Aminoguanidine-assembled functional DNA tetrahedron alleviates acute lung injury by targeting the cGAS-STING signaling pathway

Abstract

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) cause severe lung inflammation and damage, compromising respiratory function. Nanomedicine offers hope in controlling this inflammation. DNA nanostructures, as drug carriers, stand out due to their non-toxic, programmable, and precisely controllable traits. However, they face challenges in physiological stability and functionalization as carriers. Here, we introduce TET^AG, DNA tetrahedra assembled and functionalized with aminoguanidine without magnesium. Aminoguanidine not only aids tetrahedron assembly but also inhibits the cyclic GMP-AMP synthase and stimulator of interferon (cGAS-STING) pathway, a key inflammatory signal. By integrating STING siRNA into TET^AG (TET^AG-STING), we achieve synergistic anti-inflammatory effects in ALI/ARDS. Our results show that TET^AG offers improved stability and cellular uptake. Importantly, TET^AG-STING exhibits potent anti-inflammatory activity in vitro and in a Lipopolysaccharides-induced mouse model following intratracheal administration. This significantly improves the lung injury scores and pulmonary functions. These findings underscore aminoguanidine's therapeutic potential in ALI/ARDS and highlight the promise of DNA nanostructures in medical applications.

Graphical abstract

A DNA-based nanomedicine is developed for dual therapy against ALI/ARDS, integrating small interfering RNA (siRNA) and aminoguanidine. This nanomedicine features a DNA tetrahedron assembled with aminoguanidine and STING-targeting siRNAs. Both components exhibit anti-inflammatory activity in vitro and in vivo by suppressing the cGAS-STING pathway, demonstrating a significant synergistic effect.

1. Introduction

Acute lung injury (ALI) is characterized by the sudden onset of clinically significant hypoxemia and diffuse pulmonary infiltrates [1,2]. As the condition advances, it can evolve into the more severe acute respiratory distress syndrome (ARDS) with high mortality rates [3]. Uncontrolled inflammation and subsequent severe lung injury [1,2,4,5] are the key features of ALI/ARDS, for which no clinically available drugs currently exist. Nanomedicine-based combination therapy represents one of the most promising strategies for combating ALI/ARDS. For instance, Chen et al. reported an inhalable, peptide-based nanoparticle that specifically targets macrophages [6]. Such nanomedicines effectively suppressed the lung inflammation and accelerated lung regeneration in acute lung injury mouse models. In another study, poly(lactic-co-glycolic) acid (PLGA) microparticles wrapped with macrophage apoptotic body membranes were prepared with mitochondria-targeting moieties and stimulating factors for the resolution of lung injury and inflammation [7]. Despite numerous reported organic and inorganic drug delivery vehicles and drug candidates [[8], [9], [10], [11]], safe and effective nanomedicines are still highly desirable and urgently need for the treatment of ALI/ARDS.

Designer DNA nanostructures represent a novel category of nanostructured materials with applications spanning multiple research fields [[12], [13], [14], [15], [16], [17], [18]]. They exhibit key advantages, such as inherent programmability, sequence specificity, design flexibility, and excellent biocompatibility, which have led to their extensive application in both scientific research and practical uses in recent years [[19], [20], [21], [22], [23]]. Particularly, they have been successfully utilized as drug delivery vehicles for nucleic acid drugs such as antisense oligonucleotides, siRNA, and microRNA mimics or inhibitors [[24], [25], [26], [27]]. Although the presence of DNA nanostructures can protect the nucleic acid cargo, possibly due to steric effects, the vulnerability of DNA in physiological settings still limits their delivery efficiency, especially when DNA nanomedicines are administered via the bloodstream. Additionally, aside from nucleic acid drugs, only a few small molecular drugs, such as doxorubicin and porphyrin, can be incorporated into duplex DNA, which severely limits the capacity and functionality of DNA as drug carriers. Recently, the magnesium-free assembly of DNA with cationic guest molecules of diverse functionalities has emerged as an excellent approach to assemble and functionalize DNA nanostructures. The Simmel group reported that polyamines can mediate the self-assembly of DNA origami nanostructures without the need for magnesium ions [28]. Notably, the introduction of polyamines onto DNA nanostructures enabled successful electrotransfection into mammalian cells. Our group also found that cationic amino acids, such as arginine and lysine, can mediate the assembly of various DNA nanostructures in a magnesium-free manner [29]. The obtained arginine/DNA nanostructures exhibited distinct interaction behaviors with cells compared to magnesium-assembled DNA. Additionally, quantum dots and anticancer drugs, such as metformin, have also been demonstrated to be effective in assembling and functionalizing DNA nanostructures [30]. These studies collectively suggest that the magnesium-free assembly of DNA with cationic guest species is feasible and exhibits interesting properties arising from the guest species.

Aminoguanidine is an organic compound consisting of a guanidine group with an amino substituent, and it carries a positive charge in physiological environments. This compound is utilized in drug development and is being explored as a potential therapeutic agent for diseases such as diabetes and neurodegenerative disorders [31,32]. Several studies have reported that aminoguanidine inhibits inducible nitric oxide synthase (iNOS), which contributes to its anti-inflammatory effects [33,34]. Nevertheless, the precise mechanisms of its action are still not fully understood. Cyclic GMP-AMP synthase (cGAS) is a DNA sensor that transmits signals to the stimulator of interferon genes (STING) through cyclic GMP-AMP (cGAMP), initiating a series of inflammatory responses in downstream pathways [35,36]. The cGAS-STING signaling pathway is crucial in the development of various respiratory diseases, including pulmonary infections, lung cancer, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, bronchial asthma, and acute lung injury [[37], [38], [39], [40], [41], [42]]. Recently, Xia et al. reported that overexpression of iNOS leads to excessive NO production, which induces nitrative stress, mitochondrial damage, and the release of mitochondrial DNA (mtDNA) [43]. As iNOS is a critical regulator of mtDNA release, we reasoned that inhibiting iNOS expression with aminoguanidine to block excessive nitric oxide (NO) production could reduce cytoplasmic mtDNA levels, thereby suppressing the cGAS-STING pathway and exerting anti-inflammatory effects. In addition, the positively charged aminoguanidine might be able to effectively induce DNA assembly into defined nanostructures. The introduction of aminoguanidine into DNA nanostructures may also enhance the stability and biocompatibility of DNA nanostructures by altering the surface properties of DNA. However, limitations such as the binding affinity and potential side effects of aminoguanidine may also exist under certain conditions and remain to be addressed.

