目的:构建中空硫化铜纳米酶脂质复合纳米载体提升硫化铜纳米载体的内吞效率,进一步探讨其通过化学动力疗法(CDT)联合近红外二区(NIR-II)的光热治疗(PTT)杀伤黑色素瘤细胞的效果与机制。方法:构建(2,3-二油酰基-丙基)-三甲胺(氯盐)(DOTAP)阳离子脂质体包被硫化铜纳米载体(CuS@LIP),在1064 nm 激光照射下,研究不同浓度的CuS与CuS@LIP的光热性能和稳定性；通过过氧化氢(H2O2)与3,3",5,5"-四甲基联苯胺(TMB)催化活性检测体系,验证CuS@LIP介导催化H2O2的活性。最后,用一系列浓度梯度的CuS、CuS@LIP在有无1064 nm 激光处理B16-F10黑色素瘤细胞,CCK-8法检测细胞的存活率,筛选适宜的CuS@LIP浓度(20 μg/mL)用于后续实验。Calcein-AM/PI染色法检测细胞的存活情况,Annexin V-FITC/PI 染色结合流式仪测定细胞凋亡率。结果:CuS@LIP的平均粒径为178.23 ± 6.46 nm,平均Zeta电位20.47 ± 0.93 mV；在1064 nm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个激光开关循环测试CuS@LIP终点温度基本保持不变。此外,CuS@LIP与CuS 具有相同的过氧化氢酶催化活性。CCK-8结果显示CuS@LIP在20 μg/mL时对体外细胞活性没有明显影响,但经激光照射后杀伤效果显著增加58.18%(P<0.001)；活死细胞染色显示光照后死亡细胞率由2.68%上升至19.81%(P<0.001)；光照组细胞凋亡率为19.34%,较非光照组13.36%有明显提高(P<0.01)。结论:合成的CuS@LIP能够被黑色素瘤细胞高效内吞,同时保持了CuS介导催化H2O2的化学动力疗法,显示出更优异的光热杀伤抗黑色素瘤效果,因此CuS@LIP代表了一种优异的抗肿瘤治疗手段。

Objective: To construct hollow copper sulfide nanocarrier with lipid to improve the endocytosis efficiency of copper sulfide, and to further investigate the effect and mechanism of killing melanoma cells by chemodynamic therapy (CDT) combined with photothermal therapy (PTT) in near-infrared second region (NIR-II). Methods: Firstly, cationic liposome (LIP) were applied to encapsulate copper sulfide (CuS) to construct cationic liposome coated copper sulfide nanocarriers (CuS@LIP). Secondly, the photothermal properties and stability of CuS and CuS@LIP at different concentrations were studied under 1064 nm laser irradiation. The catalytic activity of CuS@LIP to catalyze H2O2 was verified by detecting system of hydrogen peroxide (H2O2) and 3,3",5,5" -tetramethylbenzidine (TMB). Finally, the cell viability of B16-F10 melanoma cells were determined with a series of CuS and CuS@LIP with various concentration gradient under 1064 nm laser via CCK-8 method, and the appropriate CuS@LIP concentration was screened for subsequent experiments, and cell viability was detected by Calcein-AM/PI method and Annexin V-FITC/PI staining and flow cytometry were used to determine the apoptosis rate. Results: The average particle size of 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 at 1064 nm laser irradiation was 65.4 ℃, which was higher than that of CuS at 63.4 ℃. The end point temperature of CuS@LIP is basically unchanged through the cyclic test of three laser on/off switches. In addition, CuS@LIP has the same catalytic activity as CuS. The results of CCK-8 showed that CuS@LIP had no significant effect on the cell activity in vitro when it was less than 20 μg/ml, but the killing effect was significantly increased by 58.18% after laser irradiation(P<0.001). The rate of dead cells increased from 2.68% to 19.81% (P<0.001). The apoptosis rate was 19.34% in the NIR group compared with 13.36% in the non-light group (P<0.01). Conclusion: CuS@LIP can be endocytosed by melanoma cells with high efficiency, and maintain the catalytic property of H2O2 used in chemodynamic therapy. At the same time, the near infrared laser irradiation showed better anti-melanoma and killing effect of photothermal therapy. Therefore, CuS@LIP represents an excellent antitumor approach towards melanoma.

国家自然科学基金项目（面上项目，重点项目，重大项目）