Inhibitory effect of fenretinide-loaded liposomes on subcutaneous transplanted tumors in nude mice inoculated with A375 melanoma cells: a preliminary study

Objective To evaluate the inhibitory effect of fenretinide-loaded liposomes (4-HPR-L) on subcutaneous transplanted malignant melanomas in nude mice. Methods A film-ultrasonic dispersion method was used to prepare 4-HPR-L. BALB/c nude mice were subcutaneously inoculated with A375 melanoma cells in the right axillary fossae to establish malignant melanoma-bearing nude mouse models. Ten nude mouse models were randomly and equally divided into 2 groups to be injected with near-infrared fluorescent cell membrane label (DiR) solution or DiR liposomes (DiR-L) at the same concentration in the caudal vein, and a live imaging system was used to observe the distribution of DiR or DiR-L in nude mice at 6, 12, 24 hours after the injection. Another 30 nude mice were randomly and equally divided into 3 groups to be injected with 5% (mass fraction) glucose solution at a single-dose of 0.2 ml (control group) , 25 mg/kg 4-HPR solution (4-HPR group) and 25 mg/kg 4-HPR-L solution (4-HPR-L group) respectively on days 8, 10, 12, 14, 16, 18, 20 and 22 after the inoculation with A375 cells. The mouse body weight and tumor volume were dynamically monitored in the above groups after the injection, and the survival situation was observed. The nude mice were sacrificed on day 2 after the final injection, and the heart, liver, spleen, lung, kidney and tumor tissues were resected. These tissues were subjected to hematoxylin-eosin staining and immunohistochemical staining to observe the metastasis of melanoma in mice, and terminal deoxyribonucleotidyl transferase-mediated nick end labelling was performed to detect the apoptosis in tumor cells. One-way analysis of variance and independent-sample t test were used to analyze measurement data. Results The live imaging system showed that DiR-L could be retained in melanoma for a long time, and strong fluorescence of DiR-L could be still observed in the tumors at 24 hours after injections. Quantitative fluorescence analysis revealed that the fluorescence intensity of DiR-L (22.85 ± 1.66) was significantly higher than that of DiR in tumor tissues (8.45 ± 0.97, t = 12.957, P < 0.01) . Compared with the control group and 4-HPR group, the resected tumor weight on day 2 after the final injection was significantly decreased in the 4-HPR-L group (F = 27.055, t = 4.768, 6.640, respectively, both P < 0.05) . Hematoxylin-eosin staining showed that liver metastasis occurred in 2 nude mice in the 4-HPR-L group, but in all the nude mice in the control group and 4-HPR group. All the nude mice in the 4-HPR-L group died within 76 days after inoculation, and the mice in the control group and 4-HPR group all died within 56 and 59 days respectively after inoculation. There were significant differences in the apoptotic index among the control group (12.14‰ ± 1.33‰) , 4-HPR group (67.17‰ ± 15.18‰) and 4-HPR-L group (152.73‰ ± 11.27‰; F = 167.588, P < 0.05) , and the apoptotic index was significantly higher in the 4-HPR-L group than in the control group and 4-HPR group (t = 18.162, 11.075 respectively, both P < 0.05) . Conclusion 4-HPR-L can effectively inhibit the growth of subcutaneous melanoma in nude mice and metastasis of melanoma cells, and prolong the survival duration of nude mice. Key words: Nevi and melanomas; Fenretinide; Liposomes; Models, animal; Apoptosis; A375 cells