In this study, we propose that aminoguanidine, bearing a positive charge, can help neutralize the negative charge of DNA and consequently mediate the folding of DNA into well-defined nanostructures such as tetrahedra. Aminoguanidine not only serves as the mediator for assembling the DNA tetrahedra but also functions as a potential therapeutic agent. Additionally, we decorated STING siRNA onto the aminoguanidine-assembled DNA tetrahedra (TET^AG-STING) to create a dual therapy nanoplatform for the treatment of ALI/ARDS (Scheme 1). Our findings revealed that TET^AG-STING exhibited a more pronounced inhibitory effect on inflammation through the cGAS-STING pathway. Moreover, we evaluated the therapeutic efficacy of TET^AG-STING, as well as its distribution after injection into the trachea in an ALI mouse model, further confirming its superior potential for both therapy and inflammation inhibition. These findings contribute to the growing body of knowledge on inflammation inhibition and may pave the way for future research and clinical applications of DNA.

Scheme 1.

Design and assembly of TET^AG-STING with a synergistic anti-inflammatory effect for the treatment of ALI/ARDS.

2. Materials and methods

2.1. Materials and reagents

DNA sequences and Cy5-tagged siRNA were purchased from Sangon Biological Engineering (Shanghai, China). The entire DNA and RNA sequences are listed in Supporting Information. Aminoguanidine Hydrochloride was purchased from Aladdin (Shanghai, China). Lipopolysaccharides (LPS) were purchased from Sigma (McLean, USA). CCK8 assay was purchased from Baoguang Biological Engineering (Chongqing, China). CellTiter 96 AQueous One Solution Cell Proliferation Assay was bought from Promega (Wisconsin, USA). Transfection Reagents were purchased from Zeta Life (Columbia, USA). TRIzol Reagent was purchased from Thermo Fisher (USA). Transcriptor First Strand cDNA Synthesis Kit and FastStart Essential DNA Green Master Mix were purchased from Roche (Basel, Switzerland). The Mouse Precoated ELISA Kit was provided by Dakewe Biotech (Shenzhen, China). Dulbecco's modified Eagle medium was acquired from Gibco (USA). FBS was purchased from Hyclone (USA). T-PER™ Tissue Protein Extraction Reagent was purchased from Thermo Fisher (USA). Polyvinylidene Fluoride (PVDF) was purchased from GVS (Suzhou, China). Skimmed Milk Powder was provided by Yuanye (Shanghai, China). Primary antibodies against iNOS, cGAS, STING, β-actin and peroxidase-conjugated goat anti-rat IgG secondary antibody were provided by Cell Signaling Technology (Boston, USA). Flow cytometry antibodies: CD11b and ly6G were purchased from Biolegend (California, USA).

2.2. Synthesis and characterization of TET^AG-STING

Component DNA strands (in a 1:1:1:1 ratio) and aminoguanidine were mixed in ultrapure water at a final aminoguanidine conxcentration of 50 mM. TET^AG were formed by annealing the mixed solution as follows: 95 °C for 5 min, 65 °C for 30 min, 50 °C for 30 min, 37 °C for 30 min, and 22 °C for 30 min. Then STING siRNA was mixed into the TET^AG solution. The molar ratio of TET^AG and STING siRNA was 1:4. The complex was incubated at room temperature for about 2 h to allow the siRNA to bind sufficiently to the overhang of TET^AG. Then TET^AG-STING were added in 6 % PAGE and run on a Bio-Rad Electrophoresis System (USA) at room temperature for 60 min. For the cell experiments, TET^AG-STING was purified using a spin column with a molecular weight cutoff (MWCO) of 30 kDa to remove excess aminoguanidine from the solution. The yields of DNA tetrahedrons were semi-quantified by calculating the percentage of the target band intensity relative to the total DNA intensity in the sample lane using ImageJ software.

2.3. AFM imaging and DLS measurements

5 μL of diluted DNA sample solution was dropped on a newly prepared mica surface. After 10 min incubation, the sample was washed off using 50 μL ultrapure water and dried by nitrogen. DNA nanoparticles were then imaged by AFM in Scanaysist-Fluid + mode on a Multimode 8 AFM system (Bruker, USA) with silicon tips on nitride levers (T: 600 nm, L: 70 μm, W: 10 μm, f₀: 150 kHz, k: 0.7 N m⁻¹). The tip-surface interaction was minimized. For DLS measurements, the DNA tetrahedrons solution was diluted to about 100 nM measured by Dynamic Light Scattering (Malvern Instruments Laser Target Designator, UK).

2.4. Stability assay of DNA tetrahedrons

The thermal stability of TET^AG-STING was conducted by electrophoresis 6 % PAGE for 60 min. Briefly, TAE/Mg²⁺ buffer solution was preheated to 37 °C and loaded into both inner and outer chambers of the electrophoresis apparatus. The electrophoresis tank was then immersed in a thermostatically controlled water bath maintained at 37 °C to initiate electrophoresis. Throughout the process, a calibrated thermometer was positioned within the system to continuously monitor the temperature of electrophoresis buffers. Precise thermal regulation was implemented to consistently maintain the buffer temperature at 37 ± 0.5 °C in both chambers during the entire electrophoretic separation. For buffer exchange: The spin column was first washed with diethyl pyrocarbonate water at 3000 RPM centrifugation for about 20 min first. Then 100 μL DNA solution and 300 μL washing buffer aminoguanidine (50 μM) or the same volume of water were added. The solution was centrifuged at 3000 RPM for about 5 min until the remaining solution in the upper tube was approximately 50 μL. Repeated the process three times. Finally, the DNA tetrahedrons were electrophoresed by 6 % PAGE gel.

