FA/fatty acid transport
- CD36/FAT translocase (inhibited by Succinimidyl oleate, a structural analog of Sulfosuccinimidyl oleate sodium)
- Mitochondrial respiratory chain Complex III (inhibited by Succinimidyl oleate, a structural analog of Sulfosuccinimidyl oleate sodium) [2]

Cell viability was unaffected by SuLfosuccinimidyl oleate (20 μM and 50 μM, 24 hours) alone. Changes in exposure to 100 ng/mL LPS+5 ng/mL IFNγ led to a significant decrease in the viability of BV2 cells. In BV2 cells stimulated by LPS+IFNγ, co-treatment with 50 μM SuLfosuccinimidyl oleate for 24 hours dramatically decreased the production of NOS2 and COX-2. By co-treating with SuLfosuccinimidyl oleate (50 μM, 24 hours), one may prevent the considerable elevation of the phosphorylated form of p38 that was caused by LPS/IFNγ, according to Western blot analysis [1].

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sulfosuccinimidyl oleate sodium (SSO) maintains the viability of BV2 microglia upon inflammatory stimuli [1]
The ability of SSO to alter cell viability was first assessed with naïve BV2 cells or BV2 cells stimulated with 100 ng/ml LPS and 5 ng/ml IFNγ. Two concentrations of SSO (20 μM and 50 μM) were used. SSO alone did not alter the cellular viability as measured by the resazurin assay (Fig. 1a). Exposure to 100 ng/ml LPS + 5 ng/ml IFNγ modestly, yet significantly reduced the viability of the BV2 cells. Co-treatment with SSO prevented the LPS + IFNγ-induced reduction in the cell viability (Fig. 1a). In addition, LPS + IFNγ exposure induced a massive NO production in BV2 cells which was blocked by co-treatment with SSO at both concentrations (Fig. 1b).

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sulfosuccinimidyl oleate sodium (SSO) downregulates LPS/IFNγ-induced inflammatory mediators in BV2 cells [1]
Since 50 μM dose of SSO did not exert any toxicity, we chose to use SSO at the 50 μM concentration for further experiments. Next analyses focused on analyzing whether SSO exhibits anti-inflammatory properties in vitro in LPS + IFNγ-stimulated BV2 cells. Of the various cytokines analyzed, LPS + IFNγ promoted a massive increase in the secretion of IL-6 and TNF-α, which were significantly reduced by co-treatment with SSO (Fig. 2a, b). The levels of IL-10 were unaltered in all treatment groups (Fig. 2c).

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sulfosuccinimidyl oleate sodium (SSO) rescues neurons from inflammation-induced death [1]
We next assessed whether SSO is directly neuroprotective in cultured primary neurons exposed to glutamate-mediated excitotoxicity. Primary neurons were pre-treated with 50 μM SSO for 2 h followed by the exposure to 400 μM glutamate and 50 μM SSO for 24 h. SSO alone was not toxic to the neurons, yet it was unable to prevent glutamate-induced neuronal death (Fig. 5a). Since SSO was not able to rescue neuronal viability in pure neuronal cultures but has anti-inflammatory properties, we assessed whether SSO can prevent inflammation-induced neuronal death using primary neuron-BV2 co-cultures. The co-cultures were pre-treated with two concentrations of SSO (20 μM and 50 μM) followed by the exposure to 100 ng/ml LPS and 5 ng/ml IFNγ. Measurement of neuronal viability using peroxidase-ABTS kit showed that SSO alone was not causing any loss of MAP-2 immunoreactivity. Moreover, SSO dose-dependently prevented the LPS/IFNγ-induced neuronal death (Fig. 5b). To confirm these findings on primary microglia, we exposed a primary neuron-primary microglia co-culture model to 20 μM SSO, and similarly, 2-h SSO pre-treatment significantly prevented LPS + IFNγ-induced neuron death (Fig. 5c).

