| Objective:To prepare N-(4-Hydroxyphenyl)retinamide loaded lipid microbubbles(4HPR-LM),explore the effect of combination of 4HPR-LM and ultrasound for the proliferation,apoptosis,cell cycle of Human keloid fibroblasts(HKFs),establish nude mice subcutaneous xenograft keloid model and examine the inhibition in tumor growth with the treatment of 4HPR-LM combined with ultrasound.Method:4HPR-loaded liposomes(4HPR-L)were prepared by film-ultrasound dispersion method,the characterization of 4HPR-L,such as drug concentration,sizes,Zeta potentials,encapsulation efficiency,drug loading,stability and leakage rates within 30 days,were evaluated by high performance liquid chromatograph(HPLC),dynamic light scattering(DLS),transmission electron microscope(TEM),respectively.Added perfluoropentane(PFP)to 4HPR liposome solution,mixtured by ultrasonic cavitation method to prepare 4HPR-LM;CCK-8 experiment was conducted to screening for ultrasound parameters,evaluating biocompatibility of blank-loaded lipid microbubbles(Blank-LM),and detecting the inhibition of free 4HPR,4HPR-L and 4HPR-LM combined with ultrasound for HKFs;Healing assay to observe anti-migration effect of 4HPR on HKFs;Applicating Flow cytometry detection the effect of 4HPR on HKFs cell cycle and apoptosis.Establish subcutaneous keloid model by HKFs inoculation,explore the efficacy of combination 4HPR-LM and ultrasound for the treatment of keloid in vivo.Results:Ultrasound probe parameter of prepared 4HPR-L is 15 min(power 200 W,ultrasonic 1 s,intermittent 2 s).(1)The 4HPR concentration in prepared 4HPR-L is exceeded 1400 ?g/ml,particle size distribution is(100.08 ± 1.33)nm,polydispersity index(PDI)is(0.169 ± 0.005),Zeta potential is(-34.27 ± 2.27)mV,the appearance of 4HPR-L showed by TEM is spherical or approximately spherical shape,size is uniform,about 100nm in diameter;entrapment efficiency is(95.8±0.2)%,4HPR loading is(8.26±0.35)%.when 4HPR-L was stored at 4? for 30 days,there is no obvious changes on outward.size and Zeta potential.The leakage rates of 7,15 and 30 days are (2.15±0.27)%,(5.91±2.7)%,(8.3±0.7)%.(2)CCK-8 experimental results show that there is no significant inhibition of ultrasound and Blank-LM on HKFs proliferation,the inhibition of 4HPR,4HPR-L and 4HPR-LM combine ultrasound have both time and dose-dependent,and compared with free 4HPR,4HPR-LM combined with ultrasound showed higher cell proliferation inhibition rate(P<0.05).(3)Healing assay showed 4HPR could inhibition of HKFs invasion;(4)Flow cytometry results showed 4HPR can induce HKFs apoptosis effectively and arrest cell cycle in the G2-M phase,the combination of 4HPR-LM with ultrasound showed significantly better effect than free 4HPR of all cell experiment;(5)Animal experiment indicate that 4HPR-LM combined with ultrasound had a significant difference on inhibit tumor growth compared with the control group(P<0.05).During our experiment,all the mice's general physical status were well,the HE staining of various organs did not show obvious pathological change.Conclusion:1.The prepared 4HPR-L had high concentration and good stability,significantly enhanced 4HPR solubility in aqueous media;2.4HPR can inhibit the proliferation and invasion of HKFs,in addition,4HPR had arrest cell cycle and induce apoptosisfunction;3.successfully establish keloid subcutaneous tumor model in nude mice by inoculated HKFs,4HPR-LM combined with ultrasound had notably anti-tumor effect in vivo;4.Lipid microbubbles combined with ultrasound can improve drug efficacy notably,has a broad clinical application. |
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