RGD多肽修饰的芬维A钱脂质体对恶性黑素瘤细胞增殖、凋亡及迁移的影响

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

目的探讨芬维A铵(4-HPR)对B16F10和A375黑素瘤细胞增殖、凋亡及迁移的影响,并观察脂质体及以RGD多肽修饰的脂质体对药物摄取及作用的影响。方法采用薄膜-水化法制备4-HPR脂质体(4-HPRL),并以RGD多肽对4-HPRL进行修饰,制备RGD-4-HPRL,对其浓度、粒径、电位、载药量及包封率进行检测:体外培养B16F10和A375细胞,分为对照组、4-HPR组、4-HPRL组和RGD-4-HPRL组,给4-HPR组分别加入含相同浓度4-HPR的4-HPR原料药、4-HPRL及RGD-4-HPRL,对照组加入等量培养液。作用不同时间后.通过CCK8细胞计数试剂盒检测细胞活性.膜联蛋白V/碘化丙锭染色检测细胞凋亡,细胞划痕实验检测药物对细胞迁移的影响:以香豆素6(C6)代替4-HPR制备C6脂质体(C6L)和RGD-C6L,流式细胞仪检测细胞对药物的摄取情况:采用SPSS22.0软件进行统计学分析.多组间数据比较采用单因素方差分析.两两组间显著性差异比较采用/检验=结果制备的4-HPRL和RGD-4-HPRL溶液可明显提高4-HPR的水溶性,并具有较高的载药量和包封率。粒径分布均匀,平均在100 nm以下。CCK8实验表明,4-HPR可明显抑制B16F10和A375细胞增殖,且相同浓度下4-HPRL对细胞增殖的抑制率高于4-HPR(P < 0.01), RGD-4-HPRL又高于4-HPRL(P < 0.01或P < 0.05)和4-HPR(P < 0.01);细胞凋亡实验表明4-HPR浓度分别为10 mg/L或20 mg/L时可明显诱导B16F10和A375细胞凋亡:4-HPRL组两种细胞凋亡率高于4-HPR组(均P<0.01),RGD-4-HPRL组高于4-HPRL组(均P<0.01)和4-HPR组(均P<0.01).细胞划痕实验显示,4-HPR可抑制两种细胞划痕愈合和细胞迁移,4-HPRL和RGD-4-HPRL抑制能力明显优于4-HPR原料药。摄取实验显示.B16F10细胞C6荧光强度在对照组为2.15 ± 0.28, C6组为&56 ± 0.36, C6L组为20.48 ± 0.13,RGD-C6L组为22.55 ± 0.07,各组间差异有统计学意义(F = 67 194.186,P< 0.01),其中,C6L组与RGD-C6L组荧光强度明显高于C6组(均P<0.01),且RGD-C6L组髙于C6L组(P<0.01)。结论4-HPR可抑制黑素瘤A375和B16F10细胞的增殖、迁移并诱导其凋亡,脂质体和RGD靶向脂质体可明显增强4-HPR对黑素瘤细胞的作用.

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 peptidemodified 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 KGD 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)%,(2&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 in tensity 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.