2.5. Cell culture

The RAW 264.7 cells were purchased from ProCell (Germany). They were cultured in a high-glucose DMEM medium, enriched with 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin. The incubation process took place at 37 °C in an environment with 95 % humidity and a 5 % CO₂ concentration. The cells were ultimately cultured within a 6-well plate at a density of 1 × 10⁶ per well. Subsequently, they were exposed to Tetrahedrons for a period of 24 h. Following this, 2 μg mL⁻¹ of LPS was introduced and the cells were further incubated for an additional 6 h.

2.6. CLSM imaging and flow cytometry

For confocal microscopy, RAW 264.7 cells were precultured in a Confocal Petri Dish at a density of 2 × 10⁴ per well. The cells were treated with DNA tetrahedrons (TET-1 was labeled with Cy5) for 1 h, 3 h, and 6 h. The cell received Cy5 concentration was 200 nM. The collected cells were washed and fixed with 4 % paraformaldehyde for 15 min. Finally, the cells were closed with a glass by antifade mounting medium with 4′6-diamidino-2-phenylindole (DAPI) band imaged by an inverted microscope (Nikon ECLIPSE TS100). For Flow cytometry, RAW 264.7 cells were seeded in 6-well plates at a density of 1 × 10⁶ per well and incubated with Cy5-labeled DNA tetrahedrons for 1 h, 3 h, and 6 h. The cell received Cy5 concentration was 200 nM. Then cells were digested by cell dissociation buffer and collected. Finally, cells were washed three times with PBS buffer and collected for flow cytometry (Backman, Gallios, USA).

2.7. CCK8 analysis

RAW 264.7 cells were cultured in 24-well plates overnight at a density of 1 × 10⁵ cell cm⁻² and incubated with different concentrations of aminoguanidine and DNA tetrahedrons separately for another 24 h. The concentration of TET^Mg, TET^AG, TET^Mg-STING, and TET^AG-STING was 25 nM. DNA mixture refers to component strands TET-1, TET-2, TET-3, TET-4, and siRNA without annealing. Finally, the cell viability was detected by CCK8 assay.

2.8. Quantitative real-time PCR

The total RNA was extracted from RAW 264.7 cells using Trizol reagent. The total RNA was reversed to cDNA by transcriptor First Strand cDNA Synthesis Kit. Then RT-qPCR was performed with FastStart Essential DNA Green Master on a Bio-Rad CFX 96 real-time PCR system. Every sample was duplicated, and GAPDH was used as an internal control. Relative mRNA expression was quantitated with the 2^-△△Ct method. The primer sequences are listed in the Supporting Information (Table S1). The cell received aminoguanidine and siRNA concentrations were 1.5 mM and 100 nM, respectively.

2.9. Western blotting analysis

Cell lysates from lung tissues and RAW 264.7 cells were extracted using RIPA buffer. Then, the protein sample was separated by 10 % or 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with 5 % skimmed milk for 2 h, followed by overnight incubation at 4 °C with the following primary antibodies: anti-iNOS, anti-cGAS, anti-STING, and anti-β-actin. After washing three times with TBST, the membrane was incubated with secondary antibodies for 2 h. Protein bands were detected using electrochemiluminescence (ECL) reagents. Band intensities were quantified using Image-Lab software and normalized to β-actin. The cell received aminoguanidine and siRNA concentrations were maintained the same as in the qPCR experiments.

2.10. LPS-induced ALI mouse model

Mice were purchased from Tengxin Laboratory Animal Co. Ltd. (Chongqing, China). All animal experiment procedures were performed in compliance with the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University. Male C57BL/6 mice (6–8 weeks) were randomly divided into six groups (at least six mice per group): PBS + PBS group, LPS + PBS group, LPS + NC group, LPS + TET^AG group, LPS + TET^Mg-STING group, and LPS + TET^AG-STING group. Mice were injected intraperitoneally with 1 % sodium pentobarbital, and 50 μL of LPS (5 mg kg⁻¹) or 50 μL of PBS buffer was injected into the trachea by Liquid Aerosol Devices (MicroSprayer Aerosolizers) after anesthesia. Two hours later, 50 μL of PBS, TET^Mg-STING NC, TET^AG, TET^Mg-STING, TET^AG-STING were injected into the trachea by Liquid Aerosol Devices according to the group. The dosage of TET carriers was 8.7 mg kg⁻¹. The concentration of siRNA was 2.5 mg kg⁻¹, and aminoguanidine was about 13.8 mg kg⁻¹.

2.11. Biodistribution study

TET^AG-STING NC was labeled by Cy5.5. After anesthesia, 50 μL LPS (5 mg kg⁻¹) was injected into the trachea through an insulin syringe. After 2 h, 50 μL TET^AG-STING NC was injected through the trachea. The concentration of Cy5.5 was 60 μg per mouse, and the dosage of TET^AG-STING NC was 12.0 mg kg⁻¹. Then the biodistribution and lung deposition were detected by an In Vivo Xtreme imaging system.

2.12. Recovery of ventilation function study

Firstly, 50 μL of LPS (5 mg kg⁻¹) or 50 μL of PBS buffer was injected into the mouse trachea. Following LPS stimulation, various DNA tetrahedra (namely TET^AG, TET^Mg-STING NC, TET^Mg-STING, and TET^AG-STING) were administered via the airways 2 h later, while PBS buffer-treated mice were set as a control group. After 24 h, under anesthesia, mice were intubated by trachea. Then the ventilation function was detected using the flexiVent FX system according to the manual and analyzed by the flexiWare v7.2 software.

