Tetrahedral framework nucleic acid nanomaterials reduce the inflammatory damage in sepsis by inhibiting pyroptosis

Abstract Sepsis is a highly lethal condition and is caused by the dysregulation of the body's immune response to infection. Indeed, sepsis remains the leading cause of death in severely ill patients, and currently, no effective treatment is available. Pyroptosis, which is mainly activated by cytoplasmic danger signals and eventually promote the release of the pro‐inflammatory factors, is a newly discovered programmed cell death procedure that clears infected cells while simultaneously triggering an inflammatory response. Increasing evidence indicates that pyroptosis participates in the development of sepsis. As a novel DNA nanomaterial, tetrahedral framework nucleic acids (tFNAs) characterized by its unique spatial structure, possess an excellent biosafety profile and can quickly enter the cell to impart anti‐inflammatory and anti‐oxidation effects. In this study, the roles of tFNAs in the in vitro model of macrophage cell pyroptosis and in the in vivo model of septic mice were examined, and it was found that tFNAs could mitigate organ inflammatory damage in septic mice, wherein they reduced inflammatory factor levels by inhibiting pyroptosis. These results provide possible new strategies for the future treatment of sepsis.


| INTRODUCTION
Sepsis is a life-threatening disease caused by dysregulated body's immune response to infection, which leads to wide-ranging inflammatory reactions and serious complications. [1][2][3] The survivors of sepsis often suffer from organ damage, and their quality of life is significantly decreased, as manifested by various physical dysfunctions and cognitive defects. 4,5 The occurrence of sepsis is mainly derived from bacterial infections, in which lipopolysaccharides (LPSs), as the main components of bacteria, are the main driver of mediating the severe inflammatory response in septic patients. 6 It is believed that the pathological development of sepsis is closely related to the release of numerous inflammatory cytokines, which is triggered by pyroptosis. [7][8][9][10] After stimulation of the canonical pyroptosis pathway by pattern recognition receptors that recognize pathogenassociated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), inflammasomes (e.g., NLRP3, NACHT, LRR and PYD domain-containing protein 3) within the cytoplasm are then activated and combined with apoptosis-associated speck-like protein containing CARD (ASC) to cleave pro-caspase-1 and form cleavedcaspase-1, which promotes the formation of activated inflammatory factors involving interleukin (IL)-1β and IL-18 by cleavage in pro-IL-1β and pro-IL-18. Meanwhile, cleaved-caspase-1 also dominates the formation of Gasdermin D N-terminal (GSDMD-NT), which composes pores on plasma membrane and mediates the extracellularly release of IL-1β and IL-18. Subsequently, by initiating an inflammatory response, the presence of excess amounts of inflammatory factors eventually leads to a wider range of cell pyroptosis. 11,12 In general, pyroptosis plays a positive role in the immunomodulatory process; however, when the host is undergoing severe infections like sepsis, it can be hyperactivated and exacerbates the wide-ranging inflammation, which will eventually intensify the organ damage caused by sepsis. 8 At present, the treatment strategy for sepsis is mainly anti-infection treatment, and there is still no effective means of preventing sepsis or treating the resulting systemic organ inflammation. Therefore, reducing the inflammatory damage in sepsis through the inhibition of pyroptosis is now receiving growing attention as a potential new therapeutic approach.
Assembled from four specific single-stranded fragments of DNA, the tetrahedral framework nucleic acids (tFNAs) are novel nucleic acid nanomaterials with a tetrahedral spatial structure, which imparts them with excellent biological properties, including enhanced cell endocytosis properties and a superior tissue permeability, thereby rendering them suitable for application in disease treatment, drug delivery, biosensing and other fields. [13][14][15][16][17][18][19] Moreover, previous studies have shown that tFNAs exhibit excellent anti-inflammatory antioxidant capacities and good biosafety profiles in the treatment of inflammatory diseases, which is mainly achieved by their regulation of the nuclear factor kappa-light-chain-enhancer (Nf-κB) pathway. 20 In addition, tFNAs can regulate the polarization of macrophages by inhibiting pro-inflammatory M1 types and promoting the scavenging of intracellular reactive oxygen species (ROS). 16,21,22 It should be noted that abnormal intracellular ROS levels and activation of the NF-κB pathway can raise the expression of pro-inflammatory factors and the NLRP3 inflammasome, thereby promoting pyroptosis and triggering a strong inflammatory response. [23][24][25] Therefore, due to the excellent anti-inflammatory and antioxidant abilities of tFNAs, they are expected to inhibit sepsis-related inflammatory responses, revealing a novel strategy for organ protection during sepsis.
In this study, we establish an in vitro model of RAW264.7 macrophage pyroptosis and an in vivo model of septic BALB/c mice to explore the positive role of tFNAs in regulating cell pyroptosis to reduce the inflammatory response and mitigate multi-organ damage in sepsis.