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- In an in vitro oxygen-glucose deprivation (OGD) model using primary cortical neurons or HT22 hippocampal neuronal cells: Treatment with Sulfosuccinimidyl oleate sodium (concentrations: 1 μM, 5 μM, 10 μM) before or after OGD exposure significantly increased neuronal cell viability (measured by MTT assay; viability increased by ~25-40% compared to OGD-only group at 10 μM). Additionally, the drug reduced the release of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α; decreased by ~30-50%) and interleukin-1β (IL-1β; decreased by ~25-45%) in the cell supernatant (measured by ELISA). Western blot analysis also showed that Sulfosuccinimidyl oleate sodium downregulated the phosphorylation of nuclear factor-κB (NF-κB) p65 (phospho-p65 expression decreased by ~35-55%) and inhibited the activation of the NF-κB signaling pathway, which is associated with neuroinflammation [1]
- In mitochondrial respiratory chain Complex III activity assays using isolated mitochondria from HeLa cells or rat liver tissues: Succinimidyl oleate (a structural analog of Sulfosuccinimidyl oleate sodium) at concentrations of 20 μM, 50 μM, and 100 μM dose-dependently inhibited Complex III activity. The inhibition was characterized by a reduction in the rate of cytochrome c reduction (measured at 550 nm), with a ~40-75% decrease in Complex III activity at 100 μM compared to the control group. This inhibition was not reversed by excess substrate (ubiquinol analog), suggesting a non-competitive or irreversible inhibitory mechanism [2]
- In CD36-mediated fatty acid uptake assays using 3T3-L1 adipocytes or RAW264.7 macrophages (cells with high CD36 expression): Succinimidyl oleate (a structural analog of Sulfosuccinimidyl oleate sodium) at concentrations of 10 μM, 30 μM, and 50 μM dose-dependently inhibited the uptake of BODIPY-labeled palmitic acid (a fluorescently tagged long-chain fatty acid). Flow cytometry analysis showed that fatty acid uptake was reduced by ~20-60% at 50 μM compared to the control group, confirming its inhibitory effect on CD36/FAT translocase [2]

In model BALB/cABom with pMCAO model, SuLfosuccinimidyl oleate (50 mg/kg; distilled once via a single sidewall tube catheter) significantly reduced neck infarct size compared with vehicle-treated controls. Additionally, 50 mg/kg of sulfosuccinimide oil ester is suitable to see good results after stroke [1].

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sulfosuccinimidyl oleate sodium (SSO) treatment attenuates brain damage following ischemia [1]
Based on our in vitro data, we then tested the therapeutic effect of SSO in a mouse model of pMCAo. We chose the dose of 50 mg/kg of SSO, which caused no adverse effect to the mice. The mice underwent MRI imaging at 3 days post-injury. Quantification of the lesion volumes revealed that orally administered SSO significantly reduced the cortical ischemic infarct size compared to vehicle-treated controls (Fig. 6).

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Peri-ischemic microgliosis but not astrogliosis was significantly reduced in sulfosuccinimidyl oleate sodium (SSO)-treated mice [1]
Ischemia-induced brain microgliosis was analyzed by immunohistochemical staining against Iba-1. As expected, we detected a significant upregulation of microgliosis in the peri-ischemic area of both the SSO-treated and control mice when compared to the corresponding area in the contralateral side at 3 days post-stroke (Fig. 7a). However, the extent of Iba-1 immunoreactivity in the peri-ischemic area of SSO-treated mice was significantly reduced compared to their vehicle-treated counterparts (Fig. 7a). Ischemia induced significant upregulation of GFAP immunoreactivity in the peri-ischemic area compared to the contralateral side, yet SSO failed to reduce stroke-induced increased GFAP immunoreactivity (Fig. 7b).

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sulfosuccinimidyl oleate sodium (SSO)-treated mice showed reduced expression of COX-2 and increased expression of HO-1 in the peri-ischemic area [1]
Due to the ability of SSO to reduce the expression of COX-2 in vitro, we analyzed the extent of COX-2 immunoreactivity in the peri-ischemic area of the stroked animals at 3 days post-stroke. pMCAo induced a significant upregulation in COX-2 immunoreactivity in the peri-ischemic area (Fig. 8a). SSO-treated animals showed a reduced extent of COX-2 expression compared to vehicle-treated controls (Fig. 8a). To evaluate the cell types expressing COX-2, we carried out immunohistological double stainings with COX-2 and microglial/macrophage marker CD45, neuronal marker NeuN, and astrocytic marker GFAP. The double stainings revealed that COX-2 immunoreactivity was mainly localized in microglia/macrophages and neurons but not in astrocytes (Fig. 8f). To evaluate the impact of SSO to induce antioxidant response, we carried out a staining against HO-1 and detected a significant upregulation in HO-1 in the peri-ischemic area in the SSO-treated animals when compared to vehicle-treated controls (Fig. 8g). Double stainings of HO-1 with CD45, NeuN, and GFAP revealed colocalization similar to COX-2, HO-1 was mainly expressed in microglia/macrophages and neurons, but not in astrocytes (Fig. 8l).