2.13. Lung wet/dry (W/D) ratio

Following the initial procedures, the lung tissues from the mice were gathered, measured for their wet weight, and then placed in an incubator set at 60 °C for a period of 72 h to determine their dry weight. Subsequently, the ratio of the wet weight to the dry weight of the lungs was calculated in order to assess the extent of lung edema.

2.14. Lung histology and lung injury scoring

For H&E staining, Lung tissues of the mice were fixed using 4 % formalin and embedded in paraffin followed by sectioning into 4-μm sections. The tissues were stained using H&E following standard procedures. The pathological characteristics of the tissues were evaluated by light microscopy. The degree of injury of lung tissue was divided into grades 0–4. Grade 0: no injury; grade 1: injury in <25 % of the field of observation; grade 2: injury in 25–50 % of the field of observation; grade 3: injury in 50–75 % of the field of observation; grade 4: injury throughout the field of observation. At least five fields of observation were randomly selected and analyzed in each sample.

2.15. Flow cytometry analysis of BALF

ALI mice were treated with PBS, TET^Mg-STING NC, TET^AG, TET^Mg-STING, and TET^AG-STING for 24 h. The dosages of STING siRNA and aminoguanidine were 2.5 mg kg⁻¹ and 13.8 mg kg⁻¹, respectively. Precooled PBS was injected into the trachea, and the alveolar lavage fluid was collected. Then, the collected BALF was centrifuged at 2000 rpm for 5 min. After centrifugation, the supernatant was stored at −80 °C, the lower cells were removed with red blood cell lysate to clear up red blood cells. Then, the cells at the bottom of the tube were collected by centrifugation again. Cells were stained with CD11b, ly6G, Data were collected by flow cytometry (Backman, Gallios, USA) and analyzed with FlowJo software.

2.16. Cytokine analysis

In vivo cytokine analysis was conducted by testing the collected BALF using a commercially available ELISA kit according to the manufacturer's instructions. Briefly, standards and samples were added in duplicate to the precoated microplate wells. The plate was then incubated at 37 °C for 90 min to allow for antigen-antibody binding. Following incubation, the wells were washed three times with the provided wash buffer to remove unbound material. Subsequently, a biotinylated detection antibody was added to each well and the plate was incubated again at 37 °C for 60 min. After a second washing step, horseradish peroxidase (HRP)-conjugated streptavidin was added and the plate was incubated for an additional 30 min. The plate was then washed thoroughly to remove any unbound conjugate. The substrate solution was added to initiate the colorimetric reaction, which was allowed to develop for 10–20 min. The reaction was stopped by adding the stop solution, and the absorbance was measured at 450 nm using a microplate reader.

2.17. Statistical analysis

All the data was analyzed by the software GraphPad Prism 7.0, and the results were presented as the mean value ± SD (standard deviation). The data was compared between two groups using t-test, and the data of more than three groups were carried out by one-way ANOVA. Significant differences between the groups were indicated by ∗ (P < 0.05), ∗∗ (P < 0.01), ∗∗∗ (P < 0.001), or ∗∗∗∗ (P < 0.0001) and # (P < 0.05), ## (P < 0.01), ### (P < 0.001), or #### (P < 0.0001).

3. Result and discussion

3.1. Design, assembly, and characterization of the TET^AG-STING

As illustrated in Scheme 1, four short DNA strands, designated TET-1, TET-2, TET-3, and TET-4, were hybridized with each other at a molar ratio of 1:1:1:1 in the presence of aminoguanidine (AG) to form a DNA tetrahedral (TET^AG) structure with single-stranded overhangs. The detailed sequences of these strands are provided in the Supporting Information. Subsequently, STING siRNA was mixed with the pre-annealed DNA tetrahedron solution at a 4:1 M ratio and incubated at room temperature for 2 h to form a functional DNA tetrahedral structure (TET^AG-STING). The protruding single-stranded DNA region at the four vertices of DNA tetrahedron is complementary to the STING siRNA sequence, thus allowing for the siRNA loading. Magnesium-assembled DNA tetrahedra (TET^Mg) and their STING.

