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The strong two-photon luminescence of gold nanorods makes them suitable to 3D imaging. Although autofluorescence of cell provides the most convenient way for cell imaging, the high laser power of 35 mW needed to induce the autofluorescence is a potential safety hazard. As shown in Fig. 4A, cells can be killed after 30 raster scans at even a lower power of 30 mW. Obvious perforation of cells was observed when the power was increased to 35 mW (Fig. 6A). A laser operating at such a high power will thus cause clinical safety concerns. The enhanced imaging in the presence of gold nanorods is due to the high two-photon excitation cross section of gold nanorods, which is about ~3 ×104 GM for a nanorod with an aspect ration of 4 [31]. This is three orders of magnitude higher than that of a fluoresceine molecule [32,33]. Therefore, compared with the conventional fluorescent molecules, gold nanorod is a superior contrast agent. It was observed that membrane blebbing occurred on both necrotic and apoptotic cells. The initiation of cell membrane blebbing has been generally regarded as the sign of apoptosis [34]. During apoptosis, the cell’s cytoskeleton breaks up causing the membrane to bulge outward [35]. The intense femtosecond laser pulses particularly the localized photothermal effects of gold nanorods produced by the femtosecond laser pulses can destroy the intracellular actin network which provides mechanical support to maintain cell shape [7]. It was observed that the staining of the necrotic cells took only a few minutes. While for apoptotic cells to get sufficient staining with Annexin V-Cy3.18, the treated cells have to be incubated for at least 2 h. The short time required for PI staining of the necrotic cells indicates that the membrane of the cells was compromised. It was observed that when the laser power is 2mW(55.6 W/cm2) and above, perforation of cell membranewas induced after only one raster scan (Fig. 6B). The sharp decrease in irradiation duration can significantly reduce the energy fluence. However, a laser operating at this power presents a potential safetyhazard. This critical power/power density should be the upper limit that can be applied. With the eduction in the power density, the thermal effect becomes dominant and thermally induced cell damage controls the death of cells. Perforation of cell membrane enhanced in the presence of gold nanorods at high energy fluence was also reported in a real-time observation of a recent work. In terms of cancer therapy, significant reduction in the energy fluence can be achieved by inducing cell apoptosis rather than necrosis. For example, at 0.5 mW, apoptosis of HeLa cells was induced after being scanned 20 times, less than 1/7 of that needed to kill the cells (150 scans). When the laser power was increased to 1.5 mW, apoptosis of cells was induced after only 2 scans, which is only 1/5 of that required to induce necrosis. As a result, at a laser power ranging from 0.5 mW to 1.5 mW, the energy fluence for apoptosis is only about 1/7 to 1/5 (less than 20%) of that for necrosis, which is also two orders of magnitude lower than the medical safety level (100 mJ/cm2) [37]. The thresholds of energy fluence that cause cells necrosis and apoptosis at different laser powers are shown in Fig. 5. Increasing the laser power, the energy fluence for both necrosis and apoptosis was reduced. This can be attributable to the more intense heat