目的：构建中空硫化铜纳米酶脂质复合载体CuS@LIP 并探讨其联合激光照射杀伤黑色素瘤B6-F10 细胞的效果与机制。方法：构建（2,3-二油酰基-丙基）-三甲胺-丙烷（氯盐）（DOTAP）阳离子脂质体包被硫化铜纳米载体CuS@LIP，研究不同质量浓度的CuS与CuS@LIP在1 064 nm激光照射下的光热性能和热稳定性，通过H2O2与3,3',5,5'-四甲基联苯胺（TMB）催化活性检测体系检测CuS@LIP 的类过氧化物活性；用系列质量浓度梯度的CuS、CuS@LIP在有/无激光条件下分别处理B16-F10细胞，CCK-8法检测细胞的存活率，Calcein-AM/PI 染色法、Annexin Ⅴ-FITC/PI 染色法结合流式细胞仪分别检测20 μg/mL CuS 或CuS@LIP 在激光照射或非激光照射条件下对B16-F10细胞活力和凋亡的影响。结果：成功制备的CuS@LIP 的平均粒径为（178.23±6.46）nm，平均Zeta 电位为（20.47±0.93）mV；在激光照射下，80 μg/mL CuS@LIP 最高温度可达65.4 ℃，比单纯CuS 的63.4 ℃更高；经3个激光开关周期测试，CuS@LIP 终点温度基本保持不变；此外，CuS@LIP 与CuS 具有相同的类过氧化物酶催化活性。低于20 μg/mL的CuS@LIP在体外对B16-F10细胞的增殖活性没有明显影响（P>0.05），但联合激光照射后细胞存活率明显降低（29.76±3.60）% vs（87.95±8.18）%，P<0.000 1，细胞凋亡率显著升高[（19.34±4.41）% vs（13.36±0.86）%，P<0.01]。结论：制备的CuS@LIP 具有符合设计要求的理化性质、良好的光热性能和优异的类过氧化物酶催化活性，其与激光照射联合后显示出更优异的杀伤B16-F10细胞的效果。

Objective: To construct a hollow copper sulfide nano-enzyme lipid composite nanocarrier CuS@LIP, and to further investigate the effect and mechanism of CuS@LIP combined laser irradiation on killing melanoma B6-F10 cells. Methods: (2, 3-dioleoyloxy-propyl) -3-trimethylammonium-propane (chloride salt) (DOTAP) cationic liposomes (LIPs) were applied to encapsulate copper sulfide (CuS) to construct cationic liposome-coated copper sulfide nanocarriers (CuS@LIP). The photothermal properties and stability of CuS and CuS@LIP at different concentrations were studied under 1 064 nm laser irradiation. The peroxidase-like activity of CuS@LIP was verified by a catalytic activity detecting system of hydrogen peroxide (H2O2) and 3, 3', 5, 5' -tetramethylbenzidine(TMB). B16-F10 cells were treated respectively with a series of mass concentration gradients of CuS or CuS@LIP with or without laser. The cell viability of B16-F10 cells was determined via CCK-8 assay. Calcein-AM/PI staining and Annexin Ⅴ-FITC/PI staining combined with flow cytometry detected the effects of 20 μg/mL CuS or CuS@LIP on the cell viability and apoptosis of B16-F10 cells under laser or non-laser irradiation, respectively. Results: The average particle size of successfully prepared CuS@LIP was (178.23±6.46) nm and the average Zeta potential was (20.47±0.93) mV. The highest temperature of 80 μg/mL CuS@LIP under laser irradiation was 65.4 ℃, which was higher than that of CuS at 63.4 ℃. The endpoint temperature of CuS@LIP was basically unchanged through the cyclic test of three laser on/off switches. In addition, CuS@LIP had the same peroxidase-like catalytic activity as CuS. CuS@LIP had no significant effect on the proliferation activity of B16-F10 cells in vitro when it was less than 20 μg/mL (P>0.05), but the survival rate was significantly reduced by 58.18% after laser irradiation ([29.76±3.60]% vs [87.95±8.18]%, P<0.000 1). The apoptosis rate was significantly increased ([19.34±4.41]% vs [13.36±0.86]% , P<0.01). Conclusion: The prepared CuS@LIP has physical and chemical properties, good photothermal properties and excellent peroxidase-like catalytic activity that meet the design requirements,and it shows a better effect of killing B16-F10 cells when combined with laser irradiation.

国家自然科学基金（No. 82072051）