Effects of RGD peptide-modified fenretinide liposomes on proliferation, apoptosis and migration of malignant melanoma cells

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