A DNA tetrahedron-based ferroptosis-suppressing nanoparticle: superior delivery of curcumin and alleviation of diabetic osteoporosis

The study addresses diabetic osteoporosis (DOP), a systemic metabolic bone disorder characterized by reduced bone mass, disrupted microarchitecture, and increased fracture risk in diabetes. Emerging evidence implicates ferroptosis—an iron-dependent, lipid peroxidation-driven form of regulated cell death—in diabetes-induced skeletal fragility, where elevated ferroptosis suppresses osteogenic transcription factors (e.g., RUNX2, OSX) and disrupts bone mesenchymal stem cell (BMSC) differentiation. GPX4 is a key enzymatic defense against ferroptosis and is diminished in the diabetic milieu, while NRF2 can inhibit ferroptosis by upregulating GPX4. Curcumin activates the KEAP1/NRF2 axis and has anti-inflammatory and antioxidant activities, but its clinical translation is limited by poor stability and bioavailability. Tetrahedral framework nucleic acids (tFNA) are biocompatible DNA nanostructures capable of efficient cellular uptake and drug delivery. The authors hypothesized that a tFNA-based nanoparticle delivering curcumin (tFNA-Cur) would activate the NRF2/GPX4 pathway, suppress ferroptosis, restore BMSC osteogenic potential in a diabetic microenvironment, and alleviate DOP in vivo.

Prior work shows patients with diabetes have a substantially higher fracture risk and mortality following fractures. Recent studies propose ferroptosis as a central mechanism in diabetes-related skeletal changes; ferroptosis involves iron accumulation and lipid peroxidation, with GPX4 as a critical defense component, whose levels decline in diabetes, promoting ROS and lipid hydroperoxide accumulation. Pharmacologic ferroptosis inhibition (e.g., ferrostatin-1) reduces bone loss in DOP models, supporting ferroptosis as a therapeutic target. NRF2 activation upregulates GPX4 and suppresses ferroptosis. Curcumin is known to activate the KEAP1/NRF2 pathway and exhibits antioxidant, anti-inflammatory, hypolipidemic, and anti-osteoclastogenic effects, but suffers from poor pharmacokinetics and stability. tFNA nanostructures can deliver nucleic acids, peptides, and natural products, traverse cell membranes, and have favorable biosafety. tFNA has been reported to ameliorate insulin resistance and lower glucose via PI3K/Akt signaling, suggesting utility in metabolic disease contexts. These lines of evidence motivate a tFNA-based curcumin delivery system targeting ferroptosis in DOP.