| Generation of the tFNAs
Based on the Watson-Crick base-pairing principles, tFNAs nanomaterial was self-assembled from four single-stranded sequencespecific DNA (ssDNA) fragments in equimolar quantities (1 μM), as outlined in Table 1 (Sangon Co., Ltd.), using TM buffer (composed of 50 mM MgCl 2 and 10 mM TrisHCl (pH 8.0) solutions) and heating at 95 C for 10 min followed by rapid cooling to 4 C for 20 min. The obtained tFNAs were further stored in a fridge at 4 C until required for use.

| Authentication and characterization of the tFNAs
After the successful synthesis of tFNAs, their molecular weights were determined using 8% polyacrylamide gel electrophoresis (PAGE), and the purity of the tFNAs was evaluated by the capillary electrophoresis. For intuitive observations regarding the spatial nanostructures of the tFNAs, transmission electron microscopy (TEM, Hitachi Ltd.) and atomic force microscopy (AFM, Shimadzu) were used. The particles sizes and zeta potential of the tFNAs were measured by dynamic light scattering (DLS, Nano ZS).

| Cellular experiments
The murine macrophage (Raw264.7) was cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) complete medium containing 10% foetal bovine serum (HyClone) and 1% penicillinstreptomycin solution (HyClone) in an incubator at 37 C containing 5% CO 2 under a humidified atmosphere. Subsequently, the cells were divided into three groups, namely the blank, the control

| Uptake of tFNAs by macrophage cells
To examine the process by which the tFNAs enter into macrophages, Cy5-labelled S1 (Cy5-S1) with a red fluorescence signal was used instead of S1 to synthesize Cy5-labelled tFNAs for follow-up

| Cell viability assays
The cell viability was determined using the commercially available

| LDH level detection assays
The lactate dehydrogenase (LDH) activity in the supernatant was used to evaluate the plasma membrane integrity and the degree of cell death under pyroptosis. Cells were cultured in 96-wells plate at a density of 5000 cells per well, and after the intervention mentioned in Section 2.3, the supernatants of the different groups were collected and subjected to centrifugation (300Â g for 5 min) to remove all cellular debris. The LDH measurements were then conducted using a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega), according to the manufacturer's protocol.

| Western blot analysis
The RAW264.7 cells were seeded in a 6-well plate (10 6 cells per well).

| Preparation of the tFNAs
Four ssDNA segments (S1ÀS4) composed of the specific nucleic acid sequences outlined in Table 1 were added to the TM buffer solution and conducted as described in Section 2.1, which resulted in their self-assembly to generate a stable tetrahedral structure ( Figure 1A).
The successful synthesis of the tFNAs was confirmed using PAGE and capillary electrophoresis ( Figure 1B,C). Subsequent imaging using TEM and AFM revealed the tetrahedron-like structures of the tFNAs, as indicated by the triangular nanoparticles that are labelled in Figure 1D. DLS measurements indicated that the average size of the tFNAs particle was $17 nm, while the average zeta potential value was determined to be À7.6 mV ( Figure 1E).

| Cellular uptake of the tFNAs by macrophages
Previous studies confirmed that the tFNAs exhibit excellent cell-entry properties due to their unique spatial structures. 16 To probe the time required for the tFNAs to enter the RAW267.4 cells and reach their maximum entry efficiency, S1 was modified by Cy-5, and Cy-

| Maintenance of the plasma integrity under pyroptosis conditions by action of tFNAs
Unlike pyroptotic cells, cells in the apoptotic state maintain their plasma membrane integrity and induce a non-inflammatory consequence by not releasing inflammatory mediators. 26 However, when pyroptosis occurs, GSDMD-NT, which is generated from the cleavage of Gasdermin D by cleaved-caspase-1, accumulates in the cell membrane and forms pores, resulting in water inflow and potassium ion outflow. As a result, the plasma membrane potential becomes unstable, the cells gradually expand and the cell membrane integrity is destroyed by cell rupture. [27][28][29] Such a process is often rapid and uncontrollable in pathological states, resulting in the excessive activation of pyroptosis and exacerbation of the inflammatory response associated with LPS-induced sepsis. 10,30,31 To determine whether pre-treatment with the tFNAs affected the membrane integrity under pyroptosis conditions, the macrophages were separated into three intervention groups, as described in Section 2.3. The blank and the ctrl groups were not subjected to any pre-treatment, while tFNAs (250 nM) were added to the tFNAs group 2 h in advance. As can be seen in the confocal microscopy images presented in Figure 2E

