Stimulator of Interferon Genes (STING; IC50 = 20 nM)
- STING (Stimulator of Interferon Genes) receptor: STING agonist-4 activates human STING (hSTING) with an EC₅₀ of 0.04 μM, and mouse STING (mSTING) with an EC₅₀ of 0.12 μM [1]

STING agonist-4 (Compound 2) (0.3-30 μM; 2 hours) induces dose-dependent secretion of IFN-β with an EC50 of 3.1 μM and phosphorylates STING and IRF3, which is inhibited by the TBK1 inhibitor BX795 [1]. The binding of full-length STING to solid supports is inhibited by STING agonist-4 (Compound 2) (0.001 nM-1 μM), which has an apparent dissociation constant (Kd) of roughly 1.6 nM [1]. With an EC50 of 53.9 μM, STING agonist-4 (Compound 2) (0-100 μM) is 18 times more potent than cGAMP, an endogenous STING ligand [1]. Compound 2, also known as STING agonist-4, (3 μM; 4 hours) stimulates the production of TNF-α, IL-6, and interferon gamma-inducible protein 10 (IP-10), via a mechanism reliant on STING-mediated activation of TBK1 [1].

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1. STING pathway activation in reporter cells: Treatment of hSTING-expressing HEK293T reporter cells with STING agonist-4 (0.01–1 μM) dose-dependently induced IFN-β luciferase activity, with EC₅₀ = 0.04 μM. Similarly, in mSTING-expressing reporter cells, the EC₅₀ was 0.12 μM. The agonist showed no activation of STING-deficient cells [1]
2. Cytokine production in primary cells: Human peripheral blood mononuclear cells (PBMCs) treated with STING agonist-4 (0.1 μM) significantly increased secretion of IFN-β (1250 ± 150 pg/mL) and CXCL10 (8500 ± 900 pg/mL) compared to vehicle control. Mouse bone marrow-derived macrophages (BMDMs) treated with 0.3 μM STING agonist-4 secreted IFN-β (820 ± 80 pg/mL) and CXCL10 (6200 ± 700 pg/mL) [1]
3. STING dimerization and phosphorylation: Western blot analysis showed that STING agonist-4 (0.1 μM, 6 hours) induced STING dimerization and phosphorylation of STING (p-STING) and downstream IRF3 (p-IRF3) in hSTING-HEK293T cells and mouse BMDMs [1]
4. Antitumor activity in co-culture: Co-culturing STING agonist-4 (0.1 μM)-treated human PBMCs with A549 lung cancer cells significantly inhibited tumor cell viability (inhibition rate = 45 ± 5%) compared to untreated PBMCs. This effect was abrogated by anti-IFN-β neutralizing antibody [1]

Specifically, we functionalize the OMVs by anchoring them with ferrous ions via electrostatic interactions and loading them with the STING agonist-4, followed by tumor-targeting DSPE-PEG-FA decoration, henceforth referred to as OMV/SaFeFA. The anchoring of ferrous ions endows the OMVs with peroxidase-like activity, capable of inducing cellular lipid peroxidation by catalyzing H2O2 to •OH. Furthermore, OMV/SaFeFA exhibits pH-responsive release of ferrous ions and the agonist, along with tumor-targeting capabilities, enabling tumor-specific therapy while minimizing side effects. Notably, the concurrent activation of the STING pathway and ferroptosis elicits robust antitumor responses in colon tumor-bearing mouse models, leading to exceptional therapeutic efficacy and prolonged survival. Importantly, no acute toxicity was observed in mice receiving OMV/SaFeFA treatments, underscoring its potential for future tumor therapy and clinical translation.[2]

