomes, while others possess ssDNA genomes[[18], [19], [20]]. There are notable differences in the sugar structure and base composition between these nucleic acids. The key distinction between DNA and RNA lies in the sugar molecules they contain. DNA contains 2′-deoxyribose, while RNA contains ribose. The difference between ribose and deoxyribose lies in the presence of an additional hydroxyl (–OH) group on ribose. Regarding base composition, both DNA and RNA include A, C, and G, but T is found only in DNA, while uracil (U) is found only in RNA.

Tetrahedral framework nucleic acids

Nucleic acids serve as a versatile material for constructing highly controllable and precise nanostructures through a “bottom-up” approach[[21], [22], [23]]. DNA nanotechnology leverages the self-assembly capabilities of DNA molecules to construct nanoscale structures. This technology is based on the Watson-Crick base pairing principle, and by designing specific DNA sequences, it allows the construction of two-dimensional and three-dimensional architectures, ranging from simple to highly complex[[24], [25], [26], [27]]. Since DNA nanotechnology was first introduced by Nadrian Seeman in the 1980 s, it has made significant progress, particularly in drug delivery, biosensors, and gene therapy[[28], [29], [30], [31]]. DNA nanomaterials are widely utilized in the biomedical field due to their programmable nature, which allows precise control over size and shape[24]. These materials can self-assemble into two-dimensional and three-dimensional structures, such as DNA nanotubes and nanocages, for applications in targeted cancer therapy, drug delivery, or serving as biosensors[24,28,32]. For example, DNA nanotubes modified with folic acid can selectively bind to cell surface receptors and facilitate cellular entry[32].

Nucleic acid molecules exhibit complex mechanical properties. Studies demonstrate that nucleic acids display mechanical flexibility at the scale of several base pairs, whereas they exhibit asymptotic rigidity at extended length scales[20]. This characteristic allows nucleic acids to withstand specific mechanical stresses without structural failure. Under loading forces, nucleic acid molecules exhibit stretching and overstretching transitions, with a twist-stretch coupling mechanism that significantly reinforces their mechanical strength[33]. Molecular dynamics simulations reveal that the structural design of tetrahedral nucleic acids confers high stability. Specifically, flat-vertex and mitered-vertex tetrahedra demonstrate enhanced dynamic motion arising from vertex fluctuations under specific length conditions, suggesting inherent structural flexibility and adaptability[34]. This inherent flexibility may improve their stability when subjected to external forces.

Chu et al.[35] reported that in 90 % human serum at 37°C, ssDNA was degraded significantly within 2 h incubation and almost completely after 4 h. In contrast, under the same conditions, standard double-stranded DNA (dsDNA) remained largely intact (<10 % degraded) even after overnight incubation. The hairpin and dumbbell structures also exhibited superior stability compared to the single-stranded form. DNA origami and other multishelix architectures exhibit superior nuclease resistance compared to linear double-stranded or plasmid DNA, attributed to their tightly packed arrangements[36]. The dense spatial organization of tFNAs minimizes the exposure of free ssDNA, thereby significantly improving nuclease resistance[37]. Molecular topology further modulates resistance, as closed-state DNA tweezers demonstrate enhanced nuclease stability relative to their open-state counterparts[38]. Increased restriction sites and their positioning also amplify nuclease resistance[[39], [40], [41]]. Additionally, DNA nanomaterials such as tFNAs also exhibit excellent serum stability, outperforming linear dsDNA in 10 % fetal bovine serum for up to 42 h[39]. Moreover, enhanced resistance to serum degradation correlates with higher DNA density, shorter chain lengths, and stabilization through extended polyethylene glycol (PEG)[42]. These structural features enable tFNAs to preserve their architectural integrity and functionality when interacting with serum components, a critical attribute for effective drug delivery and biomedical applications. Subsequent studies further confirmed tFNAs’ intracellular stability. Divita Mathur et al.[43] investigated three DNA nanostructures (nanocrosshair, octacrosshair and tetrahedron) through cytoplasmic microinjection into COS-1 monkey kidney epithelial cells. A series of precise experiments were conducted to evaluate the stability of tFNAs in the cytoplasm. Experimentsshowed that nanocrosshair and octacrosshair degrade rapidly in COS-1 cells, with almost complete structural disintegration within 10 min and a half-life of 3–4 min. In contrast, tetrahedral structures maintained their structural integrity for at least 1 h post-injection, demonstrating comparable stability across multiple cell lines, including primary dermal fibroblasts, human astrocytes, human A549 adenocarcinoma cells, and HeLa cervical cancer cells.

The programmability of tFNAs originates from their modular structural design. Researchers can engineer dsDNA edges with tunable lengths to construct size-variable DNA tetrahedra, enabling customization for specific biomedical applications[44]. Utilizing Watson-Crick base pairing or electrostatic interactions, oligonucleotides can be site-specifically conjugated to ssDNA termini[45,46]. In addition to attaching oligonucleotides to one or more vertices of tFNAs, small molecule drugs can also be encapsulated within tFNAs and efficiently delivered into live cells[45]. This structural versatility facilitates static tFNA systems to incorporate functional oligonucleotides and therapeutic payloads, in contrast to dynamic tFNA architectures that integrate stimuli-responsive DNA nanodevices. Such programmable assembly mechanisms underpin tFNAs’ emerging applications in precision medicine, including targeted drug delivery, anticancer therapy, antimicrobial interventions, and tissue regeneration[[46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60]].

Shao et al.[61] evaluated the effects of tFNAs on mouse chondrocytes. Compared with ssDNA, tFNA-treated cells showed specific uptake by chondrocytes, phenotype maintenance and proliferation. Proliferation assays demonstrated that tFNAs exhibited negligible cytotoxicity and promoted cell growth in a concentration-dependent manner. These findings highlight the excellent biocompatibility of tFNAs and their potential to enhance cell growth, with their intracellular stability and editable properties providing a solid basis for further optimization.

When compared with ssDNA and other spatial nanostructures, tFNAs demonstrate superior endocytosis efficiency, which serves as their most distinguishing feature[46]. Fan et al.[4] revealed that tFNAs are internalized via a vesicle protein-mediated pathway and trafficked to lysosomes in a microtubule-dependent manner, whereas functionalization with NLS peptides enables lysosomal escape and efficient nuclear delivery, thereby enhancing their potential in gene therapy. Furthermore, leveraging the innate ability of natural DNA to scavenge reactive oxygen species (ROS), tFNAs exhibit a remarkable capacity to mitigate cellular damage, which may contribute to the treatment of degenerative and inflammatory diseases[46].

Bone diseases

Bone diseases represent a diverse group of disorders that profoundly affect human health and quality of life. These conditions present a wide range of complex pathological manifestations, including fractures, osteoporosis, osteoarthritis, and bone tumors, causing significant patient suffering and imposing a substantial burden on healthcare resources. Traditional diagnostic and therapeutic approaches for bone diseases are often limited by insufficient precision, specificity, and safety, making it challenging to meet the increasing clinical demands. Consequently, researchers have been actively exploring novel diagnostic and therapeutic strategies. Although bone tissue possesses intrinsic remodeling and regenerative capabilities, severe bone injuries or defects often necessitate external interventions to facilitate the repair process. Bone grafting and substitute materials remain the most widely employed strategies for addressing critical bone defects; however, their clinical applications are constrained by challenges such as immune rejection, infection risks, and the limited availability of donor bone sources.

In recent years, tFNAs have shown great promise in bone tissue engineering. Existing studies have demonstrated that tFNAs promote the osteogenic differentiation of mesenchymal stem cells (MSCs) and enhance bone regeneration[62,63]. Additionally, they regulate chondrocyte phenotype, maintain cartilage tissue integrity, and stimulate cell proliferation[61,64]. Moreover, tFNAs promote osteogenic differentiation and cellular autophagy by modulating multiple specific signaling pathways, such as phosphoinositide 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR), Wnt/β-catenin, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)[[63], [64], [65], [66]]. In bone immune regulation, tFNAs contribute to an improved bone regeneration microenvironment by promoting angiogenesis and M2 macrophage polarization[63,67]. Beyond their intrinsic bioactivity, tFNAs also serve as an efficient delivery system for bioactive molecules. Researchers have employed tFNAs as carriers to achieve the targeted delivery of bioactive molecules, further enhancing bone tissue regeneration[62,63,68]. By promoting osteogenic differentiation, enhancing bone regeneration, and preventing cartilage degradation, tFNAs provide a solid foundation for developing novel bone and cartilage tissue engineering strategies. MSCs are multipotent fibroblast-like cells with remarkable immunomodulatory and tissue repair capabilities, and they can differentiate into osteoblasts, chondrocytes, and adipocytes[[69], [70], [71], [72], [73]]. Numerous studies have demonstrated that tFNAs promote the osteogenic and chondrogenic differentiation of various types of MSCs, including but not limited to bone marrow mesenchymal stem cells (BMSCs), synovial mesenchymal stem cells (SMSCs), periodontal ligament stem cells (PDLSCs), and dental pulp stem cells (DPSCs)[62,63,65,66].

Differentiation of BMSCs

Songhang Li et al.[62] designed a bioswitchable tFNA-based delivery system (stFNA-miR) to transport miR-2861 into BMSCs (Fig. 1a). Once internalized, RNase H triggered miR release, enabling inhibition of histone deacetylase 5 (HDAC5) and subsequent upregulation of runt-related transcription factor 2 (Runx2), which enhanced osteogenic differentiation. In vitro, stFNA-miR significantly increased alkaline phosphatase (ALP) activity and promoted mineralized nodule formation, while in vivo it accelerated bone defect healing with higher bone mass and collagen deposition.

Fig. 1.

a Schematic illustration of stFNA-miR promotes osteogenic differentiation. Reproduced form ref.[62] with permission from Wiley, copyright 2021. b Schematic illustration of the preparation of TDN-miR-21-5p nanocomplex and assessment of its in vitro and in vivo effects on osteogenesis and angiogenesis during the process of repairing aging bone tissues. Reproduced form ref.[63] with permission from Wiley, copyright 2024.

In a study on steroid-associated osteonecrosis of the femoral head (SAON), Donghai Li et al.[68] developed an injectable heparin lithium hydrogel for the targeted delivery of MiR335-5p-pendant TDN (MiR@TDNs/Li-hep-gel) to facilitate the repair of critical bone defects in SAON. Long-term corticosteroid (CS) use is a major contributor to SAON, with its pathogenesis linked to bone metabolism disorders, microcirculatory damage, increased osteocyte apoptosis, and abnormal differentiation of BMSCs[[74], [75], [76], [77], [78]]. The inhibition of the Wnt signaling pathway plays a pivotal role in the process[[79], [80], [81], [82]]. Moreover, CS administration can impair femoral head angiogenesis and disrupt microcirculation, while genetic regulation further influences disease onset and progression[68,83]. Currently, treatment strategies for SAON, such as core decompression combined with necrotic bone debridement, have shown some therapeutic benefits. However, the challenge of effectively repairing critical bone defects remains unresolved. BMSC transplantation and biomaterial implantation offer potential solutions, yet each approach presents inherent limitations[84,85]. MiR335-5p targets Dickkopf-1 (DKK1) to suppress its expression, thereby regulating the osteogenic differentiation of BMSCs and promoting bone formation[86,87]. Li et al.[68] developed TDNs loaded with miR-335-5p (MiR@TDNs), which were efficiently internalized by BMSCs. MiR@TDNs enhanced BMSC proliferation, reduced apoptosis under corticosteroid stress, and promoted migration. They significantly upregulated ALP activity, increased calcium nodule formation, and reduced lipid accumulation, indicating promotion of osteogenic differentiation while suppressing adipogenesis. Moreover, MiR@TDNs elevated vascular endothelial growth factor (VEGF) secretion from BMSCs, thereby stimulating endothelial cell (EC) proliferation and angiogenesis. Mechanistically, they activated the Wnt/β-catenin pathway by inhibiting DKK1 and engaged signal transducer and activator of transcription 3 (STAT3) signaling, coordinating osteogenesis and angiogenesis. These findings confirm that tFNAs function as efficient and biocompatible carriers to modulate BMSC fate, thereby promoting osteogenic differentiation and bone regeneration.

In addition to the aforementioned pathways, tFNAs may also exert their effects by activating the AKT and ERK signaling pathways. In the context of aging, the proliferation and osteogenic differentiation capacity of senescent bone marrow mesenchymal stem cells (O-BMSCs) decline, while senescent endothelial progenitor cells (O-EPCs) exhibit reduced expression of angiogenesis-related genes, impaired migration, and weakened tube formation ability, posing challenges for bone defect repair[[88], [89], [90], [91]]. The researchers successfully constructed the TDN-miR-21-5p nanocomposite. It showed higher uptake efficiency in O-BMSCs than TDNs alone and significantly enhanced osteogenic differentiation while reducing cellular senescence. Mechanistically, TDN-miR-21-5p activated AKT and ERK signaling pathways, upregulated osteogenesis-related genes (eg, ALP, BMP-2, Col-1, OPN and SATB2), and downregulated senescence-associated genes (eg, p53 and p16), thereby promoting osteogenesis and delaying senescence[63].

In the treatment of infectious bone defects, researchers constructed a 3D-bioprinted hybrid scaffold loaded with a clindamycin (CLI)-TDN complex[92]. TDN-CLI is efficiently internalized by BMSCs and promotes their proliferation, indicating excellent biocompatibility. Additionally, TDN-CLI exhibited potent antibacterial effects, potentially due to the enhanced affinity of CLI for bacteria mediated by TDN, which may help mitigate bacterial resistance. The optimized hybrid scaffold not only inhibits bacterial adhesion and colonization but also promotes BMSC proliferation and osteogenic differentiation. Furthermore, it effectively prevents and treats both early and delayed bone infections, thereby facilitating the repair of infectious bone defects.

Differentiation of chondrocytes

Articular cartilage (AC) lacks blood vessels, nerves, and lymphatic vessels, relying on diffusion from adjacent tissues for nutrition and metabolism[[93], [94], [95], [96]]. This structural characteristic endows AC with unique biomechanical and biochemical properties but also limits its self-repair capacity. The blood supply to cartilage originates from the adjacent vascularized synovium, while chondrocytes themselves exhibit resistance to angiogenesis[93,97]. Additionally, cartilage is not innervated, and consequently, under normal conditions, it does not perceive pain. However, in pathological conditions, such as osteoarthritis (OA), the surrounding tissues may become innervated and more sensitive[96]. Due to the absence of blood and lymphatic vessels, AC primarily relies on synovial fluid diffusion for nutrient exchange[94,98]. However, this diffusion-dependent nutrient supply also severely limits the repair capacity of cartilage. In cases of injury or degenerative diseases, chondrocyte metabolism and function may be significantly impaired. These intrinsic characteristics of AC underscore its essential role in maintaining joint health and function while also revealing its vulnerability and difficulty in repair under pathological conditions. Given the limited self-regenerative capacity of AC, current clinical surgical treatments face significant challenges. Tissue engineering strategies based on MSCs hold promise but require further optimization. SMSCs possess strong chondrogenic potential and play a crucial role in cartilage repair. Their ease of accessibility and cultivation makes them ideal seed cells for cartilage tissue engineering[99,100]. A cartilage regenerative system was developed using a chitosan hydrogel/3D-printed poly(ε-caprolactone) hybrid scaffold incorporating SMSCs and tFNAs, which was injected into the articular cavity (Fig. 2)[101]. During AC regeneration, in addition to mimicking appropriate mechanical strength and porosity, a favorable regenerative microenvironment is essential for promoting MSC proliferation, differentiation, and extracellular matrix (ECM) synthesis[102]. Without an appropriate microenvironment, cells within the scaffold cannot sustain optimal proliferative and differentiative capacities[103]. Chitosan is a natural polysaccharide containing amino-glucose groups that are positively charged, allowing it to bind to negatively charged tFNA through electrostatic interactions[104]. The primary components of the cartilage matrix are collagen fibers and proteoglycans, with the latter carrying a high density of fixed negative charges within the collagen fiber network[105]. When tFNAs are injected into the joint cavity, they are repelled by the negatively charged cartilage matrix but recruited by the positively charged chitosan hydrogel, enabling effective utilization of tFNAs and improving the microenvironment for cartilage regeneration[101,104,105]. Extensive experimental evidence has demonstrated that tFNAs promote cell proliferation by upregulating the Wnt/β-catenin pathway[6,106]. Following β-catenin accumulation in the cytoplasm, it is translocated to the nucleus, where it interacts with lymphoid enhancer-binding factor 1 (LEF-1). These findings indicate that tFNAs effectively promote the proliferation of SMSCs by activating the canonical Wnt/β-catenin pathway[101].