siRNA-loaded counterparts (TET^Mg-STING) were also synthesized and used as controls. We first investigated the effect of aminoguanidine concentration on DNA tetrahedron self-assembly using polyacrylamide gel electrophoresis (PAGE). Aminoguanidine effectively facilitated the synthesis of TET^AG-STING within the concentration range of 0–500 mM, with the highest yield observed at an aminoguanidine concentration of 50 mM (Fig. 1A). Notably, the yield of TET^AG increases with increasing aminoguanidine concentration, likely because, at lower concentrations, aminoguanidine is insufficient to allow all DNA strands to fold into well-defined nanostructures. However, at higher concentrations, excessive aminoguanidine leads to the formation of larger, irregular aggregates, resulting in a lower yield. To determine the tetrahedron assembly in detail, different DNA combinations were prepared and analyzed by PAGE. TET^AG and TET^AG-STING formed distinct, sharp bands in the 100–200 bp region of the DNA ladder, which corresponds to the expected molecular weight (Fig. 1B). Semi-quantification analysis indicated that the yields of TET^AG-STING and TET^Mg-STING were 73.9 ± 1.6 % and 87.0 ± 1.5 %, respectively. In comparison, TET^AG-STING migrated more slowly than TET^Mg on the gel, reflecting its larger molecular weight. Fluorescence PAGE result suggested that the optimal siRNA loading was achieved at a DNA tetrahedron to siRNA molar ratio of 1:4 (Fig. S1). There was no discernible mobility difference between TET^AG-STING and TET^Mg-STING on the gel, suggesting that TET^AG-STING likely maintains a tetrahedral structure. As shown in Fig. 1C, the Atomic Force Microscopy (AFM) imaging result revealed that individual TET^AG-STING nanoparticles were uniformly dispersed on the mica surface. Dynamic light scattering (DLS) measurements indicated that the hydrodynamic diameter of TET^AG and TET^AG-STING were approximately 13.0 and 17.0 nm, which are consistent with the presence of single-stranded and duplex overhangs (Fig. 1D and Fig. S2). It is notable that although TET^AG-STING displayed a larger hydrodynamic size than TET^AG, the difference is not significant. This is probably attributed to the fact that in solution, ssDNA handles or the DNA/RNA duplex may display a similar length. The zeta potentials for TET^AG and TET^AG-STING were determined to be −4.6 and −5.0 mV, respectively. These results collectively suggest that TET^AG-STING nanoplatforms were successfully assembled with aminoguanidine. Traditionally, DNA nanostructures are assembled in Mg²⁺-containing solutions with Mg²⁺ concentrations ranging from 1 mM to several tens of mM. Aminoguanidine, which carries one positive charge at neutral pH, can effectively bring negatively charged DNA strands together. Ideally, the concentration of aminoguanidine should match the number of base pairs in the four DNA strands, as the number of bases equals the number of negatively charged phosphate groups. However, in this study, the minimum aminoguanidine concentration required for DNA tetrahedron assembly was in the micromolar range, which is significantly higher than the number of phosphate groups. We hypothesize that the excess aminoguanidine may form hydrogen bonds with DNA bases, similar to observations in arginine-assembled DNA nanostructures from previous studies [29].

Fig. 1.

Self-assembly and characterization of TET^AG-STING. (A)To determine the optimal aminoguanidine concentration for the successful assembly of TET^AG, different concentrations ranging from 0 to 500 mM were tested. The selected aminoguanidine concentration for subsequent experiments was 50 mM. (B) PAGE analysis of self-assembled TET^AG-STING. The aminoguanidine concentration used for self-assembly was 50 mM. TET^Mg and TET^Mg-STING were also prepared as controls. (C) AFM imaging of TET^AG in liquid. TET^AG was assembled at an aminoguanidine concentration of 50 mM and diluted to 50 μM before imaging. Scale bar: 30 nm. (D) DLS measurements of TET^AG.

3.2. Stability and cellular uptake of the TET^AG-STING

In addition to evaluating the concentration of aminoguanidine, we assessed the TET^AG assembly in solutions with varying pH values. Fig. 2A shows that TET^AG was successfully assembled with yields comparable to those of TET^Mg. Interestingly, aminoguanidine could mediate the isothermal assembly of DNA tetrahedra. The isothermal assembly of TET^AG was achieved by incubating aminoguanidine and the DNA component strands at 4 °C, 22 °C, 37 °C and 45 °C for 2 h (Fig. 2B). To determine the stability of TET^AG at physiological temperature, different strand combinations and the final TET^AG were loaded onto a PAGE gel and run at 37 °C (Fig. 2C). It was found that TET^AG appeared as distinct bands on the gel, similar to those observed in the gel run at 22 °C (Fig. 1A). These results suggest that aminoguanidine can mediate DNA tetrahedra assembly over a wide pH range, and that the assembly is robust.

Fig. 2.

Stability and Cytotoxicity of TET^AG. (A) TET^AG assembly in solutions with different pH values. The yields of TET^AG were determined to be 56.3 ± 2.8 %, 57.5 ± 2.2 %, 60.0 ± 2.1 %, 58.1 ± 1.7 %, and 56.6 ± 2.1 % for pH values 5.0, 6.0 7.0, 8.0, and 9.0, respectively. (B) Aminoguanidine mediates the isothermal self-assembly of DNA tetrahedra at temperatures of 4 °C, 22 °C, 37 °C, and 45 °C. TET^AG yields increased from 70.2 ± 1.7 % at 4 °C to 74.1 ± 2.1 % at 22 °C, and then decreased to 70.0 ± 1.7 % at 45 °C. (C) PAGE gels were run at 37 °C with different DNA strand combinations. The yield of TET^AG was approximately 74.1 ± 2.3 %. (D) Serum stability analysis of TET^AG using PAGE. TET^AG and TET^Mg were incubated with 10 % FBS for 0, 3, 6, and 9 h before gel electrophoresis. Quantification analysis indicated that the remaining structure was only 58.1 ± 2.7 %, 45.8 ± 3.8 %, and 24.1 ± 2.3 % for TET^AG, while it was 64.3 ± 2.7 %, 8.0 ± 1.2 %, and 1.2 ± 1.0 % for TET^Mg. (E) The structural stability of TET^AG was examined after aminoguanidine depletion with TAE/Mg²⁺ buffer, ultrapure water, and low-concentration aminoguanidine (50 μM). After depletion, 56.0 ± 1.3 %, 58.6 ± 2.8 %, and 56.6 ± 2.2 % of TET^AG remained for TAE/Mg²⁺, ultrapure water, and aminoguanidine washing, respectively. (F) The cytotoxicity of aminoguanidine was measured using the CCK8 assay. Cells were treated with aminoguanidine for 24 h at different concentrations. The aminoguanidine concentration used for TET^AG assembly in PAGE analysis was 50 mM. All the data of Figure represent the mean value ± SD (n = 4). P-values were calculated using one-way ANOVA (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001 compared to the 0 mM group).

Stability and cytotoxicity are important parameters for the biomedical applications of DNA nanostructures. We next investigated the serum stability of TET^AG in 10 % FBS to mimic physiological conditions. The results indicated that TET^AG demonstrated a longer retention time even at the 9-h time point, while TET^Mg began to degrade at the 3-h time point (Fig. 2D). Additionally, we investigated the structural stability of TET^AG after removing excess aminoguanidine (Fig. 2E). The results showed that after being washed with TAE/Mg²⁺ buffer, ultrapure water, or low-concentration aminoguanidine (50 μM), TET^AG still exhibited sharp, dominant bands on the gel with reasonable mobility. This indicates that the binding between aminoguanidine and DNA was sufficiently strong to maintain integrity during aminoguanidine depletion and vigorous sample processing.

