基于焦脱镁叶绿酸-a不饱和脂肪酸前药的自组装纳米粒用于黑色素瘤光动力治疗中的体外研究OA

Objective This study aimed to construct a prodrug(PPA-SS-AA)by linking py-ropheophorbide-a(PPA)with arachidonic acid(AA)via a disulfide bond and to prepare self-as-sembled nanoparticles using this prodrug.The goal was to address the poor water solubility and in-sufficient targeting of PPA and to further evaluate the efficacy and mechanisms of this nanosystem for photodynamic therapy(PDT)against melanoma in vitro.Methods The PPA-SS-AA prodrug was synthesized via EDCI/DMAP-catalyzed esterification and co-assembled with DSPE-PEG2000 into nanoparticles using the nanoprecipitation method.The particle size,polydispersity index(PDI),and Zeta potential of the nanoparticles were characterized using a nanoparticle size analy-zer.The dark toxicity and phototoxicity of the nanoparticles were assessed by the CCK-8 assay in mouse melanoma B16 cells.Cellular uptake of the nanoparticles was investigated using flow cytom-etry and confocal microscopy.Apoptosis,cell death,and intracellular reactive oxygen species(ROS)generation were evaluated using Annexin V-FITC/PI double-staining flow cytometry,Cal-cein-AM/PI live-dead cell staining,and the DCFH-DA ROS probe,respectively.Results PA-SS-AA was successfully synthesized,and monodisperse nanoparticles with a particle size of 132.73 nm,a PDI of 0.102,and a Zeta potential of-23.24 mV were prepared.In vitro experiments showed that these nanoparticles could be efficiently taken up by B16 cells and exhibited a potent photodynamic killing effect under 633 nm laser irradiation,with an IC50 value of 14.43 μg/mL.Compared to the non-irradiated group,the cell viability in the irradiated group decreased to 12.3％(P＜0.001),with an apoptosis rate of 51.95％,which was 5.89 times higher than that in the non-irradiated group(P＜0.001).Mechanistic studies confirmed that the nanoparticles generated a large amount of ROS in the cells after irradiation,with levels 3.2 times higher than those in the non-irradiated group(P＜0.001),effectively triggering the apoptotic pathway.Con-clusions This study successfully constructs a self-assembled nano-delivery system based on PPA-SS-AA.This system significantly improves the delivery efficiency of PPA and efficiently generates ROS upon photoactivation,thereby inducing apoptosis and cell death in melanoma cells,demon-strating excellent potential for in vitro photodynamic therapy.This prodrug-based nanoplatform presents a promising new strategy for the precise treatment of melanoma.