Ethyl ether

VES was extracted and purified from unripe pink wax apple fruit (Syzygium samarangense (Blume) Merrill and Perry) following the reported procedure [40 (link)]. 1,4-Dithiothreit, acrylamide, aminoguanidine (AG), ammonium peroxydisulfate, D-glucose, ethyl alcohol, ethyl ether, glycine, methanol, MG, pioglitazone hydrochloride (PIO), sodium chloride, sodium dodecyl sulfate (SDS), sodium phosphate dibasic, sulfuric acid, thiourea, tris base, Triton X-100, Tween-20, and urea were purchased from Sigma (St. Louis, MO, USA).

PLGA-PEG (45k:5k, LA:GA = 50:50, Akina, Inc., West Lafayette, IN, USA) polymer was covalently labeled with fluorescent dye before nanoparticle formulation. To activate the PLGA-PEG polymer, the polymer was dissolved in a 20 mL glass scintillation vial with dichloromethane (DCM, Fisher Scientific, Pittsburgh, PA, USA) to create a 100 mg/mL polymer solution. P-nitrophenyl chloroformate (PNCF, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DCM to create a 10 mg/mL stock solution. PNCF solution was added to the polymer solution, followed by the immediate addition of pyridine (Sigma-Aldrich). The solution was stirred and reacted for 3 h at 200–300 rpm, and then the polymer reaction solution was slowly added to cold ethyl ether (Sigma-Aldrich) to stop the reaction. The solution was then centrifuged at 1000× g for 2 min and lyophilized overnight to dry the activated polymer. After polymer drying, CF647 Succinimidyl Ester (CF647^®, Biotium, Fremont, CA, USA) was dissolved in dimethylformamide (DMF, Fisher Scientific) to make a 2 mg/mL stock solution. Activated polymer was dissolved in DMF, and then the CF647^® solution was added to the polymer solution. Triethylamine (TEA, Sigma-Aldrich) was added immediately after CF647 and reacted for 4 h. The solution was lyophilized overnight to dry the dye-labeled polymer. CF647-labeled polymer was stored at −20 °C for future use.