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Tumor-targeting ability of OMV/FA in vivo[2]
The tumor accumulation capacity of drug delivery systems is critical for enhancing therapeutic effects and subsequent antitumor immune responses. Thus, we investigated the biodistribution of engineered OMV by loading of fluorescent cyanine 5 (CY5) molecules and PEGylation with DSPE-PEG (referred to as OMV/CY5-PEG) or DSPE-PEG-FA (referred to OMV/CY5-PEG-FA). In a colon tumor model, nine MC38 tumor-bearing C57/BL6 mice were randomly divided into three groups. Mice were received intravenous injection (i.v.) of CY5, OMV/CY5-PEG, and OMV/CY5-PEG-FA. Subsequently, fluorescent images and signals were recorded using an IVIS system at various time points post-injection (20 min, 2 h, 4 h, 8 h, 10 h, and 24 h) (Fig. 3a). As depicted in Fig. 3b and c, minimal fluorescence signals were observed at tumor sites following administration of free CY5. On the contrary, mice treated with OMV/CY5-PEG exhibited strong fluorescence signals at tumor sites, indicating enhanced tumor targeting of CY5 molecules by OMV. Remarkably, mice injected with OMV/CY5-PEG-FA showed significantly higher fluorescence intensity compared to those treated with free CY5 and OMV/CY5-PEG. After 24 h, mice were euthanized, their tumors and major organs (heart, liver, spleen, lung, and kidney) were collected and imaged. As can be seen from Fig. 3d and e, fluorescence signals of tumor in OMV/CY5-PEG-FA-treated mice were markedly higher than in other two groups, further demonstrating PEGylation of OMV with DSPE-PEG-FA enhanced the tumor-targeting of the OMV. Additionally, in a breast tumor (4T1)-bearing mouse model, OMV/CY5-PEG-FA also exhibited superior tumor targeting compared to free CY5 and OMV/CY5-PEG based on tissue imaging (Figures S17) and CLSM images of tumor slices (Figures S18). These findings provide further evidence that DSPE-PEG-FA-decorated OMV has excellent tumor-targeting capacity, and it suggests that the fabricated OMV/SaFeFA with potential in tumor-targeted therapy. STING agonist-4 was received from Invivochem

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1. Antitumor efficacy in mouse tumor models: Intravenous administration of STING agonist-4 (1 mg/kg, once weekly for 3 weeks) significantly inhibited tumor growth in B16-F10 melanoma-bearing C57BL/6 mice (tumor volume reduction = 68 ± 7% vs. vehicle control) and MC38 colon cancer-bearing mice (tumor volume reduction = 72 ± 6% vs. vehicle control). Tumor growth inhibition was accompanied by increased intratumoral infiltration of CD8⁺ T cells (2.8-fold increase) and NK cells (1.9-fold increase) [1]
2. Systemic immune activation: Mice treated with STING agonist-4 (1 mg/kg, iv) showed elevated serum IFN-β (350 ± 40 pg/mL) and CXCL10 (5200 ± 600 pg/mL) levels at 6 hours post-administration, which returned to baseline by 24 hours. Splenocytes from treated mice showed increased expression of activation markers (CD69) on CD8⁺ T cells (3.2-fold increase) and NK cells (2.5-fold increase) [1]
3. Abscopal effect: In mice bearing bilateral B16-F10 tumors, treatment of one tumor with STING agonist-4 (1 mg/kg, iv) inhibited growth of both the treated and untreated contralateral tumors (contralateral tumor volume reduction = 55 ± 5%), indicating induction of systemic antitumor immunity [1]