Fig. 2.

Schematic illustration of the tFNA-centered strategy for articular repair and regeneration. In this research, a cartilage regenerative system was constructed. It consists of a hybrid matrix combining chitosan hydrogel and 3D-printed poly (PCL), which encapsulates synovial mesenchymal stem cells. Then tFNAs are recruited and injected into the articular cavity to facilitate the repair and regeneration process. Reproduced form ref.[101] with permission from Elsevier, copyright 2021.

Chondrocytes, as the only cell type in cartilage tissue, play a crucial role in cell-based cartilage regeneration strategies. Beyond the structural limitations of cartilage, tissue engineering approaches involving chondrocytes are also constrained by limited cell numbers and the loss of chondrocyte phenotype[61]. Chondrocytes are prone to differentiation and phenotypic loss during in vitro expansion, significantly impairing their efficacy in tissue engineering[107,108]. The inherently low proliferative activity of chondrocytes results in poor healing capacity following injury, making cell expansion a major challenge. The regulatory effects of tFNAs on chondrocyte function are fundamental to promoting cartilage repair. tFNAs have been shown to significantly enhance chondrocyte proliferation and differentiation[109]. Additionally, they promote chondrocyte autophagy and energy metabolism by activating the autophagy pathway and reducing oxidative stress, thereby inhibiting chondrocyte apoptosis[110]. Studies have demonstrated that tFNAs effectively facilitate articular cartilage regeneration[47]. Autophagy is a key mechanism by which SMSCs maintain normal physiological functions and respond to pathological conditions. In rheumatoid arthritis (RA), SMSCs exhibit resistance to apoptosis induction. Autophagy promotes cell survival by reducing ROS levels and mitigating DNA damage, thus preventing apoptosis[111].

The migration ability of chondrocytes is considered crucial for cartilage repair, as cells need to migrate into damaged tissue spaces to promote defect healing[[112], [113], [114]]. Increasing evidence suggests that extracellular nucleotides play a crucial role in regulating cartilage physiology[115]. The study results indicate that extracellular nucleotides, particularly ADP, ADPβS, and UDPβS, stimulate the migration of differentiated ATDC5 cells. Furthermore, ADP and ADPβS specifically induce the expression of the chondrogenic marker Col2a1. Additionally, P2Y1 and P2Y13 receptors have been identified as potential regulators of chondrocyte migration and differentiation stimulated by ADP and ADPβS[116]. Therefore, future studies could explore linking tFNAs to extracellular nucleotides to regulate chondrocyte proliferation and differentiation.

Pentosan polysulfate (PPS) is a low-molecular-weight heparin-like compound derived semisynthetically from beech wood hemicellulose, exhibiting anticoagulant and fibrinolytic properties[117,118]. PPS effectively reduces cartilage degradation, enhances synovial and subchondral blood flow, and stimulates synthesis of hyaluronic acid and proteoglycans[[117], [118], [119], [120]]. In monolayer culture, PPS decreases proliferation of canine ACs by regulating cell cycle progression, maintaining significantly higher proportions of cells in the G1 phase and correspondingly fewer in the S phase, in a concentration and time dependencies. This anti-proliferative effect likely occurs via short-term suppression of cell cycle regulatory genes, particularly CDK1 and CDK4. Additionally, PPS promotes the chondrogenic phenotype of canine ACs, significantly upregulating Col2A1 mRNA expression and glycosaminoglycan (GAG) synthesis[121].

tFNAs possess excellent delivery capabilities, thus, PPS can potentially be loaded onto nucleic acid tetrahedrons to achieve targeted delivery to articular chondrocytes, thereby increasing local PPS concentrations and enhancing therapeutic efficacy. Compared to ssDNA, which cannot be taken up by cells, tFNAs are rapidly taken up via caveolin-mediated endocytosis in a microtubule-dependent manner[52,122]. Additionally, tFNAs promote proliferation of mouse L929 fibroblasts by activating the Wnt/β-catenin signaling pathway[6]. β-catenin serves as an essential positive regulator within this pathway[123,124]. Upon accumulation in the cytoplasm, β-catenin translocates to the nucleus and interacts with Lef-1, subsequently promoting cellular proliferation. Furthermore, tFNAs stimulate chondrocyte proliferation through regulation of cyclin-dependent kinases, thereby influencing cell cycle progression[61]. Type II collagen, secreted by chondrocytes into the ECM, is a critical component frequently used as a criterion for evaluating chondrocyte phenotype and function[61]. It has been reported that the Notch signaling pathway plays a significant role in regulating cellular differentiation, proliferation, apoptosis, and organ development[[125], [126], [127], [128]]. Recent studies have also identified abnormalities in the Notch signaling pathway during the in vitro dedifferentiation process of chondrocytes. Initially, tFNAs downregulated the expression of Notch 1, Notch 3, and their target gene Hes1, subsequently activating type II collagen expression, ultimately affecting the typical phenotype and function of chondrocytes in cartilage tissue engineering. The alterations in gene expression are caused by reduced type II collagen synthesis, ultimately affecting the chondrocyte phenotype and functionality. Following 24 h of exposure to tFNAs, chondrocytes exhibit a rounded morphology characteristic of their normal phenotype; this effect appears to involve mechanisms associated with the Notch signaling pathway[61]. Therefore, by regulating the microtubule cytoskeleton, upregulating genes and proteins related to the Wnt/β-catenin signaling pathway, and modulating the Notch signaling pathway, tFNAs effectively maintain chondrocyte morphology and promote their proliferation, addressing dedifferentiation and phenotype loss during in vitro culture.

Differentiation of dental-derived stem cells

PDLSCs originate from periodontal ligament tissues and consist of heterogeneous, non-clonal stromal cell populations, including stem cells with multipotential capabilities, committed progenitors, and differentiated cells[129]. PDLSCs uniquely possess the capacity to differentiate into cementum-like tissue, bone-like tissue, and periodontal ligament-like connective tissue, a characteristic not observed in other MSCs[130]. This multipotent differentiation potential is considered a distinctive characteristic of PDLSCs. They exhibit strong clonality, self-renewal ability, and multipotency, enabling their differentiation into diverse cell lineages such as neural, osteogenic, muscular, and adipogenic tissues under specific induction conditions[[131], [132], [133], [134]].

Moreover, PDLSCs offer advantages over BMSCs as they can be obtained through a simple and minimally invasive procedure and exhibit low immunogenicity[135,136]. PDLSCs demonstrate superior colony-forming ability and higher proliferation rates, making them ideal seed cells for authentic periodontal tissue regeneration in periodontal tissue engineering and repair[66,137,138]. However, the inflammatory microenvironment presents a significant challenge, as pro-inflammatory cytokines not only exacerbate periodontal tissue destruction but also impair periodontal regeneration by inhibiting the osteogenic differentiation and migration of PDLSCs[139,140]. Importantly, tFNAs have demonstrated the ability to modulate inflammatory responses, promote osteogenic and odontogenic differentiation, and enhance migration of PDLSCs and dental pulp stem cells (DPSCs), underscoring their therapeutic potential in periodontal regeneration (Fig. 3a)[65,106,141].

Fig. 3.

a TDNs regulate the biological behavior of PDLSCs/DPSCs by stimulating their proliferation and promoting odonto/osteogenic differentiation. Reproduced form ref.[65] with permission from Wiley, copyright 2019. b The effect of tFNAs on periodontal tissue under inflammatory conditions. tFNAs can improve inflammation in periodontal tissue, protect periodontal structures, particularly by reducing inflammatory infiltration and inhibiting osteoclast formation, thereby decreasing alveolar bone resorption. Reproduced form ref.[66] with permission from KeAi Communications Co, copyright 2021.

The literature indicates that inflammatory factors can activate osteoclast differentiation, enhance osteoclast function, and inhibit osteoblast activity, resulting in excessive bone resorption[142]. tFNAs exhibit excellent antioxidant and anti-inflammatory properties, mediated by the inhibition of MAPK phosphorylation and regulation of macrophage responses[143]. As key components in the MAPK/ERK signaling pathway, P38, JNK, and ERK can be activated by environmental stress, inflammatory cytokines, and G protein-coupled receptors, inducing inflammation[144]. Zhou et al.[65] reported that tFNAs promote osteogenic differentiation of PDLSCs by regulating the Wnt/β-catenin signaling pathway, indicating their potential application for PDLSC-based bone regeneration (Fig. 3a). To further investigate the effect of tFNAs on PDLSCs under inflammatory conditions, Zhou et al.[66] established lipopolysaccharide (LPS)-induced and ligation-induced inflammatory models in vitro and in vivo. Their findings showed that tFNA treatment increased gene expression levels of ALP and Runx2 and increased the protein expression of osteogenic proteins such as osteopontin (OPN) and Runx2. Furthermore, tFNAs significantly decreased the levels of ROS and inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β in PDLSCs. In vivo experiments demonstrated that tFNAs effectively prevented periodontal tissue destruction associated with periodontitis (Fig. 3b).

tFNAs inhibit the inflammatory response of PDLSCs by reducing the expression of inflammatory cytokines and suppressing the activation of the MAPK/ERK signaling pathway, exerting their anti-inflammatory effects. They restore most of the alveolar bone to its normal morphology and promote the migration and osteogenic differentiation of PDLSCs in vitro[66]. Recent evidence demonstrates that tFNAs exhibit notable anti-inflammatory and antioxidant effects through modulation of macrophage responses by inhibiting MAPK phosphorylation[143]. These findings are particularly relevant because alveolar bone defect repair typically occurs under conditions of chronic inflammation and infection[145]. In summary, tFNAs effectively promote osteogenic differentiation of PDLSCs and DPSCs under both normal and inflammatory conditions, indicating their potential as a preventive or therapeutic agent for inflammatory bone defects.

Ophthalmic disorders

Ophthalmic disorders exert a profound impact on human visual health, notably including age-related macular degeneration (AMD) and diabetic retinopathy (DR). Given the multifaceted limitations inherent in traditional treatment modalities, the necessity to explore novel therapeutic strategies becomes paramount.

Oxidative stress plays a pivotal role in the initiation and progression of retinal diseases[146,147]. Disruption of the balance between ROS and antioxidant scavengers within retinal pigment epithelium (RPE) and retinal ECs induces inflammation, mitochondrial dysfunction, and cellular degeneration, ultimately impairing blood-retina barrier integrity and retinal neuronal dysfunction[148]. DJ-1, a protein deglycase encoded by the Parkinson’s disease-associated gene 7 (PARK7), is a key redox-sensitive protein. DJ-1 protects cells from oxidative stress-induced damage by regulating transcriptional signaling, scavenging ROS, and maintaining mitochondrial function, making it a promising target for antioxidant therapies. Overexpression of DJ-1 ameliorates high glucose-induced oxidative stress and apoptosis in retinal pericytes via modulation of the PI3K/AKT/mTOR and Nrf2 signaling pathways, thus attenuating diabetic retinopathy (DR) progression[149,150]. DJ-1 demonstrates stable expression in the RPE[151]. The deletion of DJ-1 perturbs antioxidant defense mechanisms, resulting in RPE thinning and abnormal electroretinogram (ERG) responses in mice[152]. Attempts have been made to enhance the delivery of small activating RNAs (saRNAs) in vivo using delivery vectors such as lipid nanoparticles and aptamers[153]. Wu et al.[154] developed a tFNA-based delivery system for DJ-1-saRNA, which stabilized the saRNA, enhanced uptake, and improved tissue penetration. By elevating DJ-1 expression in RPE and ECs, tFNAs-DJ-1-saRNA subsequently reduced ROS levels, preserved mitochondrial integrity, enhanced cell viability, and suppressed apoptosis under oxidative stress via ERK and Nrf2 signaling (Fig. 4a).

Fig. 4.

a An illustration is provided to show the preparation of the tfnas-dj-1-sarnas complex. additionally, it demonstrates the complex’s anti-oxidative effects on both HUVECs and ARPE-19 cells. Reproduced form ref.[154] with permission from Wiley, copyright 2024. b This figure elucidates the intracellular protective mechanism of tFNAs-miR22 subsequent to its endocytosis into injured RGCs. Reproduced form ref.[171] with permission from Wiley, copyright 2024.

Glaucoma is an irreversible neurodegenerative eye disease and a leading cause of blindness, characterized by progressive damage to retinal ganglion cells (RGCs) and their axons, alongside visual field defects and optic nerve atrophy[155]. Conventional glaucoma management focuses on lowering intraocular pressure through pharmacological interventions or surgical procedures to slow disease progression and preserve visual function[156]. However, despite pressure reduction, a significant proportion of patients continue to experience vision loss, ultimately leading to blindness in the absence of effective treatment[157]. Emerging research suggests that non-coding RNAs may contribute to glaucoma pathogenesis through direct or indirect mechanisms[158]. Furthermore, the neurotrophic tyrosine kinase receptor type 2 (NTRK2) gene, which encodes the Tropomyosin related kinase (TrkB) receptor, has been shown to interact with miR-22[159,160]. A synergistic interaction between tFNAs and miR-22 enhances the neuroprotective effects of the tFNAs-miR-22 complex in damaged retinal neurons. Using tFNAs as carriers for miR-22-3p improves miR-22 delivery to retinal neurons, promotes cell proliferation, and inhibits neuronal apoptosis in the retina[161]. The B-cell lymphoma-2 (Bcl-2) family plays a central role in apoptosis regulation, comprising both the pro-apoptotic protein BCL-2-associated X (Bax) and the anti-apoptotic protein Bcl-2[162]. Caspase-3, a pivotal apoptotic effector within the caspase family, serves as a convergence point for multiple apoptotic pathways, initiating caspase cascades associated with both mitochondrial-dependent and death receptor-mediated apoptosis[163]. Notably, Bcl-2 inhibits Caspase-3-induced apoptosis[164]. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of trophic factors, plays essential roles in the development, survival, and function of a wide range of neurons[165]. tFNAs-miR22 significantly reduces N-methyl-D-aspartic acid (NMDA)-induced apoptosis and modulates the TrkB-BDNF signaling pathway in retinal neurons. It selectively activates TrkB, further influencing the TrkB-BDNF axis and facilitating ERK 1/2 activation, ultimately inhibiting apoptosis through ERK-dependent mechanisms[161]. Additionally, tFNAs-miR-22 enhances BDNF delivery, potentially augmenting TrkB activation, given BDNF’s role in promoting TrkB expression[166]. Furthermore, it activates cAMP-response element binding protein, facilitating the transcription of neuroprotective genes such as BDNF, thereby restoring its expression in damaged RGCs and exerting neuroprotective effects on retinal neurons. tFNAs-miR-22 also reverses NMDA-induced apoptosis in mouse retinal neurons through modulating the TrkB-BDNF signaling axis[161].