Prior to cellular experiments, we assessed the cytotoxicity of aminoguanidine. Our data indicated that aminoguanidine concentrations exceeding 5 mM can result in pronounced cell death (Fig. 2F). Additionally, the in vitro safety of TET^AG-STING was evaluated using the CCK8 assay, and no cytotoxicity was observed (Fig. S3). We also checked the cytotoxicity of aminoguanidine and DNA tetrahedra in two other lung epithelial cell lines A549 and BEAS - 2B using the CCK8 assay and found that they had negligible influence under the current experimental settings (Fig. S4). Overall, our findings demonstrate that the DNA tetrahedra designed in this study exhibit reliable stability in physiological environments. This stability facilitates effective siRNA delivery, thereby enhancing their potential for biological applications. To investigate the cellular uptake of DNA tetrahedra, we utilized confocal laser scanning microscopy (CLSM) to assess the uptake efficiency of TET^AG. Single-stranded DNA TET-1was labeled with the Cy5 fluorophore (Cy5-TET-1), which emits red light under fluorescence microscopy. After co-culturing cells with TET^AG and Cy5-TET-1 for 1, 3, and 6 h without any transfection reagents, we observed significant uptake in all instances. Notably, the fluorescence intensity of TET^AG was considerably higher than that of Cy5-TET-1 (Fig. 3A). Fluorescence-activated cell sorting (FACS) analysis further confirmed that the cellular uptake of TET^AG was higher than that of Cy5-TET-1 at each time point (Fig. 3B). We reasoned that the significant increase in cellular uptake could be attributed to the presence of aminoguanidine-assembled DNA tetrahedra. In addition, TET^AG also exhibited a.

Fig. 3.

Cellular uptake of TET^AG. (A) The tetrahedron was labeled with Cy5 and incubated with the cells for 1, 3, 6 h,The observations were compared to Cy5-labeled TET-1 (Cy5-TET-1). The CLSM images reveal the cellular uptake of the tetrahedron and its localization within the cells. Cell nuclei were stained with DAPI. The Cy5 concentration was kept at 200 nM. Scale bar: 25 μm. (B) Flow cytometry analysis of the internalized TET^AG and Cy5-TET-1. All data are presented as the mean value ± SD (n = 3). P-values were calculated using t-test, ∗P < 0.05, ∗∗P < 0.01, Cy5-TET-1 group versus TET^AG group.

higher cellular uptake than TET^Mg (Fig. S5). This could be attributed to the introduction of aminoguanidine onto the DNA tetrahedra, which altered the surface properties of the DNA nanostructure. Similarly, our previous study indicated that spermidine-assembled DNA prisms enhanced cellular uptake efficiency in multiple cancer cell lines [44]. Nevertheless, further studies are needed to understand how aminoguanidine in TET^AG interacts with cells. Generally, magnesium-free assembly of DNA endows DNA nanostructures with more functionalities, particularly regarding their interaction with biological interfaces. For instance, DNA nanostructures assembled with polyamines and guanidines have been demonstrated to be more stable in resisting enzymatic digestion compared to those assembled in magnesium-containing systems [45]. Additionally, other advantages such as enhanced cellular uptake have also been validated [46,47]. However, a few exceptions have been observed. For example, amino acid-assembled DNA nanostructures were found to be less stable in physiological settings and collapsed during vigorous washing with ultrapure water or low concentrations of amino acid solutions [29]. In the current study, TET^AG survived ultrapure water washing and exhibited better stability in 10 % FBS, suggesting that the binding between aminoguanidine and DNA is robust, and this tight interaction might contribute to the enzymatic resistance of TET^AG. Notably, aminoguanidine itself is an iNOS inhibitor, which endows TET^AG with functionality beyond that of a DNA nanostructure carrier.

3.3. TET^AG-STING suppresses inflammation by downregulating iNOS and the cGAS-STING pathway

Inflammatory stimuli facilitate the recruitment and activation of inflammatory macrophages, which in turn produce substantial amounts of inflammatory cytokines. LPS is a component found in the outer membrane of Gram-negative bacteria. It is commonly used to mimic bacterial infections and trigger inflammation in both in vitro and in vivo studies. We first assessed the expression of various inflammatory factors using RT-qPCR. As shown in Fig. 4A, both TET^AG and TET^AG-STING treatments led to a significant reduction in TNF-α mRNA levels compared to LPS-challenged cells, while TNF-α expression remained unchanged in the TET^Mg-NC and TET^Mg-STING groups. The TET^AG-STING treated group demonstrated greater suppression of.

Fig. 4.

TET^AG-STING downregulates iNOS to inhibit the cGAS-STING pathway and suppress the secretion of inflammatory cytokines in vitro. RT-qPCR quantitation of relative mRNA expression of TNF-α (A), IL-6 (B), IL-1β (C), which were transfected with the TET^AG-STING for 24 h and 2 μg mL⁻¹ LPS for 6 h. RT-qPCR quantitation of relative mRNA expression of iNOS (D), cGAS (E), and STING (F) in RAW 264.7 cells, which were transfected with TET^AG-STING for 24 h and 2 μg mL⁻¹ LPS for 6 h. The expression levels from A to F were normalized to Ctrl groups. (G) Western blot analysis of iNOS, cGAS, and STING expressions, β-actin was used as internal control. Cell received aminoguanidine and STING siRNA concentrations were 1.5 mM and 100 nM, respectively. All data are presented as the mean value ± SD (n = 5). P-values were calculated using one-way ANOVA, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 compared to the LPS group; #P < 0.05 indicate connected groups.