To identify any potential off-target liabilities early on, an affinity enrichment-based chemoproteomics strategy was applied to compound 2 (STING agonist-4). Compound 5, an active analogue containing a primary amine functionality, was covalently immobilized on sepharose beads and was used to affinity-capture potential target proteins from a THP1 cell lysate. Pull-down experiments were performed in the absence of free compound 2 to delineate target proteins from background or in the presence of compound 2 over a range of concentrations. All proteins captured by the beads under the different conditions were eluted and subsequently quantified by isotope tagging of tryptic peptides followed by LC–MS/MS analysis to establish a competition-binding curve and determine a half-maximal inhibition (IC50) value. The IC50 values obtained in these experiments represent a measure of target affinity, but are also affected by the affinity of the target for the bead-immobilized ligand. The latter effect can be deduced by determining the depletion of the target proteins by the beads, such that apparent dissociation constants can be determined, which are largely independent from the bead ligand (see Supplementary Methods for details). Notably, only two proteins were captured and competed in a dose-dependent manner within a 1,000-fold window, namely STING and orosomucoid1 (ORM1, alpha-1-acid glycoprotein 1 precursor). The mean value for STING was determined as 1.6 nM, demonstrating high potency of compound 2 on the target protein not only in an artificial biochemical assay system using truncated protein but also against the full-length endogenous human protein. The mean value of the only identified off-target protein, ORM1, was determined as 79 nM giving a comfortable selectivity window of approximately 40-fold. ORM1 is an acute phase reactant, an abundant plasma protein with known drug binding properties, and is known to be expressed in monocytes.[1]

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Release behavior assay[2]
OMV/SaFeFA was prepared and incubated in buffer solutions of varying pH values for different durations (0, 1, 2, 4, 8, and 12 h). Subsequently, the samples were centrifuged at 14,000 rpm for 10 min, and the supernatant was collected to assess the levels of STING agonist-4 and Fe2+ ions. The STING agonist level was determined by measuring the UV-vis spectra absorbance at 320 nm, while the Fe2+ ion level was evaluated using phenanthroline spectrophotometry. STING agonist-4 was received from Invivochem.[2]

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1. STING reporter gene assay: HEK293T cells were transfected with hSTING or mSTING expression plasmids and an IFN-β luciferase reporter plasmid. After 24 hours, cells were treated with serial concentrations of STING agonist-4 (0.001–10 μM) for 16 hours. Luciferase activity was measured, and EC₅₀ values were calculated based on dose-response curves [1]
2. STING phosphorylation assay: hSTING-HEK293T cells or mouse BMDMs were seeded and treated with STING agonist-4 (0.01–1 μM) for 6 hours. Cells were lysed, and proteins were separated by SDS-PAGE. Western blot was performed using antibodies against STING, phosphorylated STING (p-STING), phosphorylated IRF3 (p-IRF3), and GAPDH (loading control) to detect pathway activation [1]

Cell viability assay[1]
Cell Types: Human peripheral blood mononuclear cells (PBMC)
Tested Concentrations: 0.3 μM, 1 μM, 3 μM, 10 μM and 30 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: Causes phosphorylation of IRF3 and STING and induces secretion Interferon-beta.

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CCK-8 assay[2]
MC38 cells (5000) were seeded in 96-well plates and cultured overnight. Next, PBS, OMV/FA, OMV/FeFA, OMV/SaFA, and OMV/SaFeFA were added to the cell culture medium at concentrations of 15 µg/mL for OMV and 7.5 µg/mL for STING agonist-4. After 24 h, cell viability was assessed using a CCK-8 kit.[2]

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ROS and •OH imaging[2]
MC38 cells (1 × 105) were seeded in confocal small dishes and cultured overnight. Then, PBS, OMV/FA, OMV/FeFA, OMV/SaFA, OMV/SaFeFA, H2O2 (200 µM), and OMV/SaFeFA + H2O2 (200 µM) were added into the cell culture medium. The concentrations of OMV and STING agonist-4 were 15 and 7.5 µg/mL, respectively. After 24 h, the cells were stained with the DCFH-DA (ROS fluorescent probe) and BboxiProbe O26 (•OH fluorescent probe) according to the manufacturer protocols and imaged by a CLSM. The fluorescent intensity of the images was measured by an Image J software. Ferroptosis study[2]
MC38 cells (1 × 105) were seeded in confocal small dishes and cultured overnight. Subsequently, PBS, OMV/FA, OMV/FeFA, OMV/SaFA, OMV/SaFeFA, and FeCl2 (30 µg/mL) were added to the cell culture medium. The concentrations of OMV and STING agonist-4 were 15 and 7.5 µg/mL, respectively. Following a 24-h incubation period, cells were stained with the Liperfluo probe according to the manufacturer’s protocols and imaged using a CLSM.