Retinal ischemia/reperfusion (RI/R) injury is a major cause of vision impairment and irreversible blindness[167]. As a neural structure reliant on terminal vascular perfusion, the retina is particularly vulnerable to hypoxic-ischemic insults. Ischemic injury caused by vascular occlusion leads to rapid-onset tissue necrosis, often resulting in permanent visual deficits within hours[168]. Emerging evidence reveals that RI/R-related pathologies involve characteristic secondary injury cascades, in which post-reperfusion exacerbates primary damage through complex molecular pathways[169]. This paradoxical phenomenon ultimately drives progressive visual function deterioration despite the restoration of blood flow. Previous studies have identified the progressive loss of RGCs as the primary cause of visual impairment following RI/R injury[170]. By conjugating miR-22 onto tFNAs, researchers have developed a simple and efficient delivery system for RI/R injury treatment. The tFNAs-miR-22 complex demonstrates dual neuroprotective functions by inhibiting RGC apoptosis and promoting their proliferation. Additionally, it enables the safe and efficient delivery of miR-22 to damaged retinal neurons, exerting neuroprotective effects by preventing neuronal loss and facilitating retinal recovery. A synergistic interaction between tFNAs and miR-22 enhances the neuroprotective efficacy of tFNAs-miR-22, enabling a more rapid and active response in damaged retinal neurons. Mechanistically, tFNAs-miR-22 activates the ERK 1/2 signaling pathway, regulating the expression of RGC-protective proteins (eg, RPBMS and SYN) and neuroprotective genes (eg, FTL, NET1 and NUS1). Furthermore, it modulates the balance between anti-apoptotic proteins (eg, Bcl-2) and pro-apoptotic proteins (eg, Caspase-3 and BAX), thereby promoting neuronal survival and mitigating RI/R-induced damage (Fig. 4b)[171].

Pathological retinal neovascularization (RNV) diseases involve complex pathophysiological processes driven by an imbalance between pro-angiogenic factors and angiogenesis inhibitors within the retina, ultimately leading to severe visual impairment[172,173]. The retinal neurovascular unit refers to the functional coupling and interdependence among neurons, glial cells, and vascular cells within the inner retinal layers, playing a critical role in maintaining retinal homeostasis and function[174,175]. These diseases not only involve vascular pathology but are also associated with neuronal dysfunction, which can impede visual recovery even after neovascularization occurs[176]. Current therapeutic strategies present inherent limitations. While laser photocoagulation remains a therapeutic option, its clinical application is restricted by complications associated with thermal retinal injury[177]. Although intravitreal anti-VEGF agents have become the first-line therapy for retinal neovascular disorders due to their targeted pharmacological effects, emerging evidence suggests that prolonged VEGF inhibition may elicit paradoxical pathological responses[[178], [179], [180], [181], [182]]. Mechanistic studies indicate that VEGF signaling blockade disrupts physiological vascular homeostasis, impairing both developmental angiogenesis and neurovascular coupling mechanisms[181,182]. Post-treatment evaluations reveal persistent retinal ischemia, characterized by progressive vascular bed deterioration, highlighting the necessity for novel therapeutic strategies that simultaneously suppress neovascularization and preserve vascular integrity. The high recurrence rate of RNV in preterm neonates and individuals with diabetes mellitus underscores the incomplete efficacy of current pharmacotherapies in achieving long-term disease remission[181,183]. Quercetin (QUE), a natural flavonoid compound, exhibits various biological activities, including anti-inflammatory, anti-oxidant, and anti-tumor properties[184,185]. However, its clinical application is limited by poor solubility in physiological media, low bioavailability, and instability, necessitating strategies to enhance these properties. tFNAs also possess anti-inflammatory and antioxidant properties by modulating the MAPK and nuclear transcription factor-κB (NF-κB) pathways, mechanisms similar to those of QUE[186]. Jin et al.[187] developed tFNA-based quercetin delivery system (tFNA-QUE) for ischemic retinopathy. tFNA-QUE inhibited vascular endothelial cell (VEC) proliferation, migration, and tube formation in vitro, and in oxygen-induced retinopathy (OIR) models it suppressed RNV, reduced retinal non-perfusion, and preserved neuronal morphology and function. Mechanistically, it regulates the AKT/nuclear factor erythroid-derived 2-related factor 2/heme oxygenase-1 (AKT/Nrf2/HO-1) signaling pathway, to exert antioxidant and anti-angiogenic effects, thereby protecting the retinal neurovascular unit (NVU). Beyond ischemic retinopathy, tFNAs also exhibit therapeutic efficacy in promoting corneal epithelial regeneration and mitigating oxidative damage in retinal ganglion cells[188,189]. In a proof-of-concept study, Zhou et al.[190] demonstrated that tFNAs can safely and efficiently penetrate VECs under hypoxic conditions, inhibiting the proliferation, migration, and lumen formation of HUVECs, thereby suppressing pathological RNV. Additionally, tFNAs not only inhibit pathological angiogenesis but also prevent retinal vaso-obliteration (VO) and promote the normalization of damaged vessels in ischemic retinas. These effects are mediated through the regulation of the PI3K/AKT/mTOR signaling pathway, which inhibits EC growth and metabolism under hypoxic conditions.

Diabetic retinopathy (DR) is a prevalent microvascular complication, affecting approximately 22.27 % of individuals with diabetes worldwide[191]. In its early stages, DR presents with microaneurysms, intraretinal hemorrhages, and retinal edema, while advanced stages may lead to vitreous hemorrhage, visual field defects, and even blindness[170,192]. Moreover, DR remains a major cause of blindness among adults[170]. However, effective clinical strategies remain limited. Current therapeutic options include laser photocoagulation, anti-VEGF agents, and corticosteroids[193]. Nevertheless, laser photocoagulation can cause irreversible retinal damage, while anti-VEGF therapy may interfere with the physiological functions of VEGF, potentially leading to adverse effects[194,195]. DR is closely associated with oxidative stress and is characterized by excessive production of ROS[196,197]. Mitochondria serve as the primary cellular organelles responsible for ROS generation[197,198]. When damaged mitochondria fail to undergo efficient clearance, they not only impair cellular energy metabolism but also keep ROS production, further exacerbating oxidative stress[199]. Resveratrol (RSV), a natural polyphenolic phytalexin found in grapes, has attracted significant attention for its ability to regulate multiple pathways involved in mitochondrial protection[200,201]. Research has shown that RSV regulates PINK1/PARKIN-mediated mitophagy in renal cells, upregulates mitophagy, and simultaneously activates mitochondrial respiratory complexes in ECs[202,203]. However, its limited cellular uptake efficiency and lack of organelle selectivity result in poor bioavailability, restricting its clinical effectiveness and application. To address these limitations, Yao et al.[204] synthesized a trackable targeted nanosystem (PP-TRh-RSV) that not only exhibits excellent biocompatibility and bioavailability but also ensures sustained release and photochemical stability of RSV. In vitro, PP-TRh-RSV effectively escapes from endolysosomes, targets mitochondria, regulates redox homeostasis, and modulates mitophagy. Furthermore, it mitigates vascular leakage and pathological angiogenesis in DR models. In vivo, PP-TRh-RSV can be detected and monitored using clinical ophthalmic laser diagnostic instruments. Functionally, PP-TRh-RSV inhibits RNV and retinal degeneration by modulating the NF-κB/inducible nitric oxide synthase (iNOS) and NF-κB/VEGF pathways, thereby delaying DR progression. As a hallmark carrier of DNA nanotechnology, tFNAs have transitioned from proof-of-concept research to the forefront of precision medicine in ophthalmology, offering novel therapeutic strategies for addressing challenges in ophthalmic diseases treatment.

Infectious diseases

In contemporary biomedicine, the synergistic threats posed by antibiotic resistance and viral mutations are intricately linked to demographic transitions and evolving disease profiles, further exacerbating public health challenges. The accelerated aging of populations and the increasing prevalence of metabolic disorders (eg, diabetes mellitus and obesity) have significantly contributed to a rising incidence of chronic infection, while the emergence of multidrug-resistant pathogens and the rapid diversification of viral genomes have introduced new therapeutic complexities[205]. Biofilms are structured bacterial communities that adhere to the ECM secreted by bacteria on both living and non-living surfaces[206]. Within this matrix, bacteria embed themselves for protection, forming highly organized and resilient microbial assemblies. Biofilms contain various biomacromolecules, such as extracellular polysaccharides (EPS) and nucleic acids. The EPS matrix not only enhances bacterial adhesion but also promotes microbial aggregation and accumulation on surfaces, leading to the formation of dense, highly adhesive biofilms[207]. Consequently, bacteria within biofilms exhibit antibiotic resistance levels 500 to 5,000 times higher than their antibiotic-sensitive cells, including strains typically susceptible to antibiotics[208,209].

Three primary factors contribute to the high levels of antibiotic resistance observed in biofilms. Firstly, due to competition for nutrients and space, bacteria proliferating within biofilms exhibit reduced metabolic activity, making them less susceptible to growth-targeting antibiotics[210]. Secondly, the protein and polysaccharide components of the EPS matrix hinder or delay antibiotic penetration, allowing mature cells embedded deep within the matrix more time to develop resistance[210,211]. Thirdly, antibiotic-resistant bacteria secrete resistance factors that induce the entire biofilm community to acquire resistance through passive mechanisms[212]. Furthermore, the threat posed by viruses should not be underestimated. Viruses are ubiquitous, highly diverse, and rapidly adaptable, frequently establishing close associations with their hosts[[213], [214], [215], [216]]. Mutations drive sequence-level variations in viral genomes, ranging from single-base changes to large-scale genomic rearrangements[217]. Through continuous adaptation to host immune responses and environmental pressures, viruses evolve new mutant strains, consequently enhancing their survival and transmission efficiency[218,219]. Viral variation is a complex and dynamic process that not only poses a significant threat to human health but also places increasing demands on public health systems.

Traditional drug delivery systems demonstrate significant limitations in overcoming challenges associated with bacterial resistance evolution and viral mutation dynamics. Current drug delivery platforms primarily rely on liposomes or gold nanoparticles, which often present high cytotoxicity, disrupting host cell homeostasis despite their significant effectiveness. Additionally, these nanoparticles have inherent limitations in biocompatibility and structural programmability[220,221]. tFNAs demonstrate distinct advantages as next-generation drug delivery systems. Recent studies validate that tFNAs not only exert dual anti-inflammatory and antioxidant effects but also enhance mammalian cell proliferation and differentiation capacity[61,141,221]. Notably, the structural programmability of tFNAs enables their application as versatile carriers for targeted nucleic acid drug delivery[61,222,223]. Unlike conventional nanoparticles, tFNAs can penetrate live cells without the need for auxiliary molecules, demonstrating mechanical stability and resistance to nuclease degradation[39,122]. Furthermore, they are rapidly internalized by cells through clathrin-mediated endocytosis and, when functionalized with nuclear localization signals, can effectively escape from lysosomes[3,4].

Zhang et al.[224] constructed a TDN/Histatin-5 (His-5) complex, where TDNs enhanced the uptake efficiency and antifungal activity of His-5. The complex induces an increase in intracellular ROS generation. The extensive accumulation of ROS subsequently disrupts intracellular biomolecules and metabolic pathways, thereby inhibiting the growth of Candida albicans. Additionally, treatment with TDN/His-5 promotes potassium ion efflux, causing ion imbalance and disrupting intracellular and extracellular volume regulation. Ultimately, this disruption results in cell death and exhibits an antibacterial effect. Zhang et al.[225] successfully synthesized antisense oligonucleotides (ASOs)-laden tFNA (ASOs-tFNA), which efficiently penetrated Streptococcus mutans and targeted multiple extracellular polysaccharides (EPS)-related genes (gtfBCD, gbpB, ftf). Treatment with 750 nM, ASOs-tFNAs significantly downregulated these genes, reduced EPS synthesis, and disrupted biofilm architecture, yielding thinner, more porous structures without affecting bacterial morphology. This multi-targeted inhibition effectively suppressed early biofilm formation and virulence, demonstrating that tFNAs serve as precise and non-bactericidal carriers for genetic modulation (Fig. 5a).

Fig. 5.

a Graphical illustration of tFNAs’ mechanism in delivering ASOs to suppress bacterial biofilm formation by targeting EPS synthesis-related genes. Reproduced form ref.[225] with permission from Springer Nature, copyright 2020. b Mechanistic illustration of tFNA-GL13K complexes against bacteria. Reproduced form ref.[226] with permission from American Chemical Society, copyright 2020.

Antimicrobial peptides (AMPs) represent a diverse group of cationic and short peptides that naturally occur in various organisms[[227], [228], [229]]. Unlike traditional antibiotics, which primarily target specific intracellular targets, AMPs interact with negatively charged microbial membranes through electrostatic and hydrophobic interactions. This interaction leads to irreversible membrane disruption, ultimately resulting in cytoplasmic leakage[[230], [231], [232]]. Notably, AMPs exhibit strong activity against drug-resistant bacteria and show less acquired resistance in the host. However, their peptide-based nature renders them susceptible to protease degradation[[233], [234], [235]]. Liu et al.[226] combined tFNAs with the antimicrobial peptide GL13K and investigated the inhibitory effects of this complex on Escherichia coli and Porphyromonas gingivalis. The tFNA-based delivery system significantly enhanced the antibacterial efficacy of GL13K against E. coli by facilitating bacterial uptake and promoting membrane destabilization. Furthermore, tFNAs strengthened GL13K’s activity against Porphyromonas gingivalis by shielding the peptide from degradation in the protease-rich extracellular environment (Fig. 5b).

tFNAs can competitively bind to host cell receptors targeted by viral nucleic acids, thereby blocking viral infection. Kaposi’s sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is implicated in several lymphoproliferative disorders and significantly contributes to acquired immunodeficiency syndrome (AIDS)-related morbidity and mortality[[236], [237], [238]]. Similar to other herpesviruses, KSHV establishes lifelong latency by evading host immune surveillance, making it currently impossible to eradicate the virus from infected individuals[[239], [240], [241], [242]]. Consequently, disrupting the latent phase of KSHV is a crucial step towards eliminating the virus from infected host cells[240]. One of the most distinctive aspects of the KSHV genome is the terminal repeat (TR) region, which is highly enriched in guanine residues. DNA or RNA sequences with high guanine content, such as eukaryotic telomeric DNA, are known to form secondary structures called G-quadruplexes[[243], [244], [245], [246]]. The formation of G-quadruplexes in nucleic acid sequences initiates with the binding of four guanine residues to form a G-quartet, where each guanine residue interacts with another through two hydrogen bonds. The presence of a central monovalent cation (Na⁺ or K⁺) enhances the stability of the G-quadruplex. The resulting G-quartets have a strong tendency to stack, leading to the formation of a highly stable G-quadruplex structure[[247], [248], [249]]. Compounds that stabilize G-quadruplexes (eg, PhenDC3 and TMPyP4), target the GC-rich TR region of KSHV, which harbors numerous potential G-quadruplex formation sites[250]. These compounds stabilize G-quadruplex structures, leading to bidirectional stalling of replication forks, which impedes viral DNA replication processes, reduces viral genome replication, and lowers the copy number of viral episomes. Treatment with PhenDC3 triggers a stress response in KSHV-infected cells, activating additional replication forks and dormant replication origins[250].

