Effects of N-(4-hydroxyphenyl) retinamide lipid microbubble combined with ultrasound on human keloid fibroblasts / 中华烧伤杂志

ABSTRACT

Objective@#To explore the effects of N-(4-hydroxyphenyl) retinamide (4HPR), 4HPR liposome (4HPR-L), and 4HPR lipid microbubble (4HPR-LM) combined with ultrasound on proliferation, apoptosis, and cell cycle of human keloid fibroblasts (Fbs).@*Methods@#(1) 4HPR-L and 4HPR-LM were prepared by hydration ultrasonic method. The appearance morphology, particle size distribution, Zeta potential, loading drug concentration, encapsulation efficiency, and drug loading rate of 4HPR-L were investigated by high performance liquid chromatography, dynamic light scattering, and transmission electron microscope. (2) Human keloid Fbs were cultured and divided into 13 groups by random number table (the same grouping method below), with 6 wells in each group. Cells in control group were given no treatment, while cells in 12 ultrasound groups including 0.5 W 30 s group, 0.5 W 60 s group, 0.5 W 120 s group, 0.7 W 30 s group, 0.7 W 60 s group, 0.7 W 120 s group, 1.0 W 30 s group, 1.0 W 60 s group, 1.0 W 120 s group, 1.5 W 30 s group, 1.5 W 60 s group, and 1.5 W 120 s group were treated by ultrasound with corresponding parameters. The cells viability was measured by a microplate reader after 24 hours of routine culture. Another batch of human keloid Fbs were divided into 5 groups, with 6 wells in each group. Cells in control group were given no treatment, while cells in 1, 10, 20, and 50 μg/mL blank lipid microbubble groups were treated with blank lipid microbubbles in corresponding mass concentration. The cells viability was measured as before after 24 hours of routine culture. Another batch of human keloid Fbs were divided into 6 groups, with 12 wells in each group. Cells in control group were given no treatment, while cells in 1, 10, 20, 50, and 100 μg/mL 4HPR-L groups were added with 4HPR-L carrying corresponding mass concentration of 4HPR. The cells viability in 6 wells of each group was detected after 24 and 48 hours of routine culture, respectively. Another batch of human keloid Fbs were divided into 4 groups, with 6 wells in each group. Cells in control group were given no treatment, while cells in 4HPR, 4HPR-L, and 4HPR-LM+ ultrasound groups were treated with 4HPR, 4HPR-L, and 4HPR-LM (all the mass concentration of 4HPR was 20 μg/mL), respectively, and cells in 4HPR-LM+ ultrasound group were given 0.5 W 60 s ultrasound treatment immediately after drug administration. The cells viability was measured as before after 24 hours of routine culture. (3) Another batch of human keloid Fbs were divided into control group, 4HPR group, 4HPR-L group and 4HPR-LM+ ultrasound group, with 3 wells in each group, and the cells in each group were treated as before. Apoptosis of the cells was detected by flow cytometer after 24 hours of routine culture. (4) Another batch of human keloid Fbs were grouped and treated as in (3), and then the cell cycle distribution was detected by flow cytometer after 24 hours of routine culture. Data were processed with one-way analysis of variance and t test.@*Results@#(1) 4HPR-L particles had a spherical or spheroidal structure and were uniform in size, with particle size of (100.1±1.3) nm and Zeta potential of (-34.3±2.3) mV. The mass concentration of 4HPR in 4HPR-L solution was about 1 400 μg/mL, with the encapsulation efficiency of (95.8±1.2)% and drug loading rate of (8.3±0.4)%. (2) The viability of cells in the 12 ultrasound groups was higher than 93.0%, and the viability of cells in 1, 10, 20, and 50 μg/mL blank lipid microbubble groups was higher than 95.0%. The viability of cells in 1 μg/mL 4HPR-L group at administration hour 24 was similar to that at 48 (t=0.393, P>0.05). The viability of cells in 10, 20, 50, and 100 μg/mL 4HPR-L groups at administration hour 24 was significantly higher than that at administration hour 48 (t=44.593, 22.961, 32.224, 35.337, P<0.01). The viability of cells in 4HPR group, 4HPR-L group, and 4HPR-LM+ ultrasound group was (47.3±0.7)%, (42.3±1.7)%, and (38.6±0.8)%, respectively. The viability of cells in 4HPR group was significantly higher than that in 4HPR-L group and 4HPR-LM+ ultrasound group (t=4.551, 15.895, P<0.05 or P<0.01). The viability of cells in 4HPR-L group was significantly higher than that in 4HPR-LM+ ultrasound group (t=-3.360, P<0.05). (3) The percentages of total apoptotic cells in 4HPR group, 4HPR-L group, and 4HPR-LM+ ultrasound group were (32.8±2.4)%, (42.5±2.4)%, and (58.5±6.3)%, respectively, which were significantly higher than the percentage of control group [(14.9±1.6)%, t=8.748, 13.637, 9.500, P<0.01]. The percentages of total apoptotic cells in 4HPR-L group and 4HPR-LM+ ultrasound group were significantly higher than the percentage in 4HPR group (t=4.049, 5.393, P<0.05 or P<0.01), and the percentage of total apoptotic cells in 4HPR-LM+ ultrasound group was significantly higher than that in 4HPR-L group (t=3.371, P<0.01). (4) The percentage of G2/M phase cells in 4HPR group was higher than that in control group, but there was no statistically significant difference (t=2.107, P>0.05). The percentage of G2/M phase cells in 4HPR-L group was significantly higher than that in 4HPR group or control group (t=18.169, 30.026, P<0.01). The percentage of G2/M phase cells in 4HPR-LM+ ultrasound group was significantly higher than that in 4HPR-L group, 4HPR group, and control group (t=4.932, 25.854, 66.231, P<0.01).@*Conclusions@#4HPR can inhibit proliferation, induce apoptosis, and arrest G2/M phase of human keloid Fbs, and the effects of 4HPR-LM combined with ultrasound are better than those of 4HPR-L and free 4HPR.