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1. PBMC cytokine production assay: Human PBMCs were isolated and plated, then treated with STING agonist-4 (0.01–1 μM) for 24 hours. Culture supernatants were collected, and IFN-β and CXCL10 concentrations were quantified by enzyme-linked immunosorbent assay (ELISA). Mouse BMDMs were prepared similarly, treated with 0.03–3 μM STING agonist-4, and cytokines were measured [1]
2. Tumor cell-PBMC co-culture assay: A549 lung cancer cells were seeded in 96-well plates. After adherence, human PBMCs (effector cells) were added at a 10:1 effector-to-target ratio, along with STING agonist-4 (0.01–1 μM). After 72 hours of co-culture, tumor cell viability was measured using a cell proliferation assay kit. For neutralization experiments, anti-IFN-β antibody was added to the co-culture system [1]

Biodistribution[2]
All mouse experiments were implemented in accordance with protocols approved by the Animal Experimental Ethics Committee of the Fujian Normal University (No. IACUC-20220005). C57/BL6 and BALB/c mice (4–6 weeks) were used. Nine MC38 tumor-bearing C57/BL6 mice were randomly divided into 3 groups. Mice were i.v. injected with CY5, OMV/CY5-PEG, and, OMV/CY5-PEG-FA, with CY5 and OMV doses set at 50 µg and 60 µg, respectively. Then, the fluorescent signals of mice were recorded at different time points (20 min, 2 h, 4 h, 8 h, 10 h, and 24 h) using an IVIS system. After 24 h, mice were euthanized, and the fluorescent signals of tumors and major organs were recorded. For the 4T1 breast tumor model, nine tumor-bearing BALB/c mice were randomly allocated into 3 groups. These mice received i.v. injection of CY5, OMV/CY5-PEG, and, OMV/CY5-PEG-FA. At 24 h post-injection, mice were euthanized, and the fluorescent signals of tumors and major organs were recorded. Fluorescence images of tumor slices were captured using a CLSM. In vivo therapy[2]
C57/BL6 mice received an injection of 1 × 106 MC38 cells in the right hind leg flank. Ten days later, the MC38 tumor-bearing mice were randomly divided into 5 groups (n = 4) and i.v injected with PBS, OMV/FA, OMV/SaFA, OMV/FeFA, and OMV/SaFeFA on days 0 and 4. Tumor size and body weight were measured every two days throughout the treatment period, with tumor volumes calculated using the formula: V= (length × width2)/2. On day 16, mice were euthanized, and the tumors were collected. Tumors were weighted, sliced, and analyzed for H&E, TUNEL staining, and immunohistochemical staining of IFN-γ, SLC7A11, GPX4, NCOA4, and FTH1. Serum was collected for IFN-γ detection using a commercial kit. In addition, the long-term survival rates were evaluated by the same procedures except for monitoring 58-day period. Biosafety[2]
PBS, OMV/FA, OMV/FeFA, OMV/SaFA, and OMV/SaFeFA were administered via the tail vein of C57/BL6 mice. After a 16-day treatment, blood samples from C57/BL6 mice were collected for biochemical analysis. The parameters measured included blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), and gamma glutamyl transpeptidase (γ-GT), using assay kits from Solarbio (BC1555, BC1535, BC2145, BC1565, and BC1225). Subsequently, mice were sacrificed, and the major organs (heart, liver, spleen, lung, and kidney) were harvested and subjected to H&E staining for histological analysis.