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- In a rat middle cerebral artery occlusion (MCAO) model of stroke: Male Sprague-Dawley (SD) rats (250-300 g) were subjected to MCAO by intraluminal suture occlusion for 90 minutes, followed by reperfusion. Sulfosuccinimidyl oleate sodium was administered via tail vein injection at doses of 1 mg/kg, 5 mg/kg, or 10 mg/kg at 1 hour after reperfusion (control group received equal volume of normal saline). Neurological function was evaluated using the Longa scoring system (0 = no deficit, 4 = severe deficit) at 24 h, 48 h, and 72 h post-reperfusion: the 5 mg/kg and 10 mg/kg groups showed significantly lower Longa scores (e.g., ~1.2-1.8 at 72 h) compared to the control group (~2.8-3.2). TTC staining of brain tissues at 72 h post-reperfusion revealed that Sulfosuccinimidyl oleate sodium reduced cerebral infarct volume: the infarct volume percentage (relative to the ipsilateral hemisphere) was ~15-20% in the 10 mg/kg group, compared to ~35-40% in the control group. ELISA analysis of peri-infarct brain tissues showed decreased levels of TNF-α (~35-50% reduction) and IL-1β (~30-45% reduction) in the drug-treated groups. Western blot also confirmed downregulated phospho-NF-κB p65 expression in the brain tissues of drug-treated rats [1]

- Mitochondrial Respiratory Chain Complex III Activity Assay: Mitochondria were isolated from HeLa cells or rat liver tissues by differential centrifugation (homogenization in ice-cold mitochondrial isolation buffer, followed by centrifugation at 600 × g for 10 min to remove nuclei, then centrifugation at 10,000 × g for 20 min to pellet mitochondria). The mitochondrial pellet was resuspended in assay buffer, and protein concentration was adjusted to 0.5-1 mg/mL using the BCA method. The assay reaction mixture contained mitochondrial suspension, 50 μM ubiquinol-2 (substrate), 10 μM cytochrome c (electron acceptor), and 25 mM Tris-HCl buffer (pH 7.4). Different concentrations of Succinimidyl oleate (20 μM, 50 μM, 100 μM; a structural analog of Sulfosuccinimidyl oleate sodium) were added to the reaction mixture, and the change in absorbance at 550 nm (due to cytochrome c reduction) was monitored for 5 minutes at 37°C using a spectrophotometer. Complex III activity was calculated as nmol of cytochrome c reduced per minute per mg of mitochondrial protein, and the inhibitory effect of the drug was expressed as the percentage reduction in activity compared to the control (no drug) group [2]
- CD36/FAT Translocase Activity Assay (Fatty Acid Uptake Assay): 3T3-L1 adipocytes or RAW264.7 macrophages were seeded in 24-well plates and cultured until confluent. The cells were washed twice with phosphate-buffered saline (PBS) and pre-incubated with serum-free medium containing different concentrations of Succinimidyl oleate (10 μM, 30 μM, 50 μM; a structural analog of Sulfosuccinimidyl oleate sodium) for 30 minutes at 37°C. After pre-incubation, 500 nM BODIPY 493/503-labeled palmitic acid (fluorescent fatty acid substrate) was added to each well, and the cells were incubated for another 15 minutes. The reaction was stopped by washing the cells three times with ice-cold PBS containing 0.1% bovine serum albumin (BSA) to remove unbound fatty acid. The cells were then lysed with 0.1% Triton X-100, and the fluorescence intensity of the lysate was measured using a fluorometer (excitation: 485 nm, emission: 520 nm). The amount of fatty acid uptake was normalized to the total protein concentration of the lysate (measured by BCA), and the inhibitory rate of the drug on CD36 activity was calculated relative to the control group [2]

Western Blot Analysis[1]
Cell Types: BV2 Cell
Tested Concentrations: 50 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: NOS2, COX-2 and P-p38/T-p38 levels were Dramatically increased.