production from the gold nanorods at a higher laser power. Apoptosis induction is important to medical applications since proliferation of cancer cells can be inhibited with low energy fluence. This will lead to the destruction of tumors in a safer and less aggressive manner, similar to drug-induced cell apoptosis and tumor damage in chemotherapy and radiotherapy. However, photothermally induced apoptosis can offer a more localized treatment and avoid the harsh side effects that are caused by anticancer drugs and radioactive isotopes. Interestingly, it was observed in a recent work that the photothermal effects of gold nanorods could induce apoptosis of macrophages by damaging the mitochondria [38], which regulates the apoptosis of cells. Whether this mechanism also governs the apoptosis of the cancer cells in this work is an interesting topic. |
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【答案】应助回帖
wo357026239(金币+120, 翻译EPI+1): 2012-01-05 19:47:33
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因其双光子发光，金纳米棒适用于三维成像。 尽管细胞的自发萤光提供了细胞成像的最简单方法，其所需的用来激发自发光的高达35 毫瓦的激光能量为安全性带来了潜在的威胁。如图4A所示，即使激光能量低于30 毫瓦，30次光栅扫描后，细胞即可死亡。当能量被提高到35毫瓦时，可见明显细胞穿孔 （图6A）。 在如此高能量下的激光操作，将引起人们对医疗安全的担忧。 加入金纳米棒可改进成像，这是因为对一个长宽比为4的金纳米棒来说，在其横截面的双光子激发可达约 ~3 ×104 GM，这比荧光素分子高出三个数量级［32，33］。因此，和传统的荧光分子相比，金纳米棒是优秀的对比度提升剂。 在坏死的细胞和凋亡的细胞上皆可观察到细胞膜起泡。细胞膜开始气泡通常被认为是细胞凋亡的前兆［34］。 在细胞凋亡过程中，细胞骨架的破坏导致细胞膜向外鼓出。高强度的飞秒级激光脉冲，尤其是它所产生的金纳米棒区域光热效应，可破坏提供支撑以维持细胞形状的细胞间肌动蛋白网络［7］。可以观察到，坏死细胞的染色几分钟内即可完成。使用Annexin V-cy3.18染色凋亡细胞，充分的染色需要至少2小时的培养。PI染色短时间内即可完成，说明坏死细胞的膜已经受到了损坏。当激光功率为2毫瓦或更高时，一次光栅扫描后即可观察到细胞膜穿孔 （图6B）。大幅度缩短辐照时间可明显减少能量的注入。 然而，如此大功率的激光应用，可带来潜在的安全隐患。此功率／功率比应为应用中的最上限。当功率值下降时，热效应占据了主导地位，高温导致的细胞损伤成为了细胞死亡的主要因素。在近期的工作中也有报导，实时观测中可见在金纳米棒的参与下，高功率辐照引起了更多的细胞穿孔。 在癌症治疗中，与细胞坏死相比，引发细胞凋亡可明显降低所需的能量输入。比如，在0.5毫瓦下，20次扫描即导致hela细胞凋亡，少于杀死细胞所需剂量的1/7 （150次扫描）。当激光功率提高到1.5毫伏，2次扫描即可导致细胞凋亡，这是导致细胞坏死所需的1/5。因此，当激光功率处于0.5 -1.5毫伏之间时，致使细胞凋亡所需的能量只是导致细胞坏死所需能量的1/7 到1/5（少于20％），同时也比医疗安全值 (100 mJ/cm2)低两个数量级［37］。在不同激光功率下导致细胞坏死和凋亡的能量起始值如图5所示。提高激光功率，致细胞坏死或凋亡的能量即随之降低。这可归因于更高激光功率下，金纳米棒产生的热量更高。 因为低能量的注入即可阻止癌细胞的繁殖，诱使细胞凋亡对医疗中的应用非常重要。就像化学疗法和和放射疗法中，药物所导致的细胞凋亡和肿瘤破坏一样，这可以获得一种更安全和平和的方式来破坏肿瘤。然而，光热法治疗所致的细胞凋亡可更精确的控于局部，避免了抗癌药及用于辐照治疗的同位素所带来的强烈的副作用［38］。有趣的是，在近期的研究中，科学家们观察到金纳米棒的光热效应可通过破坏线粒体 （其控制细胞凋亡），导致巨噬细胞凋亡［38］。在本实验中，是否这也是控制癌细胞凋亡的机理，仍是一个有趣的研究题目。 |
2楼2012-01-05 17:01:35
terry_well
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【答案】应助回帖
wo357026239(金币+30): 2012-01-05 19:47:39
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据观察，膜出泡一般发生在坏死性和凋亡性细胞内，并且细胞膜出泡已普遍被视为细胞凋亡的先兆[34]。在细胞凋亡过程中，细胞骨架的破坏造成了细胞膜向外凸出[35]。剧烈的飞秒激光脉冲作用下，尤其是金纳米棒产生的局部飞秒激光脉冲导致的光热效应可以破坏细胞内的肌动蛋白网络，而该网络它可以为维持细胞的形状提供机械支持[7]。研究发现，只用几分钟即可破坏细胞的染色体。对凋亡的细胞来说，要采用Annexin V Cy3.18获得足够的染色处理，所处理的细胞至少需要培养2小时。若PI坏死细胞染色所需时间短则表明其细胞膜遭到了破坏。据观察，当激光功率为2MW（55.6 W/cm2时）及以上时，一个光栅扫描诱导后细胞膜发生穿孔（如图6B所示）。辐照持续时间大幅减少可以显着降低能量密度。 然而，有人提出用这个功率的激光工作存在潜在的安全隐患。这个2MW的关键电源/功率密度可视为使用上限。随着在功率密度降低，热效应成为主导，且热诱导细胞损伤成为控制细胞死亡的只要因素。最新实时检索表明，高能量通量的金纳米棒可增强细胞膜穿孔的研究已经报道。 在癌症治疗方面，显著减少能源能量密度时，可通过诱导细胞凋亡而非使其坏死。例如，在0.5毫瓦时，扫描20次后可使HeLa癌细胞发生诱导凋亡，需要杀死的细胞不到1/7（150扫描）。当激光功率的增加1.5兆瓦，只有2扫描即可诱导癌细胞凋亡，此时诱发坏死的只有1 / 5。因此，激光功率范围从0.5毫瓦到1.5兆瓦，为细胞凋亡的能源能量密度大约只有1 / 7至1 / 5（低于20％）为坏死，这也比医疗安全水平低两个数量级（安全水平为100 mJ/cm2）[37]。图5给出了在不同的激光功率下导致细胞坏死和凋亡的能量密度阈值。激光功率的增加会导致细胞坏死和凋亡的能量通量减少，这可以归因于更高功率激光金纳米棒所产生的更剧烈的热效应。 因为可以用低能量通量抑制癌细胞的扩散，诱导凋亡在医疗领域具有很重要的作用。这将获得一个更安全、更有效地破坏肿瘤的治疗方法。而类似的药物诱导癌细胞凋亡可导致肿瘤化疗和放疗损伤。然而，辐照热诱导细胞凋亡，可以提供更局部化处理并避免由抗癌药物和放射性同位素引起的附带影响。有趣的是，通过最近的工作观察，金纳米棒的光热效应通过破坏其线粒体可诱导巨噬细胞凋亡，从而调节细胞凋亡[38]。在这个作用机制是否也可以用来控制癌细胞凋亡将是一个有趣的课题。 太长了，仅供参考。  |
3楼2012-01-05 17:38:41