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1. Subcutaneous tumor models: C57BL/6 mice were subcutaneously inoculated with B16-F10 melanoma cells (1×10⁶ cells/mouse) or MC38 colon cancer cells (5×10⁵ cells/mouse). When tumors reached 100–150 mm³, mice were randomly divided into treatment and control groups. STING agonist-4 was administered intravenously at 1 mg/kg once weekly for 3 weeks, while the control group received vehicle. Tumor volume was measured every 2 days, and mice were sacrificed at the end of treatment to collect tumors and spleen for immune cell analysis [1]
2. Abscopal effect model: C57BL/6 mice were subcutaneously inoculated with B16-F10 cells in both left and right flanks. When tumors reached 100–150 mm³, STING agonist-4 (1 mg/kg) was intravenously administered once weekly for 3 weeks. Tumor volumes of both treated and contralateral tumors were measured every 2 days [1]
3. Systemic immune activation assessment: C57BL/6 mice were intravenously injected with STING agonist-4 (1 mg/kg) or vehicle. At 6, 12, and 24 hours post-administration, blood was collected to measure serum IFN-β and CXCL10 levels by ELISA. Spleens were harvested at 24 hours, and splenocytes were analyzed by flow cytometry for CD69 expression on CD8⁺ T cells and NK cells [1]

1. Plasma pharmacokinetics: After intravenous injection of STING agonist-4 (1 mg/kg) into C57BL/6 mice, the plasma half-life (t₁/₂) was 4.2 ± 0.5 h, the maximum plasma concentration (Cₘₐₓ) was 8.5 ± 1.2 μM, and the area under the curve (AUC₀₋₂₄h) was 32.6 ± 3.8 μM·h [1]
2. Tissue distribution: Two hours after intravenous injection of STING agonist-4 (1 mg/kg), it was distributed in major tissues, including the liver (6.8 ± 0.8 μg/g), spleen (5.2 ± 0.6 μg/g), lung (4.5 ± 0.5 μg/g), and tumor (3.9 ± 0.4 μg/g). Low accumulation was observed in brain tissue (0.3 ± 0.1 μg/g) [1]
3. Metabolism: In vitro liver microsomal incubation experiments showed that STING agonist-4 is metabolized slowly, with a metabolic stability half-life (t₁/₂) of 38 ± 4 minutes in human liver microsomes and 45 ± 5 minutes in mouse liver microsomes [1]

1. Acute toxicity: No deaths were observed within 14 days after intravenous injection of STING agonist-4 at doses up to 5 mg/kg (5 times the therapeutic dose) into C57BL/6 mice. Mouse weight did not change significantly, and histopathological analysis of liver and kidney tissues showed no obvious tissue damage [1]. 2. Hematological and biochemical indicators: Compared with the solvent control group, mice injected with STING agonist-4 once a week for 3 consecutive weeks showed no significant abnormalities in white blood cell count, red blood cell count, platelet count, liver function indicators (ALT, AST), and kidney function indicators (BUN, creatinine) [1].

[1]. Design of amidobenzimidazole STING receptor agonists with systemic activity. Nature. 2018 Dec;564(7736):439-443.
[2]. Metal ions-anchored bacterial outer membrane vesicles for enhanced ferroptosis induction and immune stimulation in targeted antitumor therapy. J Nanobiotechnology . 2024 Aug 9;22(1):474.

Interferon gene-stimulating factor (STING) is a receptor on the endoplasmic reticulum that mediates the recognition of cytoplasmic pathogen-derived DNA and the innate immune system. In recent years, the development of compounds capable of modulating STING has become a research hotspot in cancer and infectious disease treatment, as well as as vaccine adjuvants. To our knowledge, current research mainly focuses on developing modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these compounds have entered clinical trials for the treatment of patients with solid tumors suitable for intratumoral administration. This paper reports the discovery of a small-molecule STING agonist that is not a cyclic dinucleotide and exhibits systemic therapeutic effects against mouse tumors. We developed a linker strategy that utilizes the synergistic effect of two symmetrically related aminobenzimidazole (ABZI) compounds to construct a linked ABZI (diABZI) with enhanced STING binding capacity and cellular function. Intravenous injection of the dibenzothiazolinone STING agonist into immunocompetent mice with established homologous colon tumors induced strong antitumor activity and resulted in complete and durable tumor regression. Our discovery is a milestone in the rapidly developing field of immunomodulatory cancer therapy. [1]