TNF-α than the TET^AG group, indicating a potential combined therapeutic effect between aminoguanidine and STING siRNA. These findings show that both aminoguanidine and STING siRNA influence TNF-α expression. Similar trends were observed for IL-1β and IL-6 (Fig. 4B and C). Aminoguanidine is known to suppress inflammation by inhibiting iNOS, which relates closely to mitochondrial function, reactive oxygen species, and inflammation [34]. STING is also recognized as a key effector in inflammatory responses. After entering the cells, a small portion of TET^AG-STING escaped from the lysosomes, unwound from the tetrahedron to release the STING siRNA, and further integrated into the RNA-induced silencing complex (RISC complex) to silence its target genes. Thus, TET^AG and TET^AG-STING are expected to have anti-inflammatory effects, although the detailed mechanisms remain unclear.

To validate the underlying mechanism by which TET^AG-STING suppresses inflammation, cells treated with TET^AG, TET^Mg-STING NC, TET^Mg-STING, and TET^AG-STING were analyzed using RT-qPCR and Western blotting. TET^Mg-STING NC and TET^Mg-STING served as controls. Fig. 4D indicates that both TET^AG and TET^AG-STING downregulated the mRNA levels of iNOS. In contrast, TET^Mg-STING NC and TET^Mg-STING did not affect iNOS expression, highlighting that aminoguanidine may selectively inhibit iNOS expression. Furthermore, Fig. 4E shows that TET^AG and TET^AG-STING reduced cGAS expression, which aligns with the observed trend in iNOS expression. Meanwhile, TET^AG and TET^Mg-STING individually resulted in the inhibition of STING expression by 26.9 % and 28 %, respectively. Notably, when cells were challenged with LPS, the mRNA expression of STING significantly increased (Fig. S6). These results imply that aminoguanidine may first inhibit iNOS and subsequently downregulate the cGAS pathway. When these two agents were combined (referred to as TET^AG-STING), there was a significant reduction in STING expression, reaching up to 48.5 % (Fig. 4F). As the cGAS-STING pathway senses intracellular DNA, we reasoned that aminoguanidine might inhibit iNOS expression, alleviate mitochondrial dysfunction, reduce mtDNA release, and subsequently downregulate STING expression. We further conducted Western blotting to confirm the related protein expressions. Fig. 4G indicates that TET^AG-STING significantly decreased the protein expression levels of iNOS, cGAS, and STING. Quantitative analysis showed that STING expression was inhibited to 46.5 % in the TET^AG-STING treated group, compared to 74.0 % and 73.6 % in the TET^AG and TET^Mg-STING treated groups, respectively (Fig. S7). These results suggest that iNOS serves as an upstream target within the cGAS-STING pathway; thus, inhibiting iNOS can effectively suppress this pathway. Moreover, aminoguanidine and STING siRNA exhibit a combined therapeutic effect, collaboratively inhibiting STING expression.

3.4. Biodistribution and safety of TET^AG-STING

To investigate the biodistribution of TET^AG-STING in mice, we first administered LPS treatment for 2 h, followed by airway instillation of Cy5.5-loaded TET^AG-STING NC nanoparticles. Imaging was performed at 1, 2, 6, and 12 h after injection. Fig. 5A shows fluorescence images of ALI mice at 2 and 12 h post-administration. Red fluorescence emanating from the mouse lungs, circled with purple lines, is clearly visible. At the 12-h time point, lung fluorescence remains the strongest compared to other major organs (Fig. 5B). Quantitative analysis indicates that TET^AG-STING NC begins to accumulate in the lungs 1 h post-administration and exhibits the highest fluorescence intensities compared to the 2, 6, and 12-h groups. This suggests that TET^AG-STING NC is metabolized over time (Fig. 5C). In addition, Cy5.5-TET-1 metabolized faster than TET^AG after 12 h (Fig. S8). Other major organs such as the liver and kidney exhibit strong fluorescence signals after 6 h, indicating that TET^AG-STING NC is metabolized by these organs (Fig. S9). Furthermore, we performed H&E staining on the hearts, livers, spleens, and kidneys of the mice to assess any potential damage resulting from the airway injection of TET^AG-STING (Fig. S10). The findings indicated that these organs did not exhibit any significant damage, thus confirming the safety of TET^AG-STING in vivo.

Fig. 5.

Biodistributions of TET^AG-STING NC on the LPS-induced ALI mouse model. (A) Fluorescence images of ALI mice at 2 h and 12 h time points. Purple circles indicate the lungs. Cy5.5-labeled TET^AG-STING NC was injected into the mice via the trachea. (B) Fluorescence images of major organs from ALI mice at the 12 h time point. Mice were sacrificed, and lung, heart, liver, spleen, and kidney were dissected. (C) Lung images showing fluorescence intensity of TET^AG-STING NC at different time intervals. Quantitative analysis of the fluorescence intensities at different time points is presented in the right panel. All the data of Fig.s represent the mean ± SD (n = 4). P-values were calculated using one-way ANOVA, ∗∗P < 0.01, ∗∗∗P < 0.001 compared to the 1 h group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.5. TET^AG-STING alleviated ALI in vivo

To investigate the anti-inflammatory effects of TET^AG-STING in vivo, we established an acute lung injury (ALI) model in C57BL/6 mice through intratracheal injection of 5 mg kg⁻¹ LPS. The workflow of in vivo experiment is illustrated in Fig. 6A. Following LPS stimulation, various DNA tetrahedra (namely TET^AG, TET^Mg-STING NC, TET^Mg-STING, and TET^AG-STING) were administered via the airways 2 h later, while PBS buffer-treated mice were.

Fig. 6.