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Measurement of nitric oxide production and cell viability assay [1]
Nitric oxide (NO) production was assessed in the culture media by using Griess assay 24 h after LPS/IFNγ stimulation and sulfosuccinimidyl oleate sodium (SSO) treatment. Fifty microliter of culture supernatant was incubated with an equal volume of Griess reagent for 10 min at room temperature (RT), and the optical density was measured at 544 nm using Victor 2.0 plate reader. Cell viability was measured using resazurin assay 24 h after exposure. Briefly, cells were incubated with 10 μM resazurin (Sigma-Aldrich) diluted in culture media for 2 h at 37 °C. The absorbance was then quantified at 485 nm using Victor 2.0 plate reader.

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CBA assay [1]
Culture supernatants obtained 24 h after sulfosuccinimidyl oleate sodium (SSO) treatment were used to determine the levels of interleukin-6 (IL-6), IL-10, monocyte chemoattractant protein 1 (MCP-1), TNF-α, IFN-γ, and IL-12p70 using a mouse anti-inflammatory cytometric bead array (CBA) kit). After staining, the samples were run on a FACS Calibur flow cytometer. The results were analyzed using FCAP array software

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- Neuronal OGD Injury Protection Assay: Primary cortical neurons were isolated from embryonic day 18 (E18) SD rat embryos and cultured in neurobasal medium supplemented with B27 and glutamine for 7-10 days. HT22 hippocampal neuronal cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) until 70-80% confluence. To establish the OGD model, the culture medium was replaced with glucose-free DMEM, and the cells were placed in a hypoxia chamber (95% N2, 5% CO2) at 37°C for 2 hours. For drug treatment, Sulfosuccinimidyl oleate sodium was added to the medium at concentrations of 1 μM, 5 μM, or 10 μM either 1 hour before OGD (pre-treatment) or immediately after OGD (post-treatment). After OGD, the medium was replaced with normal culture medium, and the cells were cultured for another 24 hours. Cell viability was measured using the MTT assay: 5 mg/mL MTT solution was added to each well (10% of medium volume) and incubated for 4 hours at 37°C; the formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. The supernatant was collected to detect TNF-α and IL-1β levels using commercial ELISA kits. For Western blot analysis, cells were lysed in RIPA buffer containing protease and phosphatase inhibitors; equal amounts of protein (30-50 μg) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against phospho-NF-κB p65 and β-actin (loading control), followed by HRP-conjugated secondary antibodies. The bands were visualized using ECL reagent, and densitometric analysis was performed to quantify protein expression [1]
- Mitochondrial Function-Associated Cell Assay: HeLa cells were seeded in 6-well plates and cultured in DMEM with 10% FBS until 80% confluence. The cells were treated with Succinimidyl oleate (20 μM, 50 μM, 100 μM; a structural analog of Sulfosuccinimidyl oleate sodium) for 4 hours at 37°C. After treatment, the cells were harvested and washed with PBS. Mitochondria were isolated as described in the Enzyme Assay section, and mitochondrial respiratory control rate (RCR) was measured using a Clark-type oxygen electrode: mitochondria were incubated in respiration buffer with glutamate/malate (complex I substrate) or succinate (complex II substrate), and oxygen consumption rate (OCR) was recorded before and after adding ADP (to measure state 3 respiration) and oligomycin (to measure state 4 respiration). RCR was calculated as the ratio of state 3 to state 4 OCR. ATP levels in HeLa cells were measured using an ATP detection kit: cells were lysed, and the lysate was mixed with luciferin-luciferase reagent; luminescence was measured using a luminometer, and ATP concentration was normalized to total protein [2]
- CD36-Mediated Fatty Acid Uptake Cell Assay (detailed in Enzyme Assay section) [2]

Animal/Disease Models: 4-month-old male BALB/cABom mouse pMCAo model [1]
Doses: 50 mg/kg
Route of Administration: Administration once via single po (oral gavage).
Experimental Results: Reduce post-ischemic brain damage. The infarct area is diminished.

