Study On Preparation And In Vitro Antitumor Activity Of Iridium(?) Complex-loaded Long-circulating Liposomes

The ion-pairreversed-phasehighperformanceliquid chromatography method was established to determine the concentration of Iridium???complex?[Ir?ppy?₂?BTCP?]PF₆;abbreviated as Ir?.Determination method of encapsulation efficiency?EE?and drug loading?DL?of liposomes was established and low speed-overspeed centrifugal method was used to separate liposomes and free drugs.The equilibrium solubility of Ir in phosphate buffer saline?PBS?with pH 7.4 or in different concentrations of Tween-80 solutions was determined respectively,whichcouldprovide references for the design of formulation and preparation technic of liposomes loading Ir and the in vitro release test.Iridium???complex-loaded long circulation liposomes were preparedbythinfilmdispersion-ultrasonicmethod.The influencing factors,including the types of phospholipid,mass ratio of EPC to Chol,mass ratio of EPC to Ir,EPC concentration and other technologicalparameterswereinvestigated.Box-Behnken design with three factors and three levels was used for the optimization formulation,with the mass ratio of EPC to Ir?X₁?,the mass ratio of EPC to Chol?X₂?and EPC concentration?X₃?as independent variables and EE,LD and the diameter of liposomes as response variables.The optimized formulation was obtained and the processing factors were X₁=12.39:1,X₂=10.03:1 and X₃=9.48 mg/mL.Under the optimization formulation,the EE,DL,diameter,PDI value and Zeta potential of Lipo-Ir were 92.66±1.79%,5.50±0.85%,116.57±1.15 nm,0.19±0.02and-10.66±0.61 mV,respectively.The freeze-dried long circulation liposomes loaded Iridium???complex?FD-Lipo-Ir?werepreparatedby freeze-drying method.Differentlyophilizingprotectantswere investigated for the effective protection on liposomes and the optimal preparation of lyophilizing protectants was as following:EPC:fucose:mannitol=1:1:1?w/w?.Under the optimization formulation,the diameter,PDI value and Zeta potential of FD-Lipo-Ir were 154.20±2.57nm,0.193±0.08,-7.03±0.11 mV,respectively.The EE of liposomes was not significantly changed after freeze drying.FD-Lipo-Ir was stability within 3 months in 4?and its releasing in vitro followed the first order kinetic sequation,which was similar to Lipo-Ir.The cytotoxicity of Lipo-Ir against A549 cells was tested by MTT assay and the result showed Lipo-Ir exhibited a good antitumor activity.The morphological changes of apoptotic cells were observed through AO/EB and Hoechst 33342 staining methods.The mitochondrial membrane potential???m?,reactive oxygen species?ROS?and intracellular Ca²⁺levels were further studied,and the result showed that Lipo-Ir could induce??m depolarization,increase the ROS levels and disturb intracellular calcium homeostasis.Also,Lipo-Ir could arrest the cell growth in G0/G1 phase.These results inferred that Lipo-Ir could induce cell apoptosis effect in A549 cells via ROS-mediated mitochondria pathway.Therefore,Lipo-Ir or FD-Lipo-Ir might be a new promising approach for effective tumor therapy.