Nanoparticle synthesis and characterization: tFNA was self-assembled from four equimolar ssDNAs in TM buffer (pH 8.0) by heating to 95 °C (10 min) and cooling to 4 °C (20 min). Curcumin was loaded by incubating tFNA (200 nmol/L) with varying curcumin concentrations (5–40 µmol/L) for 3 h. Formation of tFNA and tFNA-Cur was confirmed by PAGE. Particle size and zeta potential were measured via DLS (Malvern Nano ZS). Morphology and size were observed by TEM (Zeiss Libra200) and AFM (Oxford Cypher VRS). UV–Vis spectra were collected (Mettler Toledo UV5 Nano). Drug loading and release: Encapsulation efficiency was determined at curcumin 10–160 µmol/L with fixed tFNA (200 nmol/L). In vitro release was evaluated using dialysis (30 kD) in PBS (pH 7.4) at 37 °C with agitation; curcumin concentration in the outer phase was monitored by absorbance. Stability: Serum stability was assessed by incubating tFNA and tFNA-Cur in 2% or 10% serum and analyzing integrity by gel electrophoresis over 0–12 h; curcumin stability in TM buffer vs tFNA-Cur was quantified by residual amount over time. In vivo stability of Cy5-labeled constructs was monitored up to 24 h. Cell studies: BMSCs were isolated from 4-week-old male C57 mice and cultured. Cell viability after exposure to tFNA (100–500 nmol/L), curcumin (5–25 µmol/L), and tFNA-Cur (various ratios) for 12 h was assessed by CCK-8. AGEs (25–250 µg/mL, 24 h) were used to model a diabetic microenvironment; 150 µg/mL was selected for subsequent assays based on viability and osteogenic inhibition. Cellular uptake of curcumin vs tFNA-Cur was imaged by confocal microscopy at 6 and 12 h. Ferroptosis-related assays included ROS detection (Hoechst + DCFH-DA), mitochondrial membrane potential (Rhodamine 123), and Fe2+ levels (FerroOrange), imaged by confocal microscopy. Mitochondrial morphology indicative of ferroptosis was examined by TEM. Osteogenesis assays: BMSCs pretreated with tFNA, curcumin, or tFNA-Cur (12 h) were exposed to AGEs (24 h) and then induced for osteogenic differentiation (dexamethasone, ascorbic acid, β-glycerophosphate) under continued AGEs exposure. Alkaline phosphatase (ALP) staining (day 7) and Alizarin Red S mineralization (day 14) were performed and quantified. Gene and protein analyses: RT-qPCR assessed Alp, Runx2, Osx, Opn, Gpx4, Acsl4, and Nrf2 (β-Actin control; 2−ΔΔCt). Western blot measured ALP, RUNX2, OSX, OPN, GPX4, ACSL4, NRF2, and KEAP1. Immunofluorescence (IF) visualized ALP, RUNX2, OSX, GPX4, ACSL4, NRF2; cytoskeleton stained with phalloidin; nuclei with DAPI. Molecular docking: NRF2 protein structure was predicted by AlphaFold, and docking of curcumin to NRF2 was performed using AutoDock Vina 1.2.0; interactions analyzed and visualized in PyMOL. Animal model and treatments: Type 2 diabetes was induced in male C57BL/6J mice via HFD (60% kcal fat, 4 weeks) plus low-dose STZ (35 mg/kg i.p. for 7 days). Diabetic status was confirmed by fasting glucose ≥11.1 mmol/L and symptoms. For disease characterization, body weight and glucose were monitored biweekly; IPGTT and ITT were performed; AGEs in serum and bone were measured by ELISA; micro-CT analyzed bone at 2 mm below epiphysis; histology (H&E, Masson), and TUNEL assessed bone microstructure and cell death. For therapy, groups (Control, DOP, DOP+tFNA, DOP+curcumin, DOP+tFNA-Cur; n=6) received i.p. injections three times weekly for 8 weeks: tFNA (1 µmol/L, 200 µL), curcumin (40 µmol/L, 200 µL), or tFNA-Cur (tFNA 1 µmol/L + curcumin 40 µmol/L, 200 µL); controls received saline. Outcomes included plasma glucose, AGEs, pancreatic histology, micro-CT bone parameters (BMD, BV/TV, Tb.N, Tb.Th, Tb.Sp, SMI), histology, and IF for ALP, GPX4, NRF2 in bone. Statistics: Student’s t-test or one-/two-way ANOVA with Sidak’s multiple comparisons; mean ± SD; P<0.05 significant.