Human herpesvirus 6A/B (HHV-6A/B) are two distinct DNA viruses. HHV-6B is a ubiquitous virus that infects nearly 100 % of the population. It is the causative agent of exanthema subitum, also known as the sixth disease of childhood, which manifests as a febrile rash in infants, typically between 6 and 15 months of age[251,252]. Reactivation of HHV-6B in immunosuppressed individuals has been associated with adverse clinical complications, including life-threatening encephalitis or graft rejection in transplant recipients[253]. The diseases associated with HHV-6A infection are not clearly established. BRACO-19 is a small-molecule compound that specifically binds to and stabilizes G-quadruplex structures at telomeres[254,255]. BRACO-19 binds to and stabilizes G-quadruplexes present in the single-stranded regions of telomeres. Additionally, BRACO-19 inhibits telomerase activity and causes displacement of the telomerase complex[254,255]. By binding and stabilizing telomeric G-quadruplexes, BRACO-19 impairs the interaction between telomerase and telomeric DNA, thereby inhibiting telomerase activity and reducing telomere mobility. This loss of telomere fluidity prevents homologous recombination between the viral genome and host telomeres, ultimately suppressing HHV-6A chromosomal integration[256].

Human immunodeficiency virus (HIV) is the causative agent of AIDS. HIV infection leads to the progressive collapse of the human immune system, rendering the body incapable of combating various diseases and resulting in high mortality rates. Although numerous biosensors have been developed for early HIV detection, existing methods have inherent limitations. Diao et al.[257] designed a surface plasmon resonance (SPR) sensor that combines entropy-driven strand displacement reactions (ESDRs) with bilayer DNA tetrahedra for the highly sensitive detection of HIV-related DNA molecules. In this system, target DNA initiates the ESDR amplification process, forming a dsDNA complex with terminal toe regions, which subsequently hybridizes with surface-immobilized hairpin probes. This approach exploits exposed hybridization sites within the DNA tetrahedral composite structure, effectively amplifying the SPR signal and enabling highly sensitive detection of target DNA. This strategy offers notable advantages, including high stability and low cost, making it a promising platform for the early detection of HIV-related DNA[258].

In summary, achieving high-sensitivity virus detection with a broad detection range, rapid and accurate performance, simple operation, low cost, high specificity, and strong applicability represents a significant advancement in diagnostic technology. These breakthroughs provide a reliable foundation for the early diagnosis of major infectious diseases. In the future, the integration of nucleic acid aptamers with advanced detection technologies is expected to further drive the development of portable diagnostic devices, enhancing accessibility and efficiency in disease diagnosis.

Skin and soft tissue

In the current field of dermatological disease management and soft tissue repair, conventional therapeutic strategies primarily include pharmacotherapy, physiotherapy, and surgical intervention. However, these approaches universally face inherent limitations imposed by the skin barrier, particularly in the treatment of deep cutaneous lesions, chronic conditions, and soft tissue injuries, where therapeutic efficacy is often suboptimal[259,260]. Externally applied agents (eg, corticosteroid-based formulations) may provide temporary symptomatic relief in a short term. However, their prolonged application is associated with adverse effects, including cutaneous atrophy and localized immunosuppression[261]. While laser therapy and electrostimulation-based modalities can effectively induce dermal regeneration and mitigate inflammatory responses, their effectiveness is limited in addressing complex cutaneous defects or extensive lesions, with additional concerns regarding potential complications and extended treatment durations[262,263]. Although surgical interventions can address extensive skin damage, they are often accompanied by significant trauma, prolonged recovery periods, and a higher risk of postoperative complications[264]. Compared with these conventional approaches, tFNAs demonstrate remarkable therapeutic potential in penetrating the skin barrier, achieving precise targeting, and modulating immune responses[265,266]. Due to their nanoscale three-dimensional structures, tFNAs efficiently penetrate the stratum corneum (SC), allowing for targeted delivery of therapeutic agents or genetic molecules to the dermal layer or sites of soft tissue injury, thereby overcoming the limited drug penetration associated with traditional treatments[266,267]. In addition to their high-efficiency drug delivery capability, tFNAs facilitate skin and soft tissue repair by modulating cellular functions while simultaneously minimizing the adverse effects associated with conventional therapies and enhancing treatment efficiency[268]. Consequently, tFNAs offer superior precision, controllability, and multifunctionality in dermatological therapeutics and soft tissue repair. tFNAs not only enhance drug delivery efficiency to specific lesion areas but also accelerate wound healing and tissue regeneration through coordinated mechanisms, including immune response modulation, angiogenesis promotion, and cell proliferation stimulation[268,269]. These characteristics position tFNAs as a promising therapeutic strategy for wound healing, chronic dermatosis management, and soft tissue repair.

The stratum corneum of the skin is generally a barrier that significantly impedes drug penetration, particularly in deep lesions or injured areas. As a nanomaterial, tFNAs can overcome this barrier due to their nanoscale dimensions and surface charge properties[270,271]. The surface of tFNAs can be functionally modified, for example, by conjugation with specific peptides or antibodies, enabling precise targeting of receptors on the surface of skin cells, such as the hyaluronic acid receptor (CD44)[[272], [273], [274]]. This targeting capability enables tFNAs to deliver drugs or gene molecules directly to specific skin lesions, thereby enhancing therapeutic efficacy while minimizing systemic side effects[273,275]. Beyond conventional targeted therapy, recent studies suggest that tFNAs can further refine targeted delivery by leveraging the unique microenvironment of the skin, including keratinocytes and the hair follicle matrix[[276], [277], [278]]. If tFNAs can selectively bind to specific epidermal growth factor receptor (EGFR) subtypes in the skin, they would further enhance targeting efficiency and promote drug accumulation in localized areas, especially showing significant potential in skin cancer treatment[225,279,280]. Furthermore, the self-assembling nature of tFNAs enables them to simultaneously carry and release multiple drugs or growth factors, effectively addressing the challenges associated with combining multiple therapeutic approaches in conventional treatments. For example, a dual-delivery strategy integrating anti-inflammatory drugs and growth factors not only enables rapid inflammation relief but also promotes wound healing[281,282].

Studies have shown that tFNAs modulate dendritic cell function in the skin, not only promoting local immune responses but also enhancing immune tolerance, thereby mitigating immune rejection reactions[283]. Recent research has further elucidated the influence of tFNAs on dendritic cells, revealing their ability to regulate immune responses by targeting surface molecules such as CD80 and CD86[283,284]. Through this mechanism, tFNAs play a more precise role in skin immune repair, particularly in immunomodulatory therapies, by promoting immune tolerance and reducing chronic inflammation, ultimately contributing to more sustained skin repair outcomes[[285], [286], [287]]. Additionally, tFNAs enhance the anti-inflammatory response during skin repair by modulating the expression of cytokines such as TNF-α and IL-10[288,289]. Through this precise immunomodulation, tFNAs exhibit significant therapeutic efficacy in managing skin inflammation caused by trauma, burns, or chronic skin diseases such as psoriasis[288].

Building on their ability to promote angiogenesis, the unique structure of tFNAs can also directly inhibit excessive angiogenesis by delivering siRNA targeting the VEGF receptor, thereby exerting a dual effect in the treatment of skin tumors[190,290,291]. By precisely regulating angiogenesis, tFNAs not only promote wound healing but also effectively prevent aberrant vascular formation, demonstrating significant potential for treating diabetes-related skin ulcers and promoting skin repair following radiotherapy[292]. The role of tFNAs in promoting angiogenesis is a key mechanism underlying their soft tissue repair capabilities[269,292]. Angiogenesis serves as the foundation of soft tissue repair, and tFNAs promote EC migration and proliferation, thereby facilitating neovascularization by delivering angiogenic factors such as VEGF and bFGF (Fig. 6)[63,291,292]. The formation of new blood vessels not only improves blood supply to the damaged area but also ensures an adequate supply of oxygen and nutrients necessary for the repair process[293]. Furthermore, tFNAs contribute to ECM remodeling, reduce scar tissue formation, and further enhance soft tissue regeneration[294,295].

Fig. 6.

Graphical abstract highlighting the antioxidative and angiogenesis-promoting functions of tFNAs in the context of diabetic wound healing. AGEs: advanced glycation end products. MDA: malondialdehyde. SOD: superoxide dismutase. LDH: lactic dehydrogenase. Reproduced form ref.[292] with permission from American Chemical Society, copyright 2020.

Skin cancer, particularly melanoma, has become one of the fastest-growing cancer types worldwide. According to global cancer statistics, the incidence of skin cancer has risen significantly in recent years, especially among Caucasian populations[296,297]. Skin cancer primarily comprises basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma, with melanoma being the most aggressive and associated with the highest mortality rate[298]. Although early screening and surgical intervention can effectively reduce mortality, the high recurrence rate and immune evasion mechanisms of skin cancer pose significant challenges to its treatment[[299], [300], [301]]. In recent years, the emergence of molecular targeted therapies and immunotherapy has offered new hope for skin cancer treatment. Although immune checkpoint inhibitors, such as PD-1 and CTLA-4 inhibitors, have demonstrated significant efficacy in advanced melanoma, they can also cause severe side effects, limiting their clinical application[302]. Similarly, molecular targeted therapies may induce skin toxicity, including rash and pruritus[303,304]. Consequently, there is an urgent need for the development of novel and more precise therapeutic strategies. The therapeutic application of tFNAs in skin cancer is primarily attributed to their exceptional targeting capabilities. Skin cancer cells, especially melanoma cells, frequently overexpress specific surface receptors, providing a strong basis for tFNA-based targeted therapy. Studies have shown that melanoma cells overexpress EGFR and integrin alpha-v beta-3 (αvβ3), among other receptors, making them ideal targets for tFNA-mediated delivery[[305], [306], [307], [308]]. By conjugating the DNA tetrahedron with anti-EGFR antibodies or small molecule ligands, its affinity for melanoma cells can be significantly enhanced[305,309]. The nucleic acid tetrahedron enters tumor cells through receptor-mediated endocytosis, enabling the precise delivery of encapsulated anticancer drugs or siRNA to the target site[258,310,311].

The nucleic acid tetrahedron induces apoptosis in cancer cells by modulating key molecular pathways within tumor cells. Skin cancer cells frequently evade apoptosis due to mutations in the p53 gene, making the restoration of p53 function a crucial strategy in skin cancer treatment[312,313]. Studies have demonstrated that tFNAs can serve as efficient carriers for delivering specific anticancer molecules or genes, such as the p53 gene or microRNA (miRNA)[265,314,315]. These molecules suppress tumor growth and metastasis by modulating the tumor microenvironment. This innovative therapeutic approach not only mitigates the side effects associated with conventional chemotherapy but also enhances the specificity and targeting efficiency of treatment[316]. By regulating gene expression in tumor cells, tFNAs effectively control the onset and progression of skin cancer, demonstrating groundbreaking therapeutic potential[317,318]. Furthermore, through modulation of the tumor microenvironment, tFNAs inhibit immune evasion in tumor cells and regulate immune cell polarization, thereby enhancing anti-tumor immune responses[287,317,319]. Compared to conventional treatment methods, tFNAs offer superior specificity while minimizing damage to normal tissues by precisely delivering anticancer molecules[284,314].

tFNA, as a novel nanotherapeutic carrier, provides innovative therapeutic strategies for cutaneous oncology through a multi-mechanistic approach that includes precision-targeted delivery, controlled drug release, apoptosis induction, and tumor microenvironment modulation. In the cosmetics industry, tFNAs exhibit broad application potential, primarily in anti-aging, skin whitening, skin repair, and skin barrier restoration[29,268,320]. With aging, collagen and elastin fibers gradually degrade, resulting in skin sagging, wrinkle formation, and other related issues. As an innovative nanomaterial, tFNA facilitates precise drug delivery, effectively modulating the activity of skin fibroblasts, promoting collagen synthesis, and restoring skin firmness and elasticity[266,294]. Its role in the field of anti-aging extends beyond the delivery of genes that promote collagen synthesis, such as transforming growth factor beta (TGF-β) and collagen type I alpha 1 (COL1A1). It also enhances anti-aging effects by modulating the expression of miRNAs[267,[321], [322], [323]]. For example, miR-29b, a key miRNA molecule, regulates collagen degradation, thereby preserving skin structure and elasticity[324,325]. By delivering these miRNAs or other regulatory factors, tFNAs enable more precise modulation of the skin aging process, achieving a more effective and long-lasting anti-aging effect[267,326]. Additionally, through surface modification, tFNA can bind to EGFR on skin cells, activating downstream signaling pathways such as MAPK and PI3K/Akt, thereby promoting skin cell repair and regeneration[276,[327], [328], [329], [330]]. This regulation not only enhances the skin’s self-repair ability but also accelerates skin cell proliferation by modulating the cell cycle, effectively alleviating the aging process[276,331,332].

tFNAs also demonstrate significant advantages in the field of skin whitening. By acting as carriers for small-molecule drugs that inhibit melanin synthesis (eg, hydroquinone and arbutin), tFNAs effectively reduce tyrosinase activity, thereby inhibiting melanin production and addressing issues like uneven skin tone[[333], [334], [335]]. Studies suggest that tFNAs enhance the targeting and permeability of whitening agents by binding to specific receptors on the surface of melanocytes, such as melanocortin 1 receptor (MC1R), thereby achieving a more sustained whitening effect[288,[336], [337], [338]]. This mechanism not only enhances the efficacy of whitening agents but also strengthens the skin’s resistance to external stimuli, providing more effective protection. Environmental factors contribute significantly to skin barrier disruption, which is a primary cause of skin dryness, allergies, and various dermatological conditions[339,340]. By delivering repair factors, tFNAs accelerate skin barrier restoration and significantly improve the skin’s moisture retention ability[294,341,342]. Studies have shown that tFNAs improve skin barrier function and hydration by promoting the proliferation and differentiation of keratinocytes, showing great potential in the treatment of dry skin[65,343,344]. Additionally, the antioxidant properties of tFNAs further contribute to skin barrier repair[344,345]. By delivering antioxidant molecules such as vitamin C and E, tFNAs scavenge free radicals and reduce oxidative damage, thereby delaying skin aging and preserving skin health and a youthful appearance[333,[346], [347], [348]]. These effects highlight the broader potential applications of tFNAs in skin repair and anti-aging.

Neurological and mental disorders

The global prevalence of neurological and psychiatric disorders, such as Alzheimer’s disease (AD), major depressive disorder (MDD), and epilepsy, has been rising annually, imposing a significant burden on healthcare systems. The treatment of central nervous system (CNS) diseases remains challenging due to the presence of the blood–brain barrier (BBB) and the complexity of their pathophysiological mechanisms. tFNA, owing to its unique geometric structure and nanoscale size, can effectively penetrate the BBB and precisely target the nervous system[349,350]. By adjusting the sequence and length of its building blocks, the physicochemical properties of tFNA can be optimized. Additionally, surface modifications can enhance its interactions with target cells, further improving its therapeutic potential for neurological disorders[314].

