Fluorescent dye

Glutamate may be released from microglia by mechanisms other than SxC−. To confirm that SxC− was the transporter inhibited by the novel DBT analogs, cystine uptake was measured for two of the test substances. Primary microglia were treated for 15 h, and then 35DBTA7 and 35DBTSA12 were applied during uptake of selenocystine, which can be detected after conjugation with fluorescein O,O'-diacrylate (Fig. 5). A dose-dependent inhibition of cystine uptake into microglia was observed for both compounds at IC50 values roughly similar to those for glutamate release [1].

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Another study developed fluorescent cationic and anionic nanogels linked with fluorescein O,O'-diacrylate for the visualization of cellular fluorescence and then loaded with dual therapeutic agents cisplatin and 5-fluorouracil for lung cancer (NCI-H1437) and colorectal cancer (HCT-116) pharmacotherapy and image processing functionality of the nanogel in response to pH. Both cationic and anionic nanogels had a higher fluorescence intensity in basic conditions and a substantially lower fluorescence intensity in acidic conditions [2].

Cystine uptake assay [1]
Cystine uptake was measured essentially as per Shimomura et al. using the Cystine Uptake Assay Kit (DOJINDO). Primary microglia were seeded as per glutamate release assays, and some cultures were treated the following day with 100 ng/ml LPS. After 15 h, the cultures were washed twice with Hank’s balanced salt solution (HBSS) and then incubated 5 min at 37 °C in HBSS containing fresh LPS and test substances at the indicated concentrations. The cultures were then exposed to HBSS containing selenocystine for 30 min at 37 °C, after which they were washed three times with ice-cold phosphate-buffered saline and then fixed in 50 μl methanol. A detection buffer comprising fluorescein O,O'-diacrylate and reducing agent was then added, and the plate was sealed and incubated for 30 min at 37 °C. Reaction product was then assayed in the Molecular Devices SpectraMax 3 with excitation at 490 nm and emission at 535 nm. Blanks consisted of wells containing only cells that had been washed, fixed with methanol, and incubated with the detection buffer, and the values from these wells were subtracted from the experimental values.

[1]. Design, synthesis, and characterization of novel system xC− transport inhibitors: inhibition of microglial glutamate release and neurotoxicity. J Neuroinflammation. 2023 Dec 6;20:292.
[2]. Advanced cisplatin nanoformulations as targeted drug delivery platforms for lung carcinoma treatment: a review. Journal of Materials Science. 2022，57, 16192–16227.

Neuroinflammation appears to be associated with excitotoxic release from microglia via the cysteine-glutamate antitransporter system (SxC−). To mitigate this neuronal stress and toxicity, we developed a series of SxC− antitransporter inhibitors. These compounds are based on L-tyrosine because its structural elements match those of glutamate, the primary physiological substrate of the SxC− antitransporter. In addition to 3,5-dibromotyrosine, we synthesized ten compounds via amidation reactions of this parent molecule with a series of acyl halides. We tested the ability of these compounds to inhibit glutamate release from lipopolysaccharide (LPS)-activated microglia, and eight compounds exhibited this activity. To confirm that these compounds are SxC− inhibitors, we further tested the ability of two of them to inhibit cysteine uptake. Finally, the study showed that these drugs can protect primary cortical neurons from the toxic effects of activated microglia. These drugs hold promise for mitigating the neurodegenerative effects of neuroinflammation in diseases such as encephalitis, traumatic brain injury, stroke, or neurodegenerative diseases. [1] Lung cancer is the second leading cause of cancer-related death worldwide. Cisplatin is a first-line chemotherapy drug for the treatment of lung cancer. The biggest challenges in using this drug to treat lung cancer include cell resistance, low water solubility, and adverse reactions to normal cells. To address these issues, nanotechnology-based drug delivery methods have shown encouraging results in improving drug uptake by cancer cells while minimizing adverse reactions. Studies have shown that cisplatin formulations, including polymer nanoparticles, micelles, dendritic polymers, and liposomes, are more likely to respond to changes in the tumor microenvironment and deliver sustained cisplatin to the tumor site. This review explores various in vitro and in vivo studies of cisplatin nanoparticles and covers clinical trials using nanocarrier-based cisplatin delivery. To our knowledge, this is the first comprehensive review article to provide a detailed overview of the application of cisplatin nanoparticles in the treatment of lung cancer. [2]

C26H16O7

440.40

440.089602

7262-39-7

4589514

Typically exists as solids at room temperature

160-163ºC(lit.)

4.8

0

7

6

33

788

0

C=CC(=O)OC1=CC2=C(C=C1)C3(C4=C(O2)C=C(C=C4)OC(=O)C=C)C5=CC=CC=C5C(=O)O3

GTQZGVDHWVBMOJ-UHFFFAOYSA-N

InChI=1S/C26H16O7/c1-3-23(27)30-15-9-11-19-21(13-15)32-22-14-16(31-24(28)4-2)10-12-20(22)26(19)18-8-6-5-7-17(18)25(29)33-26/h3-14H,1-2H2

(3-oxo-6'-prop-2-enoyloxyspiro[2-benzofuran-1,9'-xanthene]-3'-yl) prop-2-enoate

Diacryloyloxyfluorescein; Diacryloyloxyfluorescein; 7262-39-7; FLUORESCEIN O O'-DIACRYLATE 98; 3-Oxo-3h-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl diacrylate; (3-oxo-6'-prop-2-enoyloxyspiro[2-benzofuran-1,9'-xanthene]-3'-yl) prop-2-enoate; Fluorescein O,O'-dimethacrylate; Fluorescein O,O'-diacrylate; Fluorescein O,O inverted exclamation marka-diacrylate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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