Chinese Journal of Tissue Engineering Research ›› 2018, Vol. 22 ›› Issue (18): 2921-2926.doi: 10.3969/j.issn.2095-4344.0766
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Wang Run-sheng1, 2, Liu Yi-hao3, Mao Ke-ya1
Received:
2018-01-05
Online:
2018-06-28
Published:
2018-06-28
Contact:
Mao Ke-ya, Chief physician, Department of Orthopedics, Chinese PLA General Hospital, Beijing 100853, China
About author:
Wang Run-sheng, Doctoral candidate, Attending physician, Department of Orthopedics, Chinese PLA General Hospital, Beijing 100853, China; Department of Orthopedics, Third Affiliated Hospital of Guangxi University of Chinese Medicine, Liuzhou 545001, Guangxi Zhuang Autonomous Region, China
CLC Number:
Wang Run-sheng, Liu Yi-hao, Mao Ke-ya. Evaluation methodology of cellular uptake of magnetic nanoparticles[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(18): 2921-2926.
2.1 定性评价方法 2.1.1 普鲁士蓝染色 利用磁性纳米颗粒内含有铁元素,与普鲁士蓝试剂发生反应能生成蓝色沉淀,再通过伊红/核固红复染,增强染色对比,内吞磁性纳米颗粒的细胞被染成蓝色,而未内吞磁性纳米颗粒的细胞未出现蓝色沉淀(图1)。普鲁士蓝染色是研究磁性纳米颗粒细胞内吞最常用的定性研究方法[25-26],因磁性纳米颗粒内吞细胞后多位于细胞质的溶酶体内[27],故光学显微镜下多表现为细胞质染成蓝色,而细胞核未见蓝染[28]。Makridis等[29]采用普鲁士蓝染色观察骨肉瘤SaO-2细胞对MnFe2O4/Fe3O4和CoFe2O4/Fe3O4磁性纳米颗粒细胞内吞情况,共孵育48 h后,SaO-2细胞完全或部分变成蓝色,表明颗粒被肿瘤细胞内吞,当磁性纳米颗粒被肿瘤细胞内吞后再通过交变磁场进行细胞内热疗,提高肿瘤细胞杀伤率。普鲁士蓝染色后,利用酶标仪采用比色法比较吸光度值差异进行定量对比,检测细胞内磁性纳米含量[30],为普鲁士蓝染色定量评估细胞内吞提供新思路。但普鲁士蓝染色也存在缺点,细胞外颗粒洗脱不净可能导致假阳性结果,因此,普鲁士蓝染色评价细胞内吞需要结合荧光显微镜、透射电镜等检查。"