With the increasing incidence of neurological injuries, especially in neurodegenerative diseases, promoting nerve regeneration and repair has become a key focus of current research. The proliferation and differentiation of neural stem cells (NSCs) are crucial for nervous system development and repair. During the early stages of nervous system development, NSCs continuously proliferate, providing a sufficient cellular source for neural network formation, and gradually differentiate into various types of neural cells, such as neurons, astrocytes, and oligodendrocytes, thereby establishing a complex and precisely regulated neural network[351]. After nervous system injury, the proliferative and differentiative abilities of NSCs remain critical. They can be activated, proliferate to generate new cells, and differentiate into the corresponding neural cell types to replenish damaged areas and facilitate neural function restoration[352]. tFNAs demonstrate a distinctive and potent mechanism in promoting the proliferation and differentiation of NSCs. Ma et al.[344] systematically investigated the regulatory effects of tFNAs on NSC proliferation and differentiation, as well as the underlying molecular mechanisms. To address the challenges associated with the proliferation and differentiation efficiency of NSC transplantation for the treatment of neurological disorders, this study employed mouse neuroectodermal (NE-4C) stem cells as a model. Through material synthesis and characterization, cellular uptake experiments, and functional analyses, the study demonstrated that tFNAs can be efficiently internalized by cells and significantly enhance the proliferation and neuronal differentiation of NE-4C stem cells at a concentration of 250 nM. Further mechanistic investigations revealed that tFNAs exert their effects through dual signaling pathways. Regarding proliferation, tFNAs activate the Wnt/β-catenin pathway by specifically binding to the Wnt receptor Frizzled family, enhancing signal transduction efficiency. This interaction promotes β-catenin nuclear accumulation, where it binds transcription factors such as Lef-1 and upregulates proliferation-related genes including cyclin D1 and c-myc, thereby accelerating NSC entry into the cell cycle and promoting continuous division and proliferation[[353], [354], [355], [356]]. With respect to differentiation regulation, tFNAs exert their effects by inhibiting the Notch signaling pathway, significantly downregulating the expression of the Notch-1 membrane protein and reducing the production of inhibitory transcription factors such as Hes-1 and Hes-5, thereby relieving the inhibition of neuronal differentiation genes, such as NeuroD and Ngn[344,357,358]. Additionally, when NSCs receive differentiation signals, tFNAs can inhibit the overactivation of the Notch signaling pathway, thereby promoting NSC differentiation into neurons[357,359]. Specifically, tFNAs bind to a specific domain of the Notch receptors, preventing their interactions with ligands, thereby inhibiting Notch signaling transmission. This suppression allows the normal expression of neuronal differentiation-related genes within the cell, guiding NSCs to gradually differentiate into mature, functionally active neurons[357,358,360]. Moreover, tFNAs can indirectly influence NSC proliferation and differentiation by modulating growth factors and ECM components within the cellular microenvironment. They promote the secretion of key growth factors involved in NSC proliferation and differentiation, such as BDNF and nerve growth factor[357,[361], [362], [363]]. Upon binding to their respective receptors on the NSC surface, these growth factors activate intracellular signaling pathways, further enhancing NSC proliferation and differentiation[363,364]. Concurrently, tFNAs can also modulate ECM composition and structure, creating a more favorable environment for NSC adhesion and migration. This optimization facilitates NSC migration to appropriate locations following proliferation, thereby improving their contribution to nervous system development and repair[109,258,268,365].

In the nervous system, apoptosis is a tightly regulated physiological process essential for maintaining both structural integrity and functional balance. When the nervous system experiences external injury or internal pathological changes, a cascade of intracellular signaling events is triggered, leading to the activation or suppression of apoptosis-related genes[366,367]. tFNAs can regulate apoptosis by interacting with specific apoptosis-related genes or proteins, thereby modulating the process of apoptosis[[366], [367], [368]]. For example, tFNAs can bind to the pro-apoptotic protein Bax, inducing conformational changes that prevent its insertion into the mitochondrial membrane. This inhibition reduces cytochrome c release, thereby suppressing the downstream caspase cascade and ultimately preventing neural cell apoptosis[367]. Conversely, in situations where the clearance of abnormal or aging neural cells is required, tFNAs can promote apoptosis by activating pro-apoptotic signaling pathways, thereby maintaining neural cell population health and homeostasis[366]. Neurotransmitters are essential for maintaining the normal function and balance of the nervous system and are critical for diverse brain activities. Cui et al.[369] found that the novel tFNA oral delivery system can inhibit the production of Parkinson’s disease-related gut pathogens and their metabolites, inhibit the activation of immune cells and the release of inflammatory cytokines, and promote the secretion of the neurotransmitter 5-HT by enteroendocrine cells. This process subsequently attenuates the apoptosis of dopaminergic neurons in the striatum. These findings highlight the potential of tFNAs in modulating neural signal transmission and maintaining the functional integrity of neural networks (Fig. 7).

Fig. 7.

Schematic diagram of the mechanism by which tFNAs act on Parkinson’s disease (PD) via the microbiota-gut-brain axis. Acid-resistant exosome-like nanovesicles (Exo@tac) were prepared from ginger and were equipped with AMP-modified tFNAs. Microbiome analysis confirmed that Exo@tac normalized the gut microbiota composition in a PD mouse model and significantly improved PD symptoms upon oral administration in vivo. Reproduced form ref.[369] with permission from Elsevier, copyright 2024.

Neuroinflammation, as a core mechanism underlying various neurological and psychiatric disorders, has long been a central focus of scientific research. tFNAs play an important regulatory role in this process by effectively inhibiting neuroinflammatory responses through the modulation of multiple inflammatory pathways[366]. Among these, the NF-κB signaling pathway represents a central regulatory mechanism of inflammation and governs cellular responses to inflammatory stimuli[370]. tFNAs bind to IκB kinase (IKK), thereby inhibiting NF-κB activation and preventing the phosphorylation and degradation of IκB[288,366]. This process blocks the translocation of NF-κB from the cytoplasm to the nucleus, disrupting its ability to initiate the transcription of inflammation-related genes and thus mitigating the inflammatory response[370,371]. Additionally, tFNAs may modulate the MAPK signaling pathway by inhibiting the phosphorylation of MAPK family members, significantly reducing the expression and release of inflammatory cytokines[372]. This ingenious regulatory mechanism effectively slows the progression of neuroinflammation and protects neural cells from damage. During neuroinflammation, abnormal activation of immune cells frequently exacerbates neural damage. Specifically, tFNAs inhibit the excessive activation of microglial cells, thereby reducing the release of inflammatory mediators and ROS, which slows the progression of neural damage[350,373]. In addition, tFNAs also regulate macrophage polarization, promoting the shift from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. This transition exerts anti-inflammatory effects and further facilitates neural tissue repair and regeneration[374]. Through this series of intricate regulatory mechanisms, tFNAs effectively alleviate the burden of neuroinflammation, protect the integrity of the nervous system, and demonstrate promising potential for the treatment of neuroinflammation-related diseases.

Neurodegenerative diseases are characterized by neuronal damage, functional impairment, and aberrant immune responses. Existing treatments often fail to effectively halt disease progression, particularly in Alzheimer’s disease (AD), which has emerged as an increasingly serious global health concern. The core pathological features of AD include the accumulation of β-amyloid (Aβ) plaques and the hyperphosphorylation of tau protein, both of which disrupt neuronal signaling and contribute to progressive neurodegeneration[375]. Current AD therapies primarily aim to relieve symptoms and delay disease progression, as no curative treatment is available. The main pharmacological agents include cholinesterase inhibitors (eg, donepezil) and N-methyl-D-aspartic acid receptor antagonists (eg, memantine). The former can improve cognitive function in patients with mild to moderate AD for approximately 6–12 months but is frequently associated with side effects such as nausea and diarrhea. The latter is more effective in patients with moderate to severe AD, and combination therapy shows greater efficacy; however, the long-term therapeutic effects tend to diminish over time[[376], [377], [378]]. In recent years, disease-modifying therapies targeting AD pathology (eg, Aβ monoclonal antibodies) have achieved some breakthroughs, but their clinical benefits remain limited and are associated with risks such as cerebral edema[379,380]. Shao et al.[367] found that tFNAs can partially cross the BBB, improve the learning and memory abilities of rats, restore the abnormal morphology and number of neurons in the hippocampus, reduce the deposition of Aβ1-40, and inhibit apoptosis in the hippocampus. Besides, tFNAs show no significant toxic side effects on neural cells at specific concentrations and hold promise as a new therapeutic approach for AD.

Similarly, epilepsy treatment faces the same challenges[381,382]. Increased adenosine kinase (ADK) activity leads to adenosine deficiency in the brain in epilepsy[381,383]. Zhu et al.[384] designed a tFNA-based nanoantiepileptic drug (tFNA-ADK^ASO@AS1) carrying an antisense oligonucleotide against ADK and an A1 astrocyte-targeting peptide (AS1). The construct efficiently crossed the BBB, selectively downregulated ADK in reactive A1 astrocytes, restored endogenous adenosine levels, suppressed aberrant mossy fiber sprouting and pathological circuit reorganization, and thereby reduced seizure frequency (Fig. 8a). Long-term treatment showed no significant neurotoxicity or organ damage, providing a new direction for the treatment of epilepsy and other central nervous system diseases.

Fig. 8.

a Schematic diagram of the mechanism of the tFNA (tFNA-ADK^ASO@AS1) carrying ADK^ASO and AS1 in a chronic temporal lobe epilepsy mouse model. Reproduced form ref.[384] with permission from American Chemical Society, copyright 2023. b Schematic model of the underlying mechanism by which TDN-miR-22-3p exerts its antidepressant effects. Under physiological conditions, miR-22-3p suppresses the expression of phosphatase and tensin homologue (PTEN). This suppression enables PI3K to catalyze the conversion of PIP2 into PIP3. PIP3 then phosphorylates AKT, which subsequently inhibits the activation of nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3). Conversely, during depression, the downregulation of miR-22-3p causes an upsurge in PTEN levels. The elevated PTEN impedes PIP3 production, reactivating pathways that were initially repressed. This reactivation ultimately exacerbates the inflammatory response, contributing to the pathophysiology of depression. Reproduced form ref.[391] with permission from American Chemical Society, copyright 2023.

The treatment of psychiatric disorders is challenged by the complex imbalance of neurotransmitters and impaired synaptic plasticity. Major depressive disorder (MDD), a prevalent and highly disabling condition, affects a substantial portion of the global population. Current treatment methods are limited by delayed onset of action and a lack of efficacy in a subset of patients. The discovery of miRNA has introduced promising therapeutic prospects for depression. Among them, miRNA-22-3p, a non-coding RNA consisting of 19–24 nucleotides, is closely associated with neuroplasticity and depressive symptomatology. It also exhibits notable anti-inflammatory effects, suggesting therapeutic potential in depression[[385], [386], [387], [388]]. However, the clinical application of miRNA-22-3p is constrained by its poor in vivo stability and limited ability to cross the BBB[389,390]. To these issues, Luo et al.[391] utilized the excellent structural stability, programmability, and biocompatibility of tFNAs as carriers for miRNA-22-3p, and successfully synthesized TDN-miR-22-3p. The study results showed that TDN-miR-22-3p can effectively cross the BBB and be taken up by target cells. Moreover, it regulated molecular expression levels, reduced the release of inflammatory factors, and alleviated LPS-induced cellular inflammation, thereby alleviating depressive symptoms (Fig. 8b). This study successfully established a new miRNA delivery platform and highlighted the potential of tFNAs in enhancing miRNA-22-3p delivery across biological barriers, offering a promising direction for depression treatment.

Hepatorenal disease

The liver is a vital organ responsible for numerous essential physiological functions, and various liver diseases can significantly compromise patient health. Acute liver injury (ALI) refers to the acute damage to the liver caused by external factors such as drug toxicity, viral infections, excessive alcohol consumption, or chemical exposure[[392], [393], [394]]. Clinical manifestations of ALI include jaundice, elevated liver function tests levels, ascites, and hepatic encephalopathy. Without timely intervention, it may progress to acute liver failure (ALF), which can be life-threatening[395]. Traditional treatment approaches primarily aim to eliminate or mitigate the underlying cause of injury, yet they do not offer in-depth intervention that directly promotes hepatic repair and regeneration[396]. Upon hepatic injury, hepatocytes release large quantities of cytokines and inflammatory mediators, amplifying the local inflammatory response. This exacerbates hepatocellular damage and establishes a vicious cycle of inflammation and injury. The process is further intensified by oxidative stress responses, which trigger the excessive production of ROS and free radicals[397,398]. Excessive ROS can damage hepatocyte membranes, proteins, and DNA, leading to further cellular injury and hepatocyte necrosis[398]. Meanwhile, Kupffer cells and hepatic stellate cells contribute to the amplification of the inflammatory response. Cytokines released by these cells during the injury process not only exacerbate inflammation but also promote the progression of fibrosis[399]. In the treatment of ALI, tFNAs can be used as carriers for anti-inflammatory, antioxidative, or hepatoprotective agents, enabling targeted delivery to hepatic tissue. Targeted delivery not only minimizes drug distribution to non-target tissues, thereby reducing systemic side effects, but also ensures higher drug concentrations at sites of injury[400]. Hepatocyte proliferation and liver regeneration are crucial for the restoration of liver function following injury. As oxidative stress plays a pivotal role in the pathogenesis of ALI, tFNAs can activate multiple proliferative and pro-survival signaling pathways, creating a favorable microenvironment for hepatic recovery.

Chen et al.[401] investigated the effects of tFNAs on liver regeneration in the context of ALF. The researchers demonstrated that self-assembled tFNAs are rapidly taken up by hepatocytes and subsequently cleared via renal excretion. tFNAs promoted hepatocyte proliferation in vitro through activation of the Notch and Wnt/β-catenin pathways. Across multiple models of ALI, tFNAs alleviated hepatic damage and accelerated regeneration by driving cell cycle progression and engaging the P53 signaling pathway (Fig. 9a). Additionally, the liver possesses a certain degree of self-repair capacity following injury, particularly through the proliferative potential of hepatocytes under specific conditions[402,403]. Wei et al. developed miR-122-functionalized TDN (TDN-miR-122) to enhance stem cell-based therapy for ALF. In the first study, TDN-miR-122 effectively induced the hepatic differentiation of human mesenchymal stem cells (hMSCs) into mature hepatocyte-like cells, and transplantation of these engineered cells markedly restored liver function in ALF mice by enhancing hepatocyte function, promoting proliferation, inhibiting apoptosis, and reducing inflammation (Fig. 9b)[404]. Building on this, a subsequent study demonstrated that TDN-miR-122 was also applied to adipose-derived mesenchymal stem cells (ADMSCs), where it drove their differentiation into hepatocyte-like cells and supported the formation of hepatic spheroids with ECs, thereby improving hepatocyte maturation and liver-specific functions[405].