Synergistic anti-inflammatory effect of TET^AG-STING in the LPS-induced ALI mice model. (A) Experimental timeline in the LPS-induced ALI model. First, 50 μL of LPS (5 mg kg⁻¹) or 50 μL of PBS buffer was injected into the trachea. After 2 h, all treatments were injected into the trachea. The levels of the cytokines TNF-α (B), IL-6 (C), IL-1β (D) in the BALF were quantified using the Mouse Precoated ELISA Kit. (E) The accumulation of total protein in BALF was detected by BCA. (F) Percentages of neutrophil in BALF. (G) Total protein expressions of iNOS, cGAS, STING, and β-actin were assessed in lung tissues using western blotting. (H) Lung injury scores. (I) Peak expiratory flow (PEF) and (J) airway resistance (Rn) was investigated after treatment for 24 h. (K) Histological images of H&E-stained lung sections. Scale bar: 100 μm. All the data of Figures represent the mean value ± SD (n = 6). P-values were calculated using one-way ANOVA, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 compared to the LPS group; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared to the TET^AG-STING group.

set as control group. A cytokine storm, marked by excessive immune cell activation and overproduction of inflammatory cytokines such as TNF-α, IL-1β, and IL-6, is recognized as a major factor influencing the severity and mortality of ALI/ARDS. As shown in Fig. 6B, LPS stimulation resulted in a significant accumulation of TNF-α in the bronchoalveolar lavage fluid (BALF). TET^AG and TET^Mg-STING treatments individually suppressed TNF-α expression, while TET^AG-STING exhibited much higher suppression effects than the two single-drug groups, suggesting a combined therapeutic effect. Similarly, two other inflammation factors, IL-6 (Fig. 6C) and IL-1β (Fig. 6D), were also found to exhibit similar trends in vivo. Additionally, BALF was analyzed to assess protein accumulation indicative of lung injury, with TET^AG-STING demonstrating a notable effect compared to the other groups (Fig. 6E). After centrifugation of the BALF, the cell pellet underwent flow cytometry analysis, which revealed a substantial increase in the proportion of neutrophils in the alveolar lavage fluid following LPS stimulation. As expected, both the TET^AG and TET^Mg-STING groups exhibited a reduction in the proportion of neutrophils compared to the LPS group, with the TET^AG-STING group showing a more pronounced inhibitory effect (Fig. 6F, Fig. S11). Furthermore, the iNOS, cGAS and STING protein expressions in lung tissues were detected by WB (Fig. 6G). Quantification analysis results (Fig. S12) showed that the target proteins in the cGAS-STING pathway were significantly inhibited, aligning well with the study in vitro. Meanwhile, lung weights were measured pre- and post-drying to calculate the wet-to-dry weight ratio. The LPS group displayed a significant increase in this ratio, while the TET^AG-STING group showed the lowest ratio, indicating alleviation of pulmonary edema (Fig. S13). According to the Smith scoring method, the LPS group exhibited a higher injury score, while both the TET^AG and TET^Mg-STING groups demonstrated reductions in their scores, with the TET^AG-STING group showing the most pronounced decrease (Fig. 6H). The protective effect of TET^AG-STING was notably more robust than that of either the TET^AG or TET^Mg-STING groups. Additionally, we evaluated relevant lung function parameters in the experimental mice. Upon LPS stimulation, the mice displayed increased airway resistance and decreased peak expiratory flow rate (Fig. 6I and J). Both the TET^AG and TET^Mg-STING groups improved respiratory function indicators; however, the TET^AG-STING group showed the most substantial improvement in respiratory function. Additionally, following LPS administration, lung tissue sections were obtained for histological evaluation, which revealed several significant findings. The lung tissue displayed considerable damage characterized by collapsed and compressed alveoli, thickened alveolar walls, indistinct boundaries, and prominent infiltration of inflammatory cells, along with edema and other pathological symptoms (Fig. 6K). These results collectively demonstrate that TET^AG-STING can effectively exert superior anti-inflammatory effects in vivo and significantly improve the lung function of ALI mice. The strategy proposed in this study, which is the synergy between aminoguanidine and STING siRNA for the treatment of ALI/ARDS, holds great potential as a nanomedicine in the future.

4. Conclusions

In summary, we have developed a dual-drug nanomedicine based on DNA nanostructures that combines aminoguanidine and STING siRNA for the treatment of ALI/ARDS. Aminoguanidine specifically inhibits iNOS, thereby suppressing the cGAS-STING pathway, while STING siRNA works synergistically to inhibit the transcription and expression of STING, collectively producing anti-inflammatory effects. In addition, our findings suggest that assembling DNA tetrahedral structures with aminoguanidine represents a promising approach to functionalizing DNA and enhancing the biomedical applications of DNA nanostructures [[48], [49], [50], [51], [52]]. Nevertheless, the proposed aminoguanidine-assisted, magnetic-free DNA assembly and functionalization strategy also has limitations. For instance, only cationic molecules can neutralize DNA's negative charge and achieve correct folding of predefined nanostructures. The chemistry and function of the molecules used for DNA assembly need to be fine-tuned and customized for specific needs. Therefore, future work focusing on functionalizing DNA with rationally designed molecules (such as those conjugated with desired chemical groups) that possess unique properties and promoting the clinical translation of DNA-based nanomedicines is highly needed and desirable.

CRediT authorship contribution statement

Yue Chen: Writing – original draft, Investigation, Conceptualization. Di Wu: Validation, Investigation, Conceptualization. Quan Li: Investigation, Formal analysis. Zhenghua Wei: Investigation. Qian Liu: Validation, Investigation. Jin Li: Investigation. Daohui Gong: Investigation. Chaowang Huang: Investigation. Qianyi You: Investigation. Hang Qian: Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition, Conceptualization. Guansong Wang: Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used ChatGPT 4o to improve the readability and language of the final paper. After using this tool, the authors reviewed and edited the content as needed and took full responsibility for the content of the publication.

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.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.