Effect Of Combination 4-HPR Loaded Microbubbles And Ultrasound On The Treatment Of Keloid

Objective:To prepare 4-HPR loaded microbubbles,investigate the drug loading(DL),encapsulation efficiency(EE)and average particle size,explore the efficacy of combination of 4-HPR loaded microbubbles and ultrasound for the treatment of keloid with tumor-burdened nude mice model of keloid and keloid fibroblasts.Methods:1.4-HPR loaded micelles and microbubbles were prepared with PEG-DSPE and EPC as membrane material.Tthe film-hydration method was used for preparation of drug loaded micelles.After adding perfluoropentane(PFP)into micelle,microbubbles were prepared with ultrasound by ultrasonic cell disruptor.The morphology of drug loaded micelles prepared by optimum formulation was observed by transmission electron microscopy(TEM).The zeta potential and size distribution of the particles were also assayed.The drug loading encapsulations were determined by High Performance Liquid Chromatography(HPLC).Investigate the hemolysis of 4-HPR micelles and 4-HPR microbubbles.2.The effection of 4-HPR micelles,4-HPR microbubbles and ultrasound on human keloid fibroblasts:(1)MTT assay:were treated with 4-HPR,4-HPR micelles,4-HPR microbubbles and ultrasound.(2)observation anti-migration effect of 4 HPR loaded microbubble combined with ultrasonic impact on keloid fibroblasts.(3)cellular uptake of coumarin 6 loaded microbubble combined with ultrasonic treat on keloid fibroblasts was visualized with CLSM.3.Study of combination 4-HPR loaded microbubbles and ultrasound on the treatment of keloid:keloid fibroblasts in 0.1 ml Matrigel were implanted into the right armpit subcutaneous of nude mice to establish a tumor-burdened nude mice model of keloid.Tumors had locally injected with 4-HPR saline solution,4-HPR microbubbles and physiological saline,respectively,the dosage of 4-HPR was 12.5 mg/kg body weight.Ultrasonic irradiation on the tumor was imidiatedly followed by the injection of 4-HPR microbubbles.After the injection of 4-HPR microbubbles,tumors were sonicated 1 minute with unfocused 3-MHz ultrasound at 2 W/cm 2 power density and 20%duty cycle.Changes of tumor sizes and mice weight were regularly observed.Mice were killed 48 hours after the last treatment.The formations of subcutaneous tissues in each group were observed,and pathological features of the tissues and visceral organs were also studied.Results:1.The 4-HPR loaded micelles were spherical,drug loading and encapsulation efficiency were(14.51±0.13)%and(90.88±0.36)%,average particle size of 208.9 nm.4-HPR loaded microbubbles average particle size of 150.7 nm,potential was 42.03 mV,spread evenly.4-HPR micelles and 4-HPR microbubbles had no hemolysis.2.Investigated 4-HPR on keloid fibroblasts proliferation inhibition by cell experiment.There was concentration dependence on inhibition.4 HPR loaded microbubbles combining ultrasound can accelerate the drugs into the cell and nanoparticles drug release,better inhibition of keloid fibroblasts proliferation(P<0.05),and migration.Cellular uptake all drugs in 5 minutes.3.Keloid tumor model in nude mice could be successfully established by subcutaneously transplanted keloid fibroblasts.Combine 4-HPR loaded microbubbles and ultrasound on animal models could significant inhibit the speed of tumor growth(P<0.05)and the tumors tissue necrosis obviously.Lesions and metastases were not observed in visceral organs.Conclusion:4-HPR loaded microbubbles were successfully prepared.Combination of 4-HPR loaded microbubbles and ultrasound could significantly inhibit the proliferation of keloid fibroblasts and the tumor growthe of tumor on nude mice model of keloid.