Fig. 9.

a Schematic diagram of the mechanism of action of tFNA in a mouse model of liver injury. tFNA promotes liver cell proliferation by activating the Notch and Wnt signaling pathways, alleviating damage, and enhances liver regeneration by regulating the cell cycle and the P53 signaling pathway. Reproduced form ref.[401] with permission from American Chemical Society, copyright 2022. b Schematic illustration of ALF with TDN-miR122-hMSCs. The TDN loaded with miR122 can significantly enhance the transfection efficiency of miR122, thereby accelerating the differentiation of hepatocytes from hMSCs. The resulting TDN-miR122-hMSCs possess the functionality of mature hepatocytes, which enables them to effectively mitigate the damage caused by ALF. Significantly, TDN not only acts as a vector that induces the differentiation of hMSCs into hepatocytes but also participates in the combined treatment of ALF. Reproduced form ref.[404] with permission from KeAi Communications Co, copyright 2023.

Hepatic fibrosis (HF) and cirrhosis are irreversible pathological conditions resulting from prolonged chronic liver injury, characterized by architectural remodeling of the liver and progressive loss of hepatic function[406]. HF is typically caused by the liver’s repeated exposure to toxins, viral infections, alcohol abuse, metabolic disorders, and other risk factors, leading to excessive formation of fibrous connective tissue across various hepatic regions. This process may advance to cirrhosis and ultimately progress to liver failure[407,408]. Once cirrhosis develops, treatment options are generally limited to symptomatic supportive care or liver transplantation, due to the absence of effective anti-fibrotic therapies[409]. Current treatment strategies primarily aim to slow or halt the progression of HF. These include the administration of anti-fibrotic agents such as TGF-β inhibitors, antioxidants, and anti-inflammatory therapies. However, these approaches are often constrained by limited targeting capability and efficacy. Kim et al.[410] designed a cholesterol-conjugated DNA tetrahedron (Chol₃-Td) that exploits serum lipoprotein interactions for targeted hepatic delivery. After systemic administration, Chol₃-Td exhibited efficient liver accumulation and was preferentially taken up by hepatocytes via scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR) pathways. Utilizing this mechanism, Chol₃-Td was loaded with an antisense oligonucleotide against transforming growth factor-beta1 (TGF-β1) (ASO@Chol₃-Td), which effectively suppressed TGF-β1 expression, reduced collagen deposition, and improved liver function in fibrotic mice. Notably, ASO@Chol₃-Td achieved gene silencing comparable to the clinically validated GalNAc₃-ASO platform, underscoring its potential as a competitive and efficient strategy for liver-targeted therapy of HF.

Cirrhosis represents the advanced stage of HF and contributes significantly to the global disease burden. However, there are currently no approved pharmacological therapies available for clinical use[411,412]. Utilizing gene editing technology, tFNAs can transport miRNA or siRNA to silence fibrosis-associated genes, thereby slowing fibrosis progression, inhibiting further deterioration, and potentially improving hepatic function even after cirrhosis onset. Tian et al.[413] developed a tFNA-based siRNA delivery system (tFNA-siCcr2) to inhibit CCR2 expression for cirrhosis therapy. After intraperitoneal administration, the complex accumulated in the liver, preferentially targeting macrophages and liver sinusoidal ECs (LSECs), and showed higher silencing efficiency than conventional transfection agents. In the model of HF, tFNA-siCcr2 downregulated CCR2, reduced fibrotic progression, and reshaped the immune microenvironment by limiting pro-fibrotic macrophage and neutrophil infiltration. Mechanistically, it suppressed NF-κB signaling and decreased inflammatory mediators, thereby promoting an anti-fibrotic milieu.

Kidney diseases are also significant health issues faced in clinical practice, especially acute kidney injury (AKI). AKI is characterized by a rapid decline in renal function, typically triggered by external factors such as surgery, drug-induced nephrotoxicity, infections, hypotension, or physical trauma[[414], [415], [416]]. Clinically, AKI is manifested by decreased urine output, elevated serum creatinine levels, and electrolyte imbalances. In severe cases, it may progress to renal failure and subsequently lead to multiple organ failure, carrying a high mortality rate[417]. Although advances have been made in the clinical management of AKI, early diagnosis remains a significant challenge. Timely identification of AKI is crucial, as early intervention can dramatically improve patient outcomes and prevent irreversible kidney damage. Ding et al.[418] developed a kidney injury molecule-1 (Kim-1)-targeted tetrahedral DNA framework nanodevice (Kim-TDF) that integrates kidney targeting, biomarker recognition, and near-infrared (NIR) fluorescence imaging. Kim-TDF enabled rapid and specific accumulation in injured kidneys and detected AKI much earlier than conventional serum or urine tests, offering a sensitive and minimally invasive diagnostic strategy.

Oxidative stress plays a central role in the pathogenesis of AKI. Renal injury caused by external insults is often accompanied by excessive production of ROS. These oxidative products not only directly attack renal tubular epithelial cells, cell membranes, and organelles but also activate multiple intracellular signaling pathways, resulting in increased apoptosis, necrosis, inflammation, and aggravated tubular injury[419]. The nanoscale size of tFNAs enables them to penetrate renal tubular epithelial cells and facilitates their accumulation within the kidney[420,421]. Through surface modification, tFNAs can be directed to specific renal regions, particularly those affected by oxidative damage. The generation of ROS can damage key cellular biomolecules, including lipids, proteins, and DNA, ultimately leading to cellular dysfunction and tissue injury. In this context, antioxidant supplementation has been shown to effectively scavenge ROS and alleviate oxidative stress[422]. Superoxide dismutase (SOD) is an important antioxidant enzyme that catalyzes the conversion of superoxide anion radicals into hydrogen peroxide, thereby reducing oxidative damage to cells[422,423]. However, the intrinsic instability of SOD in biological environments complicates its direct delivery to the site of injury[424]. Recognizing the current lack of effective treatments for AKI, Yan et al.[425] constructed a tFNA-based drug delivery system by loading Tylophorine (Typ) onto tFNAs (TTC). Typ, a saponin extracted from Tylophora, exhibits anti-inflammatory, anti-tumor, and antioxidant activities[426]. TTC reduced apoptosis and oxidative stress, restored mitochondrial function, and preferentially accumulated in renal tubules to protect kidney function in AKI mice. This mitochondria-protective strategy offers a promising therapeutic approach for AKI and related renal injuries. Chen et al.[427] developed a tFNA-FG-4592 complex (TFG) that enhances the hypoxia-inducible factor-1α (HIF-1α) signaling to protect against cisplatin-induced AKI. TFG showed efficient renal accumulation, reduced tubular apoptosis, and preserved kidney function, offering a biocompatible strategy to mitigate chemotherapy-associated nephrotoxicity (Fig. 10b). However, further studies are needed to investigate the efficacy of TFG against other nephrotoxic agents and to elucidate the precise mechanisms involving HIF-1α and apoptosis-related pathways. The recovery process from AKI is multifaceted, involving the mitigation of oxidative stress, suppression of inflammation, and promotion of renal cell repair and regeneration. The regenerative capacity of renal tubular epithelial cells is regulated by a variety of biological factors, including growth factors, transcription factors, and intracellular signaling pathways[428,429]. As gene delivery carriers, tFNAs can transport genes or RNA molecules associated with cellular repair and regeneration to renal tissues, thereby promoting kidney recovery[413,430]. Hien Bao Dieu Thai et al.[430] noted that while p53 siRNA shows therapeutic potential for AKI, its clinical translation is hindered by poor cytoplasmic uptake and low stability. To overcome these barriers, they designed small-sized DNA tetrahedrons (sTds) and identified L-sTd as an efficient kidney-targeted nanocarrier. In AKI mice, L-sTd delivered p53 siRNA to renal cells, suppressed p53 expression, and restored kidney function, highlighting its promise as a precision therapy for renal diseases. In summary, by enabling the targeted delivery of antioxidants, anti-inflammatory agents, and renal repair molecules, tFNAs can effectively alleviate oxidative stress and inflammation, thereby promoting renal regeneration and functional recovery.

Fig. 10.

a Design and characterization of the Kim-TDF. The schematic illustration depicts fluorescence imaging of Kim-1, showcasing enhanced AKI-to-normal contrast achieved through the use of the Kim-TDF nanodevice. Reproduced form ref.[418] with permission from Wiley, copyright 2022. b The illustration emphasizes the synthesis of the TFG complex and elaborates on its significant anti-apoptotic function in HK-2 cells. By exerting this anti-apoptotic effect, it effectively protects the renal tissue against cisplatin-induced damage within the animal model. Reproduced form ref.[427] with permission from Wiley, copyright 2024. c sTds injected intravenously pass through the glomerular basement membrane and Bowman’s space via filtration. They then enter the tubules, and the nanoconstructs are selectively absorbed by the tubular cells. Reproduced form ref.[430] with permission from American Chemical Society, copyright 2020.

Cancer

Cancer precision therapy has long been hindered by the limitations of conventional chemotherapeutic agents, which exhibit low targeting specificity and substantial systemic toxicity. Functional modification of aptamers enables tFNAs to specifically recognize overexpressed target molecules on the surface of cancer cells, thereby achieving both targeted drug delivery and biosensing. Studies have demonstrated that this drug-loaded system delivers therapeutics with a 25-fold higher targeting efficiency compared to free aptamers. Moreover, it activates systemic anticancer immunity by inducing immunogenic cell death[431]. To date, this technology has demonstrated superior therapeutic efficacy over traditional treatments in preclinical models of breast cancer, glioblastoma, and lung cancer, and has advanced into the clinical translation phase.

Breast cancer

Breast cancer is the most prevalent malignancy among women[432]. Triple-negative breast cancer (TNBC), a subtype of breast cancer, poses a significant clinical challenge due to its aggressive nature and the absence of effective targeted therapies[433,434]. Adriamycin (Dox) is a widely used anticancer agent that mainly targets DNA and RNA synthesis[435]. However, its clinical efficacy is limited by poor cellular uptake and active efflux mediated by P-glycoprotein, resulting in insufficient intracellular accumulation in cancer cells[436]. Therefore, an efficient drug delivery system is essential to enhance the intracellular delivery of Dox to tumor cells. As drug delivery vehicles, tFNAs exhibit higher uptake in cancer cells compared to normal cells[437]. The specificity and efficacy of cancer treatment can be increased through employing targeted drug delivery systems and therapeutic agents with reduced off-target effects. A drug delivery platform was developed by loading the chemotherapeutic agent Dox onto tFNAs. At pH 5.0, the DNA tetrahedron: Dox complex (TD: Dox) at a 1:100 ratio released more drug over 24 h compared to free Dox[437]. This enhanced release may result from tFNAs entering cells via clathrin-mediated endocytosis. Once internalized, Dox intercalates into DNA and disrupts replication processes[438]. The TD: Dox system demonstrated improved cellular uptake in metastatic cells compared to free Dox and has been shown to inhibit cancer cell migration and invasion, highlighting its therapeutic value in combating metastasis[439].

Maytansine (DM1) is a microtubule inhibitor that binds to β-tubulin and restricts its movement[440]. Its anticancer potency is 100 to 1000 times greater than that of other anti-microtubule agents, such as paclitaxel (PTX)[441]. However, the non-specific toxicity and severe side effects associated with DM1 have prevented its clinical application[442,443]. DM1 was utilized in the development of antibody-drug conjugates (ADCs) because of its potent anticancer properties. The resulting conjugates demonstrated significant tumor inhibition while causing minimal harm to healthy cells[444]. Ma et al.[445] developed an anti-HER2 DNA-aptamer-modified DNA tetrahedron drug conjugate (HApt-tFNA) that precisely delivers DM1 to HER2-positive breast cancer cells (HApt-tFNA@DM1, HTD). To improve stability and therapeutic efficacy, they constructed a biomimetic nanocarrier by coating synthetic liposomes with red blood cell (RBC) membranes, which preserved natural RBC properties, prolonged circulation, and enhanced biosafety. The hybrid system also integrated pH-responsive release, ensuring selective drug delivery within the acidic tumor microenvironment (Fig. 11). When loaded with HTD, these erythrosome-based nanoparticles achieved efficient tumor targeting, strong antitumor activity, and reduced systemic toxicity compared with free drug and conventional liposomes, highlighting a promising strategy for HER2-positive breast cancer therapy.

Fig. 11.

Synthesis and antitumor mechanism of HTD encapsulated in PEOz-erythrosome hybrid nanocarriers. a Schematic representation of the stepwise synthesis protocol for HTD complex. b Construction of the PEOz-erythrosome@HTD hybrid delivery system via coextrusion methodology, combining PEOz-functionalized liposomes containing HTD with erythrosomes to form biomimetic nanocarriers with enhanced circulation stability and targeting capability. c Proposed mechanism of action: Following intravenous administration, PEOz-erythrosome@HTD selectively accumulates in the acidic tumor microenvironment due to its pH-responsive properties, triggering the controlled release of HTD. Subsequently, the released HTD complexes specifically bind to HER2 receptors on cancer cell surfaces, inducing receptor-mediated endocytosis. Within acidified endolysosomal compartments, the tFNA framework undergoes enzymatic degradation, liberating encapsulated DM1 molecules. The released DM1 binds with high affinity to β-tubulin, effectively disrupting microtubule dynamics by inhibiting both polymerization and depolymerization processes of α/β-tubulin, ultimately leading to mitotic arrest and apoptotic cell death in tumor cells. Reproduced form ref.[445] with permission from Wiley, copyright 2022.

Glioblastoma

Glioblastoma (GBM), the most common primary intracranial malignancy, arises from the neuroectoderm and has a median survival of only 15 months under the current standard of care[446]. Due to the highly aggressive growth of most GBMs, the boundary between tumor tissue and normal brain parenchyma is difficult to identify, making complete surgical resection challenging. Moreover, the cytogenetic heterogeneity of GBM hinders the efficacy of therapies targeting specific molecular pathways[447,448]. Despite recent advances in neurosurgical techniques, novel radiotherapy modalities, and the continuous development of clinical trials to optimize chemotherapy regimens, the prognosis of GBM remains poor. Temozolomide (TMZ) has served as the cornerstone of GBM treatment for decades[449]. Numerous studies have demonstrated that TMZ can induce apoptosis, autophagy, and senescence in glioma cells[[450], [451], [452]]. However, TMZ is associated with several major limitations. First, more than 50 % of GBM patients exhibit resistance to TMZ[453]. Second, prolonged or high-dose administration may lead to significant myelosuppression[446]. Third, virtually all patients eventually develop acquired resistance, resulting in tumor recurrence that is typically more aggressive than the primary glioma[454]. Extensive observations have suggested that TMZ resistance in glioma cells is primarily associated with the overexpression of O6-methylguanine-DNA-methyltransferase (MGMT)[[455], [456], [457], [458], [459]]. In the present study, Fu et al.[460] successfully synthesized a TMZ-loaded tFNA (tFNA-TMZ) that enhanced cytotoxicity in TMZ-sensitive GBM cells by inducing apoptosis and autophagy, while overcoming resistance in TMZ-resistant cells via MGMT downregulation. To optimize delivery, tFNAs were simultaneously modified with GS24 and AS1411 aptamers, enabling BBB penetration, promoting nuclear localization, and further suppressing MGMT expression. In vivo, this dual-aptamer-modified tFNA exhibited stable cerebral vascular retention, highlighting its promise as a nanocarrier platform to improve TMZ delivery and therapeutic efficacy in GBM.