2.1.2 荧光显微镜染色 磁性纳米颗粒经表面修饰后,在其表面插入荧光素,将细胞核、细胞膜或细胞骨架采用不同荧光染料进行染色,通过紫外线激发荧光,当绑定荧光素的磁性纳米颗粒、细胞膜或细胞骨架、细胞核3种荧光重叠时,可观察磁性纳米颗粒在细胞内的位置。采用针对不同细胞器的荧光探针,可观察磁性纳米颗粒内吞细胞后,在亚细胞结构中的位置。Zhang等[27]采用溶酶体荧光探针Lysotracker将溶酶体标记后,可观察绑定荧光素TRITC的磁性纳米颗粒位于溶酶体内。利用溶酶体荧光探针Lysosensor随pH变化的特征,可实时观察动态磁场作用下,由于溶酶体膜破裂导致溶酶体pH变化后绿色荧光的强弱变化情况,判断磁性纳米颗粒在动态磁场中诱导肿瘤细胞凋亡的原因可能与溶酶体膜破裂有关。Shah等[31]将壳核结构磁性纳米颗粒与线粒体靶向促凋亡两性锚定肽连接,再在表面修饰靶向配体iRGD,并绑定荧光素(Alexa-594),通过荧光显微镜观察其对整联蛋白高表达的人胶质瘤细胞系U87具有靶向性,并可增加内吞量,联合热疗作用,可诱导肿瘤细胞通过线粒体途径凋亡。磁性纳米颗粒表面绑定荧光素是荧光显微镜观察磁性纳米颗粒内吞的前提条件,荧光素绑定率可能会影响观察结果。 2.1.3 共聚焦显微镜 磁性纳米颗粒、细胞膜、细胞核荧光标记后,通过激光激发荧光探针发出不同颜色荧光,观察磁性纳米颗粒在细胞内的位置,所得图像较荧光显微镜更为清晰,而且可通过断层扫描、三维重建观察细胞及亚细胞结构。共聚焦显微镜是研究磁性纳米颗粒内吞、颗粒细胞内示踪、磁性纳米颗粒热疗、载药等治疗后细胞特征形态变化的重要观察手段[32]。Sanz等[7]将磁性纳米颗粒采用Alexa 488荧光探针标记,细胞核采用DAPI荧光探针标记,通过共聚焦显微镜观察磁性纳米颗粒内吞入细胞后,主要位于细胞质中,细胞核中无磁性纳米颗粒,磁热疗后细胞核出现形态改变,且磁热疗较水浴热疗细胞核形态变化(核固缩)更明显。Connord等[33]设计微型动态磁场,将微型磁场置于细胞培养皿中,通过共聚焦显微镜动态观察内吞荧光标记磁性纳米颗粒的肿瘤细胞死亡,可能与溶酶体膜的通透性改变、ROS释放有关,而且观察到内吞入肿瘤细胞的磁性纳米颗粒,在微型动态磁场的变化下由散乱分布而数分钟内变为线性排列现象。 2.1.4 透射电镜 透射电镜是观察磁性纳米颗粒与细胞关系最直接的评价方法[34],可观察到磁性纳米颗粒在细胞内的分布及与细胞器的关系,并可观察治疗前后磁性纳米颗粒在细胞内的聚集、分散情况。Di Corato等[35]通过透射电镜观察5种常用磁性纳米颗粒在4-37 ℃下与细胞的关系,发现4 ℃时磁性纳米颗粒主要位于细胞膜上,而37 ℃时磁性纳米颗粒被肿瘤细胞内吞进入细胞内,在溶酶体内聚集,从而验证细胞内吞磁性纳米颗粒是主动内吞,需消耗能量。Guardia等[2]通过透射电镜可观察到与细胞共孵育后,磁性纳米颗粒主要位于细胞溶酶体内,且在溶酶体内聚集,进行磁热疗后,磁性纳米颗粒随着溶酶体膜的破裂而逐渐分散(图2)。Zhang 等[27]将磁性纳米颗粒荧光标记并连接溶酶体膜相关蛋白1抗体,内吞入细胞后与溶酶体膜特异性紧密结合,在低频动态磁场作用下使磁性纳米颗粒发生旋转,通过机械力撕裂溶酶体膜导致溶酶体内容物释放,用透射电镜观察到在动态磁场施加前后,细胞内的磁性纳米颗粒由聚集状态变成分散状态。"