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Activating ferroptosis is a multifunctional strategy to enhance the antitumor immune response in cancer treatment. However, developing ferroptosis inducers with both high biocompatibility and therapeutic efficiency remains a challenge. In this study, we propose a novel approach for tumor therapy using bionanoparticles derived from Escherichia coli outer membrane vesicles (OMVs) to activate ferroptosis and stimulate the immune response. Specifically, we anchor ferrous ions to OMVs via electrostatic interactions and load them with the STING agonist-4, followed by tumor-targeting DSPE-PEG-FA modification, thereby functionalizing OMVs, hereinafter referred to as OMV/SaFeFA. The anchoring of ferrous ions endows OMVs with peroxidase-like activity, enabling them to induce cellular lipid peroxidation by catalyzing the generation of •OH from H₂O₂. Furthermore, OMV/SaFeFAs also exhibit pH-responsive release of ferrous ions and agonists, as well as tumor-targeting capabilities, thereby achieving tumor-specific therapy and minimizing side effects. Notably, the simultaneous activation of the STING pathway and ferroptosis elicited a strong antitumor response in a mouse model of colon cancer, resulting in superior therapeutic efficacy and prolonged survival. Importantly, no acute toxicity was observed in mice treated with OMV/SaFeFA, highlighting its potential for future cancer therapy and clinical translation. [2]

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1. STING agonist-4 is an amide-benzimidazole-derived STING receptor agonist with higher selectivity for STING than other innate immune receptors (TLR4, TLR9, RIG-I). [1]
2. The antitumor mechanism of STING agonist-4 includes activation of the STING-IRF3 pathway, induction of type I interferon and pro-inflammatory cytokines, and enhancement of antitumor immune response by increasing the infiltration of cytotoxic T cells and NK cells into tumors. [1]
3. STING agonist-4 has good pharmacokinetic properties (long half-life, good tissue distribution) and low acute toxicity, supporting its potential as a systemic antitumor therapy. [1]

C34H38N12O4

678.7435

678.313

C, 60.16; H, 5.64; N, 24.76; O, 9.43

2138300-40-8

132000066

Typically exists as white to gray solids

2

4

8

13

50

1150

0

ICZSAXDKFXTSGL-UHFFFAOYSA-N

InChI=1S/C34H38N12O4/c1-5-45-27(15-19(3)41-45)31(49)39-33-37-23-17-21(29(35)47)9-11-25(23)43(33)13-7-8-14-44-26-12-10-22(30(36)48)18-24(26)38-34(44)40-32(50)28-16-20(4)42-46(28)6-2/h9-12,15-18H,5-8,13-14H2,1-4H3,(H2,35,47)(H2,36,48)(H,37,39,49)(H,38,40,50)

1-[4-[5-carbamoyl-2-[(2-ethyl-5-methylpyrazole-3-carbonyl)amino]benzimidazol-1-yl]butyl]-2-[(2-ethyl-5-methylpyrazole-3-carbonyl)amino]benzimidazole-5-carboxamide

STING agonist-4; 2138300-40-8; diABZI STING agonist-2; STING agonist 2; 1,1'-(1,4-butanediyl)bis[2-[[(1-ethyl-3-methyl-1H-pyrazol-5-yl)carbonyl]amino]-1H-benzimidazole-5-carboxamide; CHEMBL4440744; STING agonist diABZI compound 2; 1-[4-[5-carbamoyl-2-[(2-ethyl-5-methylpyrazole-3-carbonyl)amino]benzimidazol-1-yl]butyl]-2-[(2-ethyl-5-methylpyrazole-3-carbonyl)amino]benzimidazole-5-carboxamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~10 mg/mL (~14.73 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: 1 mg/mL (1.47 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 1 mg/mL (1.47 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)