Lapotko Dmitri O, Lukianova Ekaterina, Oraevsky Alexander A
Luikov Heat and Mass Transfer Institute, Minsk, 220072, Belarus.
Lasers Surg Med. 2006 Jul;38(6):631-42. doi: 10.1002/lsm.20359.
Previously reported studies on laser nano-thermolysis of cancerous cells demonstrated insufficient efficacy and specificity of malignant cell damage. Safety, that is, absence of damage to normal cells in the course of the laser thermolysis was also low due to less than optimal protocol of cancer cell targeting with nanoparticles (NP). The objective of this study was two-fold: to optimize NP targeting to real tumor (human) cells and to better understand physical mechanisms of cell damage for improved control of the laser ablation.
STUDY DESIGN/MATERIALS AND METHODS: We have suggested (1) two-stage targeting method to form clusters of light-absorbing gold NPs selectively in target cells, and (2) the cell damage mechanism through laser-induced generation of vapor bubbles around NP clusters. Experimental investigation of laser nano-thermolysis of leukemia cells was performed using 30 nm spherical gold nanoparticles as a light absorbing agent, and photothermal and fluorescent microscopies as well as flow cytometry as methods to monitor microbubble formation and resulting damage of leukemia cells in human bone marrow specimens.
NP clusters were formed and visualized using fluorescence microscopy at cell membranes and in cytoplasm of B-lymphoblasts. Laser irradiation of cells (532 nm, 10 nanoseconds, 0.6 J/cm2) induced microbubbles selectively in leukemia cells with large clusters, but not in cells with single NPs or small clusters. Quantitative analysis demonstrated that only 0.1%-1.5% of tumor cells and 77%-84% of normal bone marrow cells survived laser pulse.
Two-stage cell targeting method permits formation of NP clusters selectively in diagnosis-specific tumor cells. The clusters serve as effective sources of photothermally-induced microbubbles, which kill individual target cells after a single laser pulse. The laser fluence threshold for generation of microbubbles is inversely proportional to the volume of NP clusters.
先前关于癌细胞激光纳米热解的研究表明，恶性细胞损伤的疗效和特异性不足。由于纳米颗粒（NP）对癌细胞的靶向方案不够理想，激光热解过程中对正常细胞无损伤的安全性也较低。本研究的目的有两个：优化NP对实际肿瘤（人类）细胞的靶向作用，并更好地理解细胞损伤的物理机制，以改善激光消融的控制。
研究设计/材料与方法：我们提出了（1）两阶段靶向方法，以在靶细胞中选择性地形成吸光金NP簇，以及（2）通过激光诱导在NP簇周围产生蒸汽泡的细胞损伤机制。使用30nm球形金纳米颗粒作为吸光剂，对白血病细胞进行激光纳米热解的实验研究，并采用光热和荧光显微镜以及流式细胞术作为监测人骨髓标本中微泡形成和白血病细胞由此产生的损伤的方法。
使用荧光显微镜在B淋巴母细胞的细胞膜和细胞质中形成并观察到NP簇。对细胞进行激光照射（532nm，10纳秒，0.6J/cm²），在具有大簇的白血病细胞中选择性地诱导微泡，但在具有单个NP或小簇的细胞中则不会。定量分析表明，激光脉冲后仅0.1%-1.5%的肿瘤细胞和77%-84%的正常骨髓细胞存活。
两阶段细胞靶向方法允许在诊断特异性肿瘤细胞中选择性地形成NP簇。这些簇作为光热诱导微泡的有效来源，在单个激光脉冲后杀死单个靶细胞。产生微泡的激光能量密度阈值与NP簇的体积成反比。