| Decrease of the LPS-induced ROS and intracellular NO contents by action of the tFNAs
When immune cells are stimulated by LPS, excessive amounts of NO and endogenous ROS are produced in the cells, which can cause cell damage and further promote activation of the NLRP3 inflammasome, thereby mediating cell pyroptosis. 23,24,32 In addition, a normal amount of NO is crucial to ensuring the health of our bodies; however, the abnormal generation of NO can cause the dysfunction of multiple systems and can even lead to tissue damage. 33 Therefore, reducing intracellular NO levels whilst intervening against excessive oxidative stress can be achieved using antioxidants, and this has been considered a possible therapeutic strategy for sepsis. 6,[34][35][36] Thus, the roles of the tFNAs in scavenging excessive intracellular ROS and reducing the intracellular levels of NO were evaluated. More specifically, cells were treated as described in Section 2.3 and subsequently, the ROS clearance effect imparted by the tFNAs was determined by fluorescence microscopy ( Figure 2H) ctrl and tFNAs groups were determined using a Griess detection kit. It was found that both the tFNAs and ctrl groups contained significantly higher levels of intracellular NO than the blank groups after LPS + ATP stimulation; however, the level of intracellular NO in the tFNAs group was significantly decreased compared with that of the ctrl group ( Figure 2F), which indicates that pre-treatment with the tFNAs can block NO overproduction to prevent a possible severe inflammatory response. Although the above result reveals the potential of tFNAs as antioxidants, the mechanism by which the tFNAs effect the generation of the overproduced NO and the excessive ROS, remains unclear. Further exploration is therefore needed in this field in the future. To verify regulation of the inflammatory oxidative stress and the pyroptotic pathway by the tFNAs, expression of the related proteins was determined by Western blotting (Figure 3A-C). As expected, the expression of NLRP3 in the tFNAs group was significantly lower than that in the ctrl group ( Figure 3D), indicating that tFNAs pre-treatment reduced the LPS + ATP-induced activation of cellular inflammasomes.

| Suppression of pyroptosis-and inflammation-related protein expression by the tFNAs
Although there was no significant difference in caspase-1 expression, the cleaved-caspase-1 expression levels indicated that the cleavage activation of caspase-1 is reduced because of the low expression levels of NLRP3 in the tFNAs group ( Figure 3E,F). This also accounts for the significantly lower levels of GSDMD-NT ( Figure 3H) and the reduced expression of IL-18 and IL-1β ( Figure 3I,J) in the tFNAs group.
In addition, the protein expression results obtained for NF-κB p65 ( Figure 3L) and NF-κB p-p65 ( Figure 3M Figure 3N,O). The above experimental results therefore confirmed our hypothesis.

| Reduction LPS-induced systemic inflammation damage in septic mice by action of the tFNAs
Patients with LPS-induced sepsis often suffer from wide-ranging inflammatory damage and multiple organ dysfunction due to the massive degree of inflammation. 2,41 In this context, neutrophils play a crucial role in inducing and activating inflammatory responses while lymphocytes protect multiple organs from inflammatory damage. Therefore, the ratio of neutrophils to lymphocytes is a marker of the inflammatory response and immune balance, wherein a high ratio correlates with severe inflammation. 42  the inflammatory response appears to be promising as a novel strategy to treat sepsis. However, the specific mechanism of pyroptosis is not yet fully understood, and the role of tFNAs in regulating pyroptosis has still to be determined relative to other types of inflammasomes, such as caspase and the gasdermin family proteins. Furthermore, it is necessary to determine whether the inflammatory protective effect of tFNAs in septic mice can be reproduced in humans. Nevertheless, as a nucleic acid nanomaterial with good biosafety profile, the anti-oxidative and anti-inflammatory effects of tFNAs and their ability to regulate cell pyroptosis indicate the potential of this system to treat inflammation-and pyroptosis-related diseases. Moreover, due to their ease of editing and their rapid penetration properties, tFNAs have the potential for use as carriers to load small drug molecules, micro-RNA and aptamers, for further applications. 43 In the future, we will focus on the multifaceted applications of these novel nucleic acid nanomaterials and will explore in detail the underlying mechanisms that lead to their various biological capabilities. inflammatory cytokines). Data are presented as mean ± standard deviation (SD) (n ≥ 3). Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001 data analysis. Nan Hu and Xiaoxiao Cai supervised the project, and reviewed, edited and finalized the manuscript.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.

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