2.1.5 光热显微镜 光热显微镜又称光热单粒子显微镜,可观察非荧光标记的纳米颗粒,主要依赖于检测粒子光热吸收特性的不同检测粒子,其信号强弱与激光束强度和纳米颗粒光热吸收率呈正比例[36],是将宏观的光热光谱扩展到纳米级领域,具有高灵敏度和选择性,这项技术目前主要用于观察金属纳米颗粒,如金、银、量子点、单畴碳纳米管。Bogart等[37]采用光热显微镜观察右旋糖酐包埋的磁性纳米颗粒细胞内吞过程,并与荧光显微镜、普鲁士蓝染色、透视电镜观察结果进行对比,结果提示光热显微镜观察的结果与以上传统方法观察结果一致,不但可观察固定的细胞、冰冻切片,而且可用于观察活细胞。除定性观察外,又根据光热信号的差异进行定量分析,与电感耦合等离子体质谱分析仪和普鲁士蓝染色比色法两种定量分析结果一致。光热显微镜具有以下优势:对细胞无损伤,可进行活细胞观察;具有高敏感性和分辨率,可精确观察低剂量磁性纳米颗粒在细胞内的位置。 2.1.6 原子力显微镜 原子力显微镜主要用于包括绝缘体在内的固体材料表面结构分析,通过检测样品表面和微型力敏感元件之间的极微弱原子间相互作用力来研究物质的表面结构及性质,可纳米级分辨率获得表面形貌结构信息及表面粗糙度信息,也用于分析细胞表面特征及生物大分子。Silva等[38]通过原子力显微镜动态观察聚乙二醇2000修饰磁性纳米颗粒内吞入NIH-3T3细胞的过程,将磁性纳米颗粒与NIH-3T3共孵育30 min后,可实时观察到细胞膜表面由于磁性纳米颗粒内吞逐渐形成的内吞坑和凹陷(图3),为磁性纳米颗粒细胞内吞提供实时、直观的观察方法。 2.2 定量评价方法 2.2.1 电感耦合等离子体 包括电感耦合等离子体质谱分析仪和电感耦合等离子体原子发射光谱仪,前者是检测离子质谱,后者是检测光学光谱,通过电感耦合等离子体检测,可明确内吞磁性纳米颗粒内金属元素的含量,联合细胞计数可分析单个细胞平均磁性纳米颗粒内吞量,进行定量评价,电感耦合等离子体是目前检测磁性纳米颗粒内吞最常用、最为准确的定量方法。Datta等[22]采用电感耦合等离子体质谱分析仪研究人乳腺癌细胞MDA-MB 231对壳聚糖包覆MnFe2O4磁性纳米颗粒的内吞作用,分别检测24,48,72 h内吞量,发现内吞量呈时间依耐性。Kyoung等[39]采用电感耦合等离子体联合透射电镜、共聚焦显微镜研究脉冲磁场、永磁场、无磁场作用下,磁性纳米颗粒内吞量的变化,发现脉冲磁场可减少磁性纳米颗粒在细胞膜表面聚集,较永磁场和无磁场更利于磁性纳米颗粒的内吞。 2.2.2 磁化强度测定 先测定单位质量磁性纳米颗粒的磁化强度,当磁性纳米颗粒被细胞内吞后,将细胞外磁性纳米颗粒冲洗后,再将内吞磁性纳米颗粒的细胞消化、离心,通过磁强计测定标本中磁性纳米颗粒的磁化强度,计算磁性纳米颗粒的质量,结合细胞计数进行定量分析。Zysler等[40]采用磁化强度测定方法可对组织内的磁性纳米颗粒进行定量分析,其测量精度可达1 μg。Silva等[38]采用磁化强度测量方法比较3种细胞系对聚乙二醇350和聚乙二醇2000包覆超顺磁性纳米颗粒的内吞量差异进行定量分析。但该方法对磁强计的测量精度要求高,如细胞数量少、内吞的磁性纳米颗粒总质量少会导致误差较大。 2.2.3 普鲁士蓝染色比色法 根据细胞内吞磁性纳米颗粒量不同,细胞内含有铁元素质量不同,利用普鲁士蓝试剂反应后细胞悬液色阶差异,采用分光光度计或酶标仪进行检测,可间接得到细胞内吞磁性纳米颗粒的含量变化趋势。Yallapu等[30]研究磁性纳米颗粒与血清蛋白共孵育后理化及生物特性的变化,普鲁士蓝染色后采用酶标仪观测发现,人前列腺癌C4-2B和胰腺癌细胞Panc-1对与血清共孵育的磁性纳米颗粒内吞量,随血清浓度增高内吞量增大,从而验证了磁性纳米颗粒与血清混合后会影响细胞对磁性纳米颗粒的内吞。 "
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