Accumulating data suggests that platelet-derived growth factor receptor β (PDGFRβ), a marker of the basement membrane, strongly drives tumor progression and is closely associated with tumor cell proliferation, migration, and angiogenesis[461]. The Gint4.T aptamer, a high-affinity ligand and inhibitor of PDGFRβ, has been employed to deliver polymer nanoparticles, miRNAs, and CL4 aptamers to Uppsala 87 Malignant Glioma (U87MG) cells[[461], [462], [463]]. Gint4.T significantly inhibited proliferation and migration of U87MG cells and blocked tumor growth in vivo by its interaction with PDGFRβ. Based on the observation that ECs express PDGFRβ, Franchini et al.[462] suggested that Gint4.T may have the capacity to cross the BBB. PTX is an anticancer agent that stabilizes microtubule polymerization and disrupts mitotic processes and is widely used in the treatment of malignant gliomas[[464], [465], [466]]. However, its clinical application is limited by its high lipophilicity, cytotoxicity, and poor selectivity. Moreover, BBB prevents PTX from effectively penetrating brain tissue[467]. Shi et al.[372] synthesized a dual-aptamer-modified tFNA carrying GMT8 and Gint4.T (Gint4.T-tFNA-GMT8, GTG), which specifically target U87MG glioma cells and PDGFRβ-expressing ECs. GTG efficiently crossed an in vitro BBB model and, when loaded with PTX (GPC), markedly inhibited glioma cell proliferation, migration, and invasion while inducing apoptosis (Fig. 12a). This strategy demonstrates the promise of aptamer-modified tFNAs as targeted nanocarriers for glioma therapy.

Fig. 12.

a The preparation of GPC and its mechanism for inducing apoptosis in U87MG cells involve GPC targeting these cells by crossing the BBB. Reproduced form ref.[372] with permission from Elsevier, copyright 2019. b Precision-targeted gene therapy for pancreatic ductal adenocarcinoma: a novel tFNA-based delivery system incorporating CEBPA-saRNA and hTfR aptamer. Reproduced form ref.[316] with permission from BioMed Central, copyright 2024.

Pancreatic cancer

Pancreatic cancer is among the most lethal malignancies worldwide, with pancreatic ductal adenocarcinoma (PDAC) accounting for approximately 85 % of all cases[468]. CCAAT/enhancer binding protein α (CEBPα), a member of the leucine zipper transcription factor family, is prominently expressed under normal physiological conditions and functions as a tumor suppressor. Mutations or downregulation of the CEBPα gene have been identified as key contributors to tumor initiation and progression in various cancer types[[469], [470], [471]]. Moreover, elevated expression of CEBPα has been shown to inhibit tumor progression and reduce the metastatic potential of PDAC[472,473]. RNA activation (RNAa) is an epigenetic regulatory mechanism in which gene expression is upregulated by a novel class of small RNAs known as saRNAs. These saRNAs can be engineered with tail tags to specifically target promoter regions of tumor suppressor genes, thereby inducing their transcriptional activation[[474], [475], [476], [477]]. Thus, the process inhibits cancer cell proliferation, invasion, and migration. Notably, saRNAs designed to target CEBPα have previously been shown to enhance CEBPα expression along with its downstream effector, cyclin-dependent kinase inhibitor 1 (P21), thereby inducing anti-tumor responses and reducing burden in a mouse PDAC model[473,478]. Wang et al.[316] designed a tFNA-based gene therapy agent, termed aptFNAsa, which utilizes CEBPα-saRNA as the gene effector and a human transferrin receptor (hTfR) aptamer as the targeting ligand for PDAC treatment. This engineered construct demonstrated excellent structural stability, efficient cellular uptake, and intracellular release of saRNA assisted by endogenous RNase H. Consequently, it significantly activated the tumor suppressor gene CEBPα and its downstream effector P21, resulting in marked inhibition of PDAC cell proliferation in vitro and substantial suppression of tumor growth in PDAC mouse models (Fig. 12b).

Lung cancer

Lung cancer is one of the most prevalent malignancies and remains the leading cause of cancer-related mortality worldwide. Several immunotherapeutic agents have been approved by the U.S. Food and Drug Administration (FDA) as first-line treatments for lung cancer, especially for patients with small cell lung cancer (SCLC)[479]. However, traditional delivery methods for immunosuppressive agents, such as intravenous or intraperitoneal administration, often fail to achieve sufficient tumor-specific accumulation and may result in off-target exposure, thereby reducing therapeutic efficacy and increasing the risk of adverse effects. Additionally, the presence of immunoglobulin G (IgG) in alveolar macrophages and pulmonary mucus can recognize and clear inhaled exogenous agents, further limiting their bioavailability[480,481]. Cytosine-phosphate-guanosine (CpG)-loaded TDNs (C@TDN) need to be internalized by immune cells to activate antigen-presenting cells via the Toll-like receptor 9 (TLR9) pathway, significantly promoting bone marrow-derived dendritic cells (BMDCs) maturation and inducing macrophage phagocytosis and M1 polarization more effectively than free CpG or TDN alone[[482], [483], [484]]. Building on this, Fan et al. [284] designed an inhalable nanoplatform (CP@TDN) co-delivering CpG and a PD-L1 aptamer, which not only activates innate immune responses but also blocks the PD-1/PD-L1 checkpoint to enhance T cell activity. CP@TDN activated pro-inflammatory immune responses, enhanced intratumoral accumulation, and in a lung melanoma metastasis model, markedly inhibited tumor growth and prolonged survival, demonstrating strong anti-tumor immunity (Fig. 13).

Fig. 13.

A diagram presents the design of an immunomodulatory transmucosal delivery system based on tFNA. This system is intended for the treatment of metastatic lung cancer and functions by triggering a potent antitumor immune response. Reproduced form ref.[284] with permission from Elsevier, copyright 2023.

Non-small cell lung cancer (NSCLC) accounts for about 80 % of all types of lung cancer and is the leading cause of cancer-related death worldwide[485]. PTX has significant efficacy against cancers such as lung, ovarian, and breast cancer[486]. Despite the clinical efficacy of PTX, its multidrug resistance limits its drug application[487]. Xie et al.[48] successfully synthesized TDNs and loaded PTX into these TDNs to construct a PTX/TDN drug delivery system. PTX/TDNs can significantly inhibit the proliferation of both multidrug resistant cells and wild-type cells due to the efficient internalization of PTX by the drug system. PTX/TDNs overcame resistance by downregulating mdr1 and P-gp expression, bypassing efflux pumps via endocytosis/exocytosis pathways, and enabling intracellular PTX accumulation. This led to microtubule bundle formation, mitotic arrest, and apoptosis, confirmed by altered caspase-3, Bax, and Bcl-2 expression. These findings highlight PTX/TDNs as a promising strategy to reverse multidrug resistance and enhance chemotherapy efficacy.

Comparative analysis of tFNA/TDN variants across disease contexts

To provide a more integrated understanding beyond individual studies, we compared the different tFNAs/TDNs reported across various disease fields (Table 1 and Table 2). Despite structural diversity, several common mechanisms emerge. First, almost all tFNA variants exhibit efficient cellular uptake, predominantly via caveolin- or clathrin-dependent endocytosis, often followed by endosomal trafficking, with nuclear localization signal (NLS) modification further enhancing nuclear delivery [4,61,63,154,204,224,226]. Second, many studies confirm an intrinsic anti-inflammatory and antioxidative effect of tFNAs, independent of payloads, primarily through modulation of NF-κB and MAPK pathways[143,186,372]. Third, tFNAs act as versatile protective and delivery platforms, improving the stability and intracellular availability of nucleic acid drugs (eg, RNAs and ASOs) and bioactive molecules (eg, flavonoids and antimicrobial peptides)[154,187,225,226,256,372]. Fourth, convergent signaling pathways including Wnt/β-catenin, PI3K/AKT, ERK, and Notch are repeatedly engaged across diverse applications[63,65,68,154,401].

Table 1.

Representative examples of tFNAs for disease therapy.

Name	Structural Composition	Molecular Target	Function Description	Disease Treatment Effect/Application	Ref.
PTX/TDNs	TDNs loaded with PTX	mdr1 gene/P-gp protein	Enables efficient intracellular PTX delivery, inhibits drug efflux pumps, downregulates mdr1/P-gp, and induces microtubule aggregation and apoptosis	Overcomes multidrug resistance in NSCLC and other tumors, improving antitumor efficacy	[48]
stFNA-miR	loaded with miR–2861	HDAC5 protein	Inhibits the expression of HDAC5 protein and enhances osteogenic differentiation	Treatment of bone defects and osteoporosis	[62]
TDN-miR-2-5p	loaded with miR-21-5p	AKT and ERK signaling pathways	Activates the AKT and ERK pathways and enhances the osteogenic differentiation ability of aged BMSCs	Treatment of bone defects and delay of cell senescence	[63]
MiR@TDNs	loaded with miR335-5p	DKK1 gene/Wnt pathway	Inhibits DKK1 expression, activates the Wnt pathway, and promotes bone formation and angiogenesis	Treatment of SAON of the femoral head	[68]
TDN-CLI	bound to clindamycin	Bacterial cell membrane	Enhances antibacterial ability and promotes osteogenic differentiation	Treatment of infected bone defects	[92]
PPS-tFNA	bound to pentosan polysulfate	Articular chondrocytes	Targeted delivery of PPS, inhibits cartilage proliferation, and promotes cartilage regeneration	Treatment of osteoarthritis and cartilage damage	[121]
DJ-1-saRNA-tFNA	bound to DJ-1-saRNA	DJ-1 protein	Increases DJ-1 expression and reduces oxidative stress	Treatment of diabetic retinopathy and macular degeneration	[154]
tFNA-miR-2	loaded with miR-22	TrkB-BDNFpathway	Activates the TrkB-BDNF and ERK pathways to protect retinal neurons	Treatment of retinal ischemia–reperfusion injury and glaucoma	[161]
tFNA-QUE	bound to quercetin	AKT/Nrf2/HO-1 pathway	Inhibits pathological angiogenesis and improves retinal function	Treatment of ischemic retinopathy	[187]
TDN/His-His5	bound to the antimicrobial peptide His-His 5	Base sequence	Enhance antifungal activity, promote ROS production and cell destruction	Treatment of fungal infections	[224]
ASOs-tFNA	bound to ASOs	Biofilm-related genes	Inhibits the formation of pathogenic biofilms	Treatment of bacterial infections and biofilm-related diseases	[225]
GL13K-tFNA	bound to the antimicrobial peptide GL13K	Bacterial cell membrane	Enhances the stability and activity of the antimicrobial peptide and inhibits bacterial infections	Treatment of Gram-negative and periodontal pathogen infections	[226]
Double-Layer DNA	ESDR with a double-layer DNA tetrahedron structure	HIV-related DNA target sequence	The target DNA initiates the ESDR reaction to form a dsDNA complex, which then hybridizes with surface-immobilized hairpin probes. The exposed hybridization sites in the DNA tetrahedron	An early detection platform for HIV-related DNA, with advantages of high stability and low cost	[257]
aptFNAsa	tFNA loaded with CEBPα-saRNA, conjugated with hTfR aptamer	CEBPα tumor suppressor/P21 pathway	Targeted delivery via hTfR aptamer, RNase H-mediated saRNA release, activates CEBPα expression, and inhibits PDAC cell proliferation	Inhibits PDAC tumor growth and progression in mouse models by restoring tumor suppressor gene function.	[316]
tFNA-ADKASO-AS1	Bound to ADKASO and linked to an A1-type	ADK	Crosses the BBB, targets A1-type astrocytes, inhibits ADK expression, increases adenosine levels in the brain, improves neural circuits, and reduces the frequency of epileptic seizures	Treatment of epilepsy and potential central nervous system diseases, with good targeting and biosafety	[384]
TDN-miR-22-3p	loaded with miR-22-3p	Inflammatory pathway factor	Cross the BBB, take up by target cells, regulate molecular expression, inhibit the release of inflammatory factors, reduce inflammatory response, and relieve depressive symptoms	Alleviate LPS-induced neuroinflammation and depressive symptoms, a novel miRNA delivery platform was constructed	[391]
FNA-miR-122	Functionalized miR-122	Gene associated with hepatocyte differentiation	Induce ADMSCs differentiation to hepatocyte like cells, construct functional liver-like spheres, improve liver function, and relieve liver injury and inflammation	Treatment of ALF, promote liver regeneration, reduce inflammation and apoptosis, improve stem cell engineering efficiency	[405]
ASO@Chol3-Td	Chol3-Td binding to TGF-β1 mRNA ASO	SR-B1 and LDLR	Interact with serum lipoprotein, preferentially taken up by hepatocytes, targeted deposited in liver, effectively downregulating TGF-β1 expression, alleviating liver fibrosis, and reducing serum AST and ALT levels	Treat liver fibrosis, reduce liver collagen deposition and liver enzyme levels, which could be comparable to GalNAc3-ASO	[410]
tFNA-siCcr2	loaded with CCR2	CCR2 gene	Targeting macrophages and hepatic sinusoidal endothelial cells, effectively inhibited CCR2 expression, reshaped the liver immune microenvironment, and inhibited the aggregation of pro-fibrotic cells	Improve liver fibrosis, significantly reduced the expression of inflammatory factors and extended the retention time of drugs in the liver during preventive and therapeutic intervention	[413]
Kim-TDF	bound to kidney TDF	Kim-1	Targeting Kim-1 to detect early AKI, with good photo stability and renal clearance efficiency, NIR imaging and urine analysis can improve the sensitivity of early diagnosis	Early diagnosis of AKI and enables significant differentiation between diseased and healthy tissues	[418]
tFNA-Typ	bound to the Chinese medicine component Typ	Mitochondrial related	Targeting renal tubules, inhibiting ROS generation, protecting mitochondrial function, and reducing ischemia–reperfusion induced apoptosis	Alleviate AKI related renal injury, improve renal function, and provide a new strategy for the treatment of mitochondrial-related diseases	[425]
TFG	combined with HIF-1α stabilizer FG-4592	HIF-1α regulatory pathway	Improve the uptake efficiency of HK-2 cells, relieve cisplatin-induced cytotoxicity and apoptosis, reduce renal tubular damage, and promote renal function recovery	As a drug delivery platform for the treatment of kidney diseases, cisplatin-induced AKI therapy provides a protective strategy for chemotherapy-related nephrotoxicity	[427]
sTd-p53 siRNA	bound to p53 siRNA	p53 mRNA	Low immunogenicity, optimized nanosize, promote specific endocytosis, target kidney effective delivery of siRNA, inhibit p53 expression, improve renal injury	Promise renal targeted siRNA delivery platform for the treatment of AKI and the promotion of renal function recovery	[430]
HApDC	Anti-HER2 aptamer-DM1 conjugate	HER2 protein	Targets HER2-positive tumor cells, releases drug in acidic tumor microenvironment and prolongs circulation time for enhanced tumor accumulation	Treats HER2-positive breast cancer with high efficacy and safety, outperforming free drugs and traditional liposomes	[445]
tFNA-TMZ	tFNA loaded with TMZ, functionalized with GS24 aptamer	BBB penetration/MGMT pathway	Enhances TMZ delivery across BBB, induces apoptosis/autophagy in glioblastoma cells, and downregulates MGMT to overcome drug resistance	Treats TMZ-sensitive and resistant GBM by improving drug brain penetration and antitumor efficacy	[460]

Table 2.

Comparative analysis of tFNA variants.

