Objective To prepare a new type of lipid nanoprobes(HD@P-NPs),and to investigate the effects of ultrasound imaging in vitro,migration inhibition as well as amplification therapy on keloid fibroblasts(KFs).Methods HD@P-NPs were prepared through ultrasonic emulsification with perfluoroethane as the core lipid shell and loading of hematoporphyrin monomethyl ether(HMME)and doxorubicin(DOX).The morphology,structure,particle size and Zeta potential of HD@P-NPs were measured,and the encapsulation efficiency and loading rate of corresponding drug were calculated.The imaging effects of two-dimensional ultrasound and contrast-enhanced ultrasound(CEUS)under the irradiation of low intensity focused ultrasound(LIFU)were explored.The hemolysis rate of HD@P-NPs at different concentrations were calculated.KFs in the logarithmic growth phase were cultured under different experimental conditions and divided into the following 5 groups:control group(no treatment),LIFU group,D@P-NPs group,HD@P-NPs group and HD@P-NPs+LIFU group.The cell survival rate was detected by MTT assay,the cell activity in each group was observed by Calcein-AM/PI staining method.Cell migration rate in each group was detected by cell migration experiment.Reactive oxygen species fluorescence staining was applied to observe the intracellular reactive oxygen species(ROS)production,and the fluorescence intensity was obtained.Results HD@P-NPs were successfully prepared with uniform dimensions.The average particle size was(170.36±6.03)nm,the average Zeta potential was(-36.91±3.56)mV,respectively.The encapsulation efficiency and loading rate of HMME and DOX were 67.41%,5.18%and 72.80%,11.20%,respectively.Microbubbles were generated by the phase transition of HD@P-NPs after LIFU irradiation.The optimal imaging efficacy was achieved under the irradiation conditions of 3 W/cm2 for 2 min and these parameters were subsequently adopted for further experiments.The hemolysis rate of HD@P-NPs at different concentrations(25,50,100,200 µg/ml)were(2.48±0.02)%,(4.87±0.06)%,(5.03±0.03)%,(6.10±0.04)%,respectively.Cell survival rate in the control,LIFU,D@P-NPs,HD@P-NPs,and HD@P-NPs+LIFU groups were 100%,(96.87±0.71)%,(77.94±2.83)%,(77.11±3.53)%,(49.75±1.25)%,respectively.A statistically significant difference was observed between the HD@P-NPs+LIFU group and the control group(P＜0.05).There was obvious red fluorescence while minimal green fluorescence in the HD@P-NPs+LIFU group.The migration rate in the control,LIFU,D@P-NPs,HD@P-NPs,and HD@P-NPs+LIFU groups were(29.96±3.20)%,(28.62±2.56)%,(18.13±0.89)%,(17.46±0.20)%,(10.04±1.62)%,respectively.The migration rate in the HD@P-NPs+LIFU group was significantly lower than that in other groups(all P＜0.05).The ROS in the HD@P-NPs+LIFU group was 22.43±3.10,the difference was statistically significant compared with other groups(all P＜0.05).Conclusion The HD@P-NPs were successfully prepared in this experiment.These nanoprobes can increase the effective drug concentration in the target area and realize cascade amplification therapy of KFs under LIFU irradiation and ultrasound visualization.
NETL
NSTL
万方数据
