Pirarubicin-loaded PLGA Nanoparticle Synthesis

A PLGA NP was generated as described previously^{18 (link)} Briefly, 25 mg of PLGA (#B6017–1 50:50 DL-PLG, Corefront, Inc.) and 4 mg of pirarubicin (Sigma-Aldrich) were dissolved in 1 ml of acetone. The solution was then placed in 20 ml of a 2% polyvinyl alcohol (PVA)/distilled water mixture and irradiated with an ultrasonic wave at a power level of 1.0 (Sonicator 3000, Misonix Inc.) for 10 minutes followed by stirring for 16 h to evaporate the acetone. Then, the supernatant was collected after centrifugation at 200 RCF for 10 minutes and further centrifuged at 15000 RCF for 30 minutes with additional distilled water for washing. The washing step was repeated 3 times, and PBS was added to the pellet and stirred with a homogenizer at 10,000 rpm for 10 minutes to disperse the NPs. Finally, the solution was freeze-dried using a lyophilizer (VD-550R, TAITEC, Tokyo, Japan) for over 48 h. The freeze-dried powder was used for all the experiments. We generated rhodamine-labeled Pir-PLGA NPs the same method by mixing rhodamine with pirarubicin as a landmark of incorporated NPs to AdSCs.