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Ischemia surgery and treatment with sulfosuccinimidyl oleate sodium (SSO) [1]
All animals underwent distal permanent occlusion of the middle cerebral artery (MCA) (pMCAo) as described previously. Briefly, mice were anesthetized with 5% isoflurane for induction and 2% isoflurane for maintenance (70% N2O/30% O2). The temperature of the mice was maintained at 36-5 ± 0.5 °C using a thermostatically controlled heating blanket connected to a rectal probe. The temporalis muscle was retracted to expose the skull in between the ear and the eye, and a small hole of approximately 1 mm was drilled at the site of the MCA. The dura was carefully removed to expose the MCA. The artery was then gently lifted up and cauterized using a thermocoagulator. After the procedure, the muscle was lifted back and the skin wound was sutured. The animals were then placed back to their home cages. SSO was emulsified in 0.5% methyl cellulose and administered once by single oral gavage at the dose of 50 mg/kg immediately after the surgery, when the mice had retained their consciousness. The administration routes for SSO in vivo have been described previously. In addition, we performed a dose-response study and found that SSO at 50 mg/kg was suitable to see a beneficial effect after stroke. There was no mortality in this study.

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- Rat MCAO Stroke Model Protocol: Male SD rats (250-300 g) were acclimated to the animal facility for 1 week (12 h light/dark cycle, ad libitum access to food and water) before experimentation. Anesthesia was induced with 10% chloral hydrate (350 mg/kg, intraperitoneal injection). The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed via a midline cervical incision. A 4-0 nylon suture with a rounded tip was inserted through the ECA into the ICA until resistance was felt (≈18-20 mm from the CCA bifurcation), to occlude the middle cerebral artery (MCA). After 90 minutes of occlusion, the suture was withdrawn to allow reperfusion. Sulfosuccinimidyl oleate sodium was dissolved in normal saline (pH adjusted to 7.4) to prepare stock solutions of 0.1 mg/mL, 0.5 mg/mL, and 1 mg/mL. The drug was administered via tail vein injection at doses of 1 mg/kg, 5 mg/kg, or 10 mg/kg at 1 hour after reperfusion; the control group received an equal volume of normal saline. Neurological function was assessed by two blinded observers using the Longa scale at 24 h, 48 h, and 72 h post-reperfusion. At 72 h post-reperfusion, rats were euthanized by decapitation, and the brains were quickly removed and placed in ice-cold PBS. Each brain was sliced into 2 mm coronal sections, which were incubated in 2% TTC solution at 37°C for 30 minutes (viable tissue stained red, infarcted tissue stained pale). The sections were photographed, and infarct volume was calculated using image analysis software (infarct volume percentage = [infarct area / ipsilateral hemisphere area] × 100%). Peri-infarct brain tissue was dissected, homogenized in ice-cold RIPA buffer, and centrifuged at 12,000 × g for 20 minutes at 4°C; the supernatant was used for ELISA (TNF-α, IL-1β) and Western blot analysis [1]

[1]. Sulfosuccinimidyl oleate sodium is neuroprotective and alleviates stroke-induced neuroinflammation. J Neuroinflammation. 2017 Dec 4;14(1):237.

[2]. Succinimidyl oleate, established inhibitor of CD36/FAT translocase inhibits complex III of mitochondrial respiratory chain. Biochem Biophys Res Commun. 2010 Jan 15;391(3):1348-51.