Type	Structure/Modification and Loading	Uptake/Targeting Mechanism	Mechanism of Action	Applications	Advantages	Limitations	Ref.
Standard tFNA	Four ssDNA strands self-assemble into structures with tunable edge lengths	Endocytosis; partial nuclear import facilitated by NLS	Antioxidant effects, modulation of Wnt/Notch/MAPK/NF-κB signaling pathways, promotion of cell proliferation and differentiation	Tissue repair, anti-inflammatory, neuroprotection	Good biocompatibility, multifunctional	Poor in vivo stability and limited loading capacity	[4,35,61]
miRNA/siRNA@tFNA (eg, miR-2861 and siCcr2)	miRNA/siRNA modifications at vertices/edges	Endocytosis	Regulating genes and pathways	Bone regeneration, skin anti-aging, fibrosis disease	Easy preparation, good biocompatibility, improved hybridization efficiency	Poor mechanical properties, RNA exposure is prone to off-targeting, and the bioswitchable delivery system (BDS) is costly	[68,314,405,413]
stFNA	Designed for triggered release	Endonuclease −triggered	Targeted release, regulating related genes or pathways	Bone regeneration	Precise genetic regulation	Environment-dependent trigger (needs in vivo validation)	[62]
Drug-loaded (eg, QUE, DOX and Baicalin)	Small molecule/ Chemotherapy drug coordination or embedding	Increase cell uptake and local concentration	Drug effect and tFNA synergy	Cancer, inflammatory disease and ischemic retinopathy	Improving pharmacokinetics, high biocompatibility, strong targeting and high stability	Release from serum/environmental effects and differential drug release	[187,272,374,509]
AMP complex (eg, GL13K and His-5)	AMP-tFNA conjugate	Enhance cell/bacterial membrane action	Enhance ROS induction and membrane damage	Antibacterial, potentially used to inhibit microorganisms and related infections	Improve AMP stability	The safety of the host cell has not been verified and further studies are needed	[224,226]
Lipid-modified type (eg, Chol3-Td)	Cholesterol modification	SR-B1 and LDLR	Promotes hepatic uptake	HF and ALF	Natural liver targeting	The accumulation varies across different tissues.	[410]
ASO@tFNA	Loaded with and functionalized by antisense oligonucleotides	Enhancement of ASO uptake efficiency	Genetic silencing inhibits disease pathways	HF, epilepsy and Candida albicans	Multiple targets	Toxicity and off-target effects were not evaluated	[224,384,410]
Aptamer modifications (eg, GS24 and AS1411)	Add aptamers to the surface of tFNA	Receptor-mediated binding	Improved tissue/cell specificity	BBB penetration and targeted drug delivery	Targeting specificity and reduced off-target side effects	Restriction of receptor heterogeneity	[460]
Spherical nucleic acids (SNA) (eg, tDF-SNAs)	A SNA architecture constructed on tFNA surface to load siRNA	The SNA shell promotes efficient cellular uptake; the tFNA structure optimizes the spatial interface	Enhanced siRNA effect	siRNA gene silencing therapy	High loading capacity	Complex construction steps	[290]

Beyond these shared features, each variant demonstrates disease- or context-specific mechanisms. For instance, bioswitchable stFNA-miR enable microenvironment-triggered release of miR-2861 to enhance osteogenic differentiation via HDAC5/Runx2 signaling, highlighting their value in bone regeneration[62]. Chol3-Td exploits lipoprotein transport pathways for intrinsic liver targeting, making them promising for HF therapy[410]. Ligand-functionalized TDNs achieve tumor-specific or BBB-penetrating delivery, thereby expanding therapeutic windows in oncology and neurology[272]. The AMP-loaded complex GL13K-tFNA enhances antibacterial efficacy by improving peptide stability and promoting bacterial membrane disruption with subsequent cytoplasmic leakage, thereby exhibiting stronger bactericidal activity against pathogens[226]. Collectively, this comparative analysis illustrates that while tFNAs possess universality as protective, biocompatible carriers with inherent bioactivity, their performance and limitations are closely tied to structural modifications and disease context.

Conclusion and perspective

This review provides a comprehensive overview of tFNA self-assembly principles, structural optimization approaches, and multifunctional modification strategies. It also summarizes recent research advances across diverse medical domains, such as cancer therapy, bone and cartilage tissue engineering, treatment of infectious and neurological disorders, liver and kidney repair, and skin and soft tissue regeneration. Special attention is placed on the unique advantages and promising potential of tFNAs in targeted drug delivery and therapeutic applications. Nevertheless, despite these advances, multiple challenges remain before tFNA-based systems can be successfully translated into clinical practice.

Limited long-term structural stability

A persistent obstacle is the limited stability of tFNAs in vivo. While their tetrahedral structure confers improved mechanical strength and partial nuclease resistance compared to linear DNA, they remain vulnerable to sustained enzymatic degradation in complex physiological environments[52,269]. This compromises their drug delivery and therapeutic reliability. Recent studies suggest that backbone modifications such as phosphorothioation, together with PEGylation, can enhance nuclease resistance and prolong circulation time[[488], [489], [490]]. However, the long-term biocompatibility and safety of such chemical modifications require systematic preclinical validation.

Limited drug loading capacity

The intrinsic spatial constraints of tFNAs limit their drug loading efficiency and controlled release capability. Excessive surface modification may also hinder target interactions and impair bioactivity. Clinically, high drug-to-carrier ratios are essential to minimize off-target effects and systemic burden[491]. Simpler structures with fewer modifications may be more suitable for clinical translation, as they reduce complexity, costs, and nonspecific interactions. Innovative approaches, such as exploiting like-charge attraction to enhance membrane penetration may inspire new strategies for improving drug loading while maintaining delivery precision[492].

Biocompatibility, biosafety and ethical concerns

Although tFNAs demonstrate excellent cell permeability and therapeutic potential, their long-term toxicity and immunogenicity remain poorly characterized. Data on cumulative toxicity, repeated dosing, and organ-specific accumulation are limited, underscoring the need for rigorous safety assessments before clinical application. Additionally, ethical issues warrant careful consideration. The environmental risks of large-scale production and disposal of DNA nanomaterials should be monitored. High costs may also restrict access to tFNA-based personalized therapies, raising concerns about fairness and equity. Transparent informed consent and robust regulatory oversight are therefore essential to ensure that the development of tFNA-based therapies is both safe and socially responsible.

Limited adaptability to dynamic microenvironments

Current tFNA-based drug delivery strategies predominantly rely on static physical or chemical modifications. However, pathological microenvironments are dynamic, with variable pH values and enzyme activities that may impair efficacy. Designing stimuli-responsive systems capable of environment-adaptive release (eg, pH- or enzyme-triggered) represents a promising direction to improve therapeutic precision and expand clinical applicability[[493], [494], [495]].

Challenges in multifunctional theranostic platforms

tFNAs offer unique opportunities for integrated diagnosis and therapy. Their combination with CRISPR-Cas gene editing, photothermal or photodynamic agents, and other modalities could enable multifunctional theranostic platforms with high precision. Yet, significant barriers remain, including the modular assembly of multiple components, temporal control of release, and synergistic coordination among modalities[336,496]. Future studies should aim to elucidate the interaction mechanisms among different functional modules and clarify the synergistic effects between various therapeutic modalities. This necessitates the development of more robust modular design principles and optimization strategies to ensure that tFNA-based multifunctional theranostic platforms function with greater efficiency, precision, and safety under actual clinical conditions, thereby accelerating their translation from laboratory research to clinical application.

Barriers to large-scale production

Large-scale clinical application of tFNAs is hampered by the high cost and quality control issues associated with conventional enzymatic amplification and purification methods[190]. Recent advances in microfluidic chip technology enable rapid, reproducible, and scalable tFNA synthesis, while enzyme-free amplification strategies offer cost-effective alternatives[497,498]. Future research and development should prioritize the further integration and optimization of microfluidic chip technologies and enzyme-free amplification methods to establish a standardized, economically viable, and quality-consistent production framework. These advancements are expected to improve the controllability and scalability of large-scale tFNA manufacturing. The successful implementation of such advanced fabrication strategies will be essential for enabling industrial-scale production, accelerating clinical translation, and ultimately extending the therapeutic benefits of tFNA-based platforms to a broader patient population.

Opportunities in interdisciplinary integration

The profound interdisciplinary integration and innovative application of related technologies are also expected to open broader avenues for the future development of tFNA-based systems. Emerging fields such as synthetic biology and artificial intelligence are opening new avenues for advancing tFNA structural design and functional optimization. For example, unnatural nucleic acid backbones (eg, phosphorothioate-modified tFNAs) enhance nuclease resistance and stability, while AI-based modeling can predict folding pathways and optimize drug-loading strategies[499,500]. However, the safety and long-term biosafety of such innovations require thorough evaluation.

Methodological limitations

Although the above findings demonstrate the versatility of tFNAs across disease models, several methodological limitations warrant attention. First, differences between in vitro and in vivo outcomes remain underexplored. While many in vitro studies show efficient uptake and robust pathway activation, in vivo efficacy can be compromised by serum protein adsorption, nuclease degradation, and immune clearance[501]. Besides, the applicability of animal models is limited, as metabolic and immunological differences, which may reduce translational relevance from rodents to humans[502,503]. More importantly, lack of standardization in tetrahedron size, edge length, assembly methods, and functionalization hampers reproducibility and inter-study comparisons. Finally, quantitative evaluation of drug loading and pharmacokinetics is insufficient[504].

To address these issues, future work should incorporate comprehensive pharmacokinetics, toxicology, and human-relevant models such as organoids or non-human primates to bridge preclinical and clinical research. Regulatory policies, manufacturing costs, and quality control frameworks should also be addressed, drawing lessons from other nanomedicines that have advanced into clinical use.

Clinical Translation: Experiences and Lessons Learned

To date, several therapeutic nanomedicine products have received regulatory approval from the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA)[502,503]. The clinical authorization of these nanomedicines has been primarily attributed to their superior therapeutic efficacy and improved safety profiles compared with conventional treatment modalities[505]. Representative success stories include the anticancer formulations Doxil®/Caelyx® and Abraxane®, which are widely regarded as landmark cases in the field[502,506]. Unlike traditional chemotherapeutics that exert non-selective cytotoxicity toward both malignant and healthy cells, these nanomedicine platforms achieve targeted delivery to tumor tissues and a significantly higher dose, a feature that has been central to their clinical and commercial success[505]. PEGylation has played a pivotal role in the clinical success of nanomedicines by markedly prolonging systemic circulation and mitigating immune clearance[507]. This type of modification forms the basis of many approved nanomedicines[508]. Such examples underscore the potential of nanomedicine to deliver significant clinical benefit beyond standard therapies. However, despite these achievements, the majority of nanomedicine candidates have failed to meet therapeutic expectations in clinical trials, and consequently, few have successfully advanced to commercialization.

The development of nanomedicines requires sustained efforts in clinical translation and commercialization. However, the majority of nanomedicine candidates have failed to demonstrate superior therapeutic efficacy and improved safety, often exhibiting limited targeting capability, which hinders their successful clinical translation and market entry[506]. In many cases, formulations that achieved remarkable efficacy in animal models failed to replicate these outcomes in clinical trials. The principal factors underlying such translational failures include difficulties in establishing robust and cost effective large scale manufacturing, the absence of comprehensive regulatory guidelines, insufficient characterization methodologies, safety concerns, inadequate stability in complex biological environments, and a limited understanding of patient-specific disease heterogeneity[[501], [502], [503], [504], [506]]. Consequently, a thorough understanding of the multidimensional attributes of approved nanomedicines, including their fundamental principles, physicochemical characterization, clinical performance, and regulatory requirements, is critical for precisely guiding the research and development process and enhancing the likelihood of successful translation.

Future directions: precision and personalized medicine

Looking ahead, tFNAs hold great promise for advancing precision and personalized medicine. Their integration with high-sensitivity sensing and therapeutic technologies could yield multifunctional platforms for both diagnosis and treatment. Personalized tFNA formulations tailored to individual patient profiles may enhance therapeutic precision and efficacy. Furthermore, synergy with existing modalities such as immunotherapy and gene editing could broaden their therapeutic impact. Continued efforts should focus on optimizing fabrication, drug loading, safety, and cost-effectiveness, while ensuring equitable access. With sustained interdisciplinary collaboration and responsible innovation, tFNAs are poised to become an important tool in clinical medicine, contributing meaningfully to human health worldwide.

CRediT authorship contribution statement

Zhenhong He: Investigation, Writing – original draft, Writing – review & editing. Yi Liu: Investigation, Writing – original draft, Writing – review & editing. Yihuang Chen: Investigation, Writing – original draft, Writing – review & editing. Yuanqun Zhang: Investigation, Writing – review & editing. Yuyao Zhang: Investigation. Zhihong Chen: Investigation. Dingsu Bao: Supervision, Project administration, Funding acquisition, Writing – review & editing. Weihu Yang: Conceptualization, Supervision, Project administration, Writing – review & editing. Huan Liu: Conceptualization, Supervision, Project administration, Funding acquisition, Writing – review & editing.

Funding

This work was supported by Key research and development projects of Sichuan Science and Technology Plan Project (2024YFFK0135), Fujian Provincial Natural Science Foundation of China (2024 J011450) and the China Postdoctoral Science Foundation (2023 M732927).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Zhenhong He is a master’s student of Southwest Medical University, Luzhou 646000, Sichuan, China. He is mainly working with his supervisor on tendon-bone healing and sports medicine. hzhhzhhzh7@163.com

Yi Liu is an undergraduate student majoring in clinical medicine in Southwest Medical University, Luzhou 646000,Sichuan, China. 18981636587@163.com

Yihuang Chen is an undergraduate student majoring in clinical medicine, School of Basic Medical Sciences,Putian 351100, Fujian,China. 18876343873@163.com

Yuanqun Zhang is an undergraduate student majoring in clinical medicine, School of Basic Medical Sciences,Putian 351100, Fujian,China. 15059617484@163.com

Yuyao Zhang is a master's student of Southwest Medical University, Luzhou 646000, Sichuan, China. She is mainly working with her supervisor on tissue engineering and Lumbar degenerative disease. 937858379@qq.com

Zhihong Chen is an undergraduate student majoring in clinical medicine, School of Basic Medical Sciences,Putian 351100, Fujian,China. 15159819075@163.com

Dingsu Bao is an associate chief orthopedic-joint surgeon and associate professor at the orthopedics department of the Affiliated Hospital of Traditional Chinese Medicine of Southwest Medical University, Luzhou 646000, Sichuan, China. He is currently focusing research on tendon-bone healing and scaffolds for tissue engineering and has published lots of scientific research articles associated with this field. 332639420@qq.com

Weihu Yang graduated in 2009 from Sichuan University with a Ph.D. in Biomedical Engineering. Principal investigator of two projects funded by the National Natural Science Foundation of China and two provincial-level projects including the Chongqing Natural Science Foundation. Has published over 30 papers in SCI journals such as Chemical Engineering Journal, Biomaterials Science, and Acta Biomaterialia, with more than 200 cumulative citations.

Huan Liu holds a Ph.D. in Chongqing University and a post-doctoral position in Southern Medical University. She is currently working mainly on bone tissue engineering, stem cells, biological 3D printing, and other related research at the Affiliated Hospital of Traditional Chinese Medicine of Southwest Medical University, Luzhou 646000, Sichuan, China. She has published several scientific research articles related to the orthopedics. 20016040@163.com