Abstract: 【Abstract】 Objective To assess the effect of fenretinide （4-HPR） on proliferation, apoptosis and migration of B16F10 and A375 melanoma cells, and to evaluate the effect of liposomes and RGD peptide-modified liposomes on its uptake and therapeutic effects. Methods A film-hydration method was used to prepare 4-HPR liposomes （4-HPRL）, which were modified with RGD peptide to prepare RGD-4-HPRL, and the concentration, particle size, electric potential, drug loading capacity and encapsulation efficiency were measured for 4-HPRL and RGD-4-HPRL. In vitro cultured B16F10 and A375 cells were divided into several groups: 4-HPR group, 4-HPRL group and RGD-4-HPRL group treated with Dulbecco′s minimum essential medium （DMEM） containing 4-HPR bulk drug, 4-HPRL and RGD-4-HPRL respectively at the same concentration of 4-HPR, and control group treated with culture solution at the same volume. After different durations of treatment, cell counting kit-8 （CCK8） assay was performed to evaluate cellular proliferative activity, annexin V/propidium iodide staining to detect apoptosis, and scratch wound healing assay to evaluate the effect of drug treatment on cell migration ability. Then, 4-HPR was replaced by coumarin 6 （C6） to prepare C6 liposomes （C6L） and RGD-C6L, and flow cytometry was conducted to evaluate C6 uptake by B16F10 cells. Statistical analysis was carried out with SPSS22.0 software by one-way analysis of variance （ANOVA） for the comparison among several groups and t test for the comparison between two groups. Results The concentration of 4-HPR in the prepared 4-HPRL solution was over 1 300 mg/L. The encapsulation efficiency and drug loading capacity of 4-HPRL were （95.51 ± 1.22）% and （7.27 ± 0.11）% respectively, and those of RGD-4-HPRL were （95.82 ± 0.81）% and （7.14 ± 0.13）% respectively. The particle size distribution of 4-HPRL and RGD-4-HPRL was uniform, and their average particle size was below 100 nm. CCK8 assay showed that 4-HPR could markedly inhibit the proliferative activities of B16F10 and A375 cells. The cell proliferation inhibition rate of 4-HPRL was higher than that of 4-HPR at the same concentration of 4-HPR （P < 0.01）, and the inhibition rate of RGD-4-HPRL was higher than that of 4-HPRL （P < 0.01 or P < 0.05） and 4-HPR （P < 0.01）. As annexin V/propidium iodide apoptosis assay showed, when the concentration of 4-HPR was 10 mg/L, the total apoptosis rates of B16F10 cells in the control group, 4-HPR group, 4-HPRL group and RGD-HPRL group were （4.44 ± 0.35）%, （28.33 ± 0.66）%, （46.43 ± 0.77）% and （51.33 ± 0.37）% respectively. When the concentration of 4-HPR was 20 mg/L, the total apoptosis rates of A375 cells in the above 4 groups were （4.97 ± 0.62）%, （16.68 ± 3.81）%, （32.62 ± 1.24）% and （44.85 ± 4.92）% respectively. The apoptosis rates of B16F10 and A375 cells were significantly higher in the 4-HPRL group than in the 4-HPR group （both P < 0.01）, and higher in the RGD-4-HPRL group than in the 4-HPRL group （both P < 0.01） and 4-HPR group （both P < 0.01）. Scratch wound healing assay showed that 4-HPR could inhibit scratch healing and migration of B16F10 and A375 cells, and the inhibitory effects of 4-HPRL and RGD-4-HPRL were distinctly superior to those of 4-HPR bulk drug. C6 uptake assay revealed that the fluorescence intensity of C6 in B16F10 cells in the control group, C6 group, C6L group and RGD-C6L group were 2.15 ± 0.28, 8.56 ± 0.36, 20.48 ± 0.13 and 22.55 ± 0.07 respectively, and there were significant differences between the 4 groups （F = 67 194.186, P < 0.01）. Additionally, the fluorescence intensity of C6 was significantly higher in the C6L group and RGD-C6L group than in the C6 group （both P < 0.01）, and higher in the RGD-C6L group than in the C6L Group （P < 0.01）. Conclusions 4-HPR can inhibit the proliferation and migration of A375 and B16F10 cells, and induce their apoptosis. Liposomes and RGD-targeted liposomes can markedly enhance the effect of 4-HPR on melanoma cells.

Key words: Melanoma, Cell line, tumor, Fenretinide, Liposomes, Integrin alphaVbeta3, Cell proliferation, Apoptosis, Cell migration assays