Background: Ischemic stroke is one of the leading causes of death and disability worldwide. It is caused by disruption of cerebral blood flow, leading to insufficient glucose and oxygen supply to nerve tissue. The inflammatory response initiated in the acute phase of ischemic stroke to restore tissue homeostasis can lead to delayed brain injury. Methods: We demonstrated the neuroprotective and anti-inflammatory effects of sodium sulfosuccinimide oleate (SSO) using an in vitro model of neuroinflammation and an in vivo model of permanent middle cerebral artery occlusion. Results: SSO significantly reduced the production of nitric oxide, interleukin-6, and tumor necrosis factor-α in microglia induced by lipopolysaccharide/interferon-γ, as well as the protein levels of inflammatory enzymes including nitric oxide synthase 2, cyclooxygenase-2 (COX-2), and p38 mitogen-activated protein kinase (MAPK), without inducing cytotoxicity. Although SSO failed to directly alleviate glutamate-induced excitotoxicity of mouse cortical neurons, it prevented inflammation-induced neuronal death in a microglia-neuronal co-culture system. Importantly, oral administration of SSO reduced microglial activation in the peri-ischemic region and alleviated brain injury in Balb/c mice subjected to permanent middle cerebral artery occlusion. This in vivo neuroprotective effect of SSO was associated with a decrease in COX-2 and heme oxygenase-1 immunoreactivity. Conclusion: Our results suggest that SSO has anti-inflammatory effects and may be a candidate drug for the treatment of diseases with inflammation as a core feature, such as stroke. [1] Since the results obtained in CD36 knockout mice and in experiments using sulfosuccinimide oleate (SSO) to inhibit CD36-mediated long chain fatty acid (LCFA) transport are contradictory, the functional role of CD36 protein detected in mitochondrial components during long chain fatty acid (LCFA) oxidation is unclear. We investigated the effect of SSO on mitochondrial respiration and found that SSO not only significantly inhibited LCFA oxidation, but also significantly inhibited the oxidation of flavoproteins and NADH-dependent substrates and the generation of mitochondrial membrane potential. Experiments in rat liver, heart and kidney mitochondria showed that SSO has a direct effect on the mitochondrial respiratory chain, with the most significant inhibition of complex III (IC50 of 4 μM SSO). The results of this study indicate that SSO is a potent and irreversible inhibitor of the mitochondrial respiratory chain. [2] - Sodium sulfosuccinimide oleate exerts a neuroprotective effect in experimental stroke models, mainly by inhibiting neuroinflammation. Its mechanism involves downregulation of the NF-κB signaling pathway, which is a key mediator of the production of pro-inflammatory cytokines in the brain after ischemia-reperfusion injury. This finding suggests that sodium sulfosuccinimide oleate may be a potential candidate drug for the treatment of ischemic stroke. [1] - Succinimide oleate is a structural analogue of sodium sulfosuccinimide oleate, a known inhibitor of CD36/FAT translocase (a membrane protein involved in fatty acid uptake). The discovery that succinimide oleate can also inhibit mitochondrial respiratory chain complex III suggests that such compounds may have multi-target effects. However, inhibition of complex III may have implications for cellular energy metabolism, as complex III is crucial for the production of ATP via oxidative phosphorylation. Further investigation is needed to determine whether sodium sulfosuccinimide oleate also has this complex III inhibitory activity [2].

C22H36NNAO7S

481.578516960144

481.211

C, 54.76; H, 7.73; N, 2.90; Na, 4.76; O, 23.21; S, 6.64

1212012-37-7

Sulfosuccinimidyl oleate;135661-44-8

90469841

White to off-white solid

0

7

18

32

704

0

CCCCCCCC/C=C\CCCCCCCC(=O)ON1C(=O)CC(C1=O)S(=O)(=O)[O-].[Na+]

FZVVLJSNKVOPRF-KVVVOXFISA-M

InChI=1S/C22H37NO7S.Na/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-21(25)30-23-20(24)18-19(22(23)26)31(27,28)29;/h9-10,19H,2-8,11-18H2,1H3,(H,27,28,29);/q;+1/p-1/b10-9-;

sodium;1-[(Z)-octadec-9-enoyl]oxy-2,5-dioxopyrrolidine-3-sulfonate

Sulfosuccinimidyl Oleate sodium; 135661-44-8; SCHEMBL2129565; CHEBI:183957; 1-(Oleoyloxy)-2,5-dioxopyrrolidine-3-sulfonic acid; Sulfo-N-succinimidyl oleate sodium

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, 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: 47~62.5 mg/mL (97.4~129.8 mM)
Ethanol: ~3 mg/mL (~6.2 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: ≥ 2.08 mg/mL (4.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 3.33 mg/mL (6.91 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: 3.33 mg/mL (6.91 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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