• Successful synthesis and characterization of tFNA-curcumin nanoparticles (tFNA-Cur): tFNA size 12.03 ± 1.499 nm; tFNA-Cur 40.23 ± 6.41 nm; zeta potentials 4.93 ± 2.74 mV (tFNA) and −13.5 ± 2.28 mV (tFNA-Cur). TEM/AFM confirmed 3D structure and 40–50 nm size.
• High drug loading and sustained release: At fixed tFNA 200 nmol/L, encapsulation efficiencies at curcumin 10, 20, and 40 µmol/L were 91.825%, 84.288%, and 82.226%, respectively; sustained release maintained ~40% cumulative release at 48 h.
• Enhanced stability and uptake: Free curcumin degraded rapidly in buffer, with only 27.459% remaining at 12 h vs 74.566% for tFNA-Cur. tFNA-Cur exhibited superior serum stability (2% and 10% FBS) and remained detectable in vivo up to 24 h. Cellular uptake of tFNA-Cur by BMSCs was higher than free curcumin at 6 and 12 h.
• DOP model validation: HFD+STZ mice showed rising plasma glucose and weight loss, impaired IPGTT/ITT, elevated AGEs in serum and bone, and osteopenic trabecular changes with decreased BV/TV, Tb.N, Tb.Th and increased Tb.Sp and SMI; BMD not significantly different. Increased TUNEL-positive cells were observed in bone.
• In vitro osteogenesis rescue: AGEs impaired BMSC osteogenesis (reduced ALP activity, mineralization, and Alp, Runx2, Osx, Opn expression). Pretreatment with tFNA-Cur significantly restored osteogenic markers at mRNA and protein levels and increased ALP, RUNX2, and OSX IF signals, outperforming tFNA or curcumin alone.
• Ferroptosis inhibition via NRF2/GPX4: AGEs increased ROS and Fe2+ (FerroOrange), and reduced mitochondrial membrane potential with ferroptotic mitochondrial morphology. tFNA-Cur markedly reduced ROS and Fe2+, preserved mitochondrial membrane potential and morphology. GPX4 and NRF2 expression were upregulated (WB, IF, RT-qPCR), while ACSL4 showed opposite trends consistent with reduced lipid peroxidation. Docking supported curcumin–NRF2 binding (hydrogen bond with NRF2 Asp408; binding energy −7.1 kcal/mol).
• In vivo efficacy and safety: In DOP mice, tFNA-Cur reduced plasma glucose and AGEs, preserved pancreatic islet morphology, restored trabecular structure, improved micro-CT parameters (increased BV/TV, Tb.N, Tb.Th; decreased Tb.Sp and SMI), and elevated ALP, GPX4, and NRF2 IF in bone. No detectable toxicity was observed in major organs (lung, liver, kidney).

The findings support the central hypothesis that targeting ferroptosis can mitigate diabetic bone fragility. tFNA-Cur activated the NRF2/GPX4 antioxidant axis to suppress ferroptosis, thereby lowering ROS and iron-dependent lipid peroxidation, protecting mitochondrial function, and restoring the osteogenic capacity of BMSCs in an AGEs-rich diabetic microenvironment. In vivo, these molecular and cellular effects translated into improved trabecular integrity and bone formation, despite the absence of significant BMD changes typical of type 2 DOP within the study timeframe. The data underscore AGEs accumulation as a key driver of oxidative stress and ferroptotic signaling in diabetic bone. By using tFNA to overcome curcumin’s poor solubility, stability, and bioavailability, the study demonstrates a synergistic nanodelivery approach that enhances therapeutic efficacy at lower doses with favorable safety. Overall, suppressing ferroptosis via NRF2/GPX4 activation emerges as a viable therapeutic strategy for DOP and potentially other ferroptosis-related skeletal and metabolic diseases.

This work introduces a DNA tetrahedron-based ferroptosis-suppressing nanoparticle (tFNA-Cur) that efficiently delivers curcumin to bone marrow, enhances its stability and bioavailability, activates the NRF2/GPX4 pathway, inhibits ferroptosis, and restores BMSC osteogenic differentiation in diabetic conditions. In a type 2 DOP mouse model, tFNA-Cur reduced hyperglycemia and AGEs, improved trabecular microarchitecture, and increased osteogenic and antioxidant markers without evident toxicity. The study provides mechanistic and preclinical evidence for ferroptosis-targeted nanotherapy in DOP and highlights DNA nanostructures as versatile carriers for natural products. Future research should optimize serum stability, control and quantify natural product loading on DNA frameworks, and incorporate bone- and BMSC-targeting ligands or biomimetic coatings to further enhance specificity and translational potential.

• Serum stability, while improved, still requires further enhancement to maximize in vivo absorption and effectiveness.
• Precise and controllable quantification of natural product loading on DNA frameworks remains challenging and limits clinical translation.
• In vivo targeting specificity to BMSCs needs improvement; proposed strategies include adding bone-targeting peptide SDSSD via click chemistry and encapsulating the nanoparticle with BMSC membrane for enhanced targeting.