药学学报  2015, Vol. 50 Issue (8): 980-985   PDF    
金属特性碳纳米管的筛选分离及对人类乳腺癌细胞的光热效应
侯进1, 弥曼1, 魏明1, 易文辉2     
1. 西安医学院基础医学部药理学与毒理学教研室, 陕西 西安 710021;
2. 西安交通大学电信学院陕西省信息光子技术重点实验室, 陕西 西安 710049
摘要:分离并研究金属碳纳米管 (m-SWNTs) 的光热效应及其对乳腺癌细胞的抑制作用。采用PmPV (poly [(m-phenylenevinylene)-alt-(p-phenylenevinylene)]) 从单壁碳纳米管 (SWNTs) 中分离得到具有金属特性碳纳米管, 测定其光热效应, 与MCF-7乳腺癌细胞共同培养, 在808 nm波长下, 用红外光 (NIR) 以2 W·cm-2的强度照射细胞3 min, MTT比色法测定m-SWNTs对MCF-7细胞的杀灭作用; 流式细胞术测定m-SWNTs对MCF-7细胞凋亡的影响。结果表明, 用PmPV可有效分离m-SWNTs, 其光热效应明显增强; 可以抑制MCF-7细胞的存活率, 促进MCF-7细胞凋亡, 其效果强于未经分离的SWNTs。通过PmPV分离的m-SWNTs具有更高的光热转化效率, 并且对MCF-7细胞具有更强的杀灭效果。
关键词碳纳米管     MCF-7细胞     光热效应    
Photothermal effects of metallic carbon nanotubes on human breast cancer cells
HOU Jin1, MI Man1, WEI Ming1, YI Wen-hui2     
1. Department of Pharmacology, School of Basic Medical Sciences, Xi'an Medical College, Xi'an 710021, China;
2. Department of Electronic Science and Technology, School of Information and Electronics Engineering, Xi'an Jiaotong Univeristy, Xi'an 710049, China
Abstract: SWNTs are a mixture of 1/3 metallic SWNTs (m-SWNTs) and 2/3 semiconducting SWNTs (s-SWNTs). It is desirable to separate the metallic SWNTs from the semi-conducting ones. In this study m-SWNTs was separated by using a poly[(m-phenylenevinylene)-alt-(p-phenylenevinylene)] (PmPV) derivative and used as photo-thermal media instead of SWNTs. The separation effects of m-SWNTs were evaluated by Raman spectra, molecular modeling and TEM images. The effects of m-SWNTs on MCF-7 cell proliferation and apoptosis were evaluated with MTT assay and flow cytometry, respectively. m-SWNTs were separated with high purity. A strong inhibition of MCF-7 cell growth was observed with the m-SWNTs under near-infrared (NIR) light irradiation. Our results will be helpful for the potential applications of m-SWNTs in clinical photo- thermal cancer therapy.
Key words: carbon nanotube     MCF-7 cell     photo-thermal effect    

乳腺癌是女性最常见的恶性肿瘤之一。发病率居女性癌症第一位。全世界每年约有130万新发病例, 50万死亡病例。近年来,我国乳腺癌发病率增长明显,目前国内患者人数已超过50万。传统的乳腺癌治疗方法包括手术切除、化疗和内分泌治疗。但是很多乳腺癌患者 (包括早期患者)经过严格治疗之后,仍然会发生复发和转移[1,2]

近年来以碳纳米管 (carbon nanotubes, CNTs)为媒介的光热疗法 (carbon nanotube-mediated thermal therapy, CNMTT)受到广泛关注。研究表明, CNMIT对于肿瘤细胞可进行有效杀灭,能够有效降低肿瘤治疗过程中的抵抗,防止复发[3,4,5]

CNTs是一层或者多层sp2碳原子组成的石墨片无缝卷绕而形成的圆柱状结构,具有较宽的电磁波频谱吸收范围,涵盖生物组织的近红外窗口 (NIR窗口, 700~1 100 nm)以及无线电频段和微波频段。CNTs在生物医药领域和临床上具有广阔的应用前景: ① CNTs特殊的一维空间结构,使其具有“纳米针”作用 (nano-needle),很容易刺穿细胞膜而进入细胞内[6]。CNTs的穿刺作用和细胞的内吞作用相结合,使CNTs可以作为理想的载体,将药物、核酸、荧光标记等传递给组织和细胞。②由于CNTs具有增强渗透和滞留效应 (enhanced permeability and retention effect, EPR),即在血液循环系统中长时间存留并且通过肿瘤周围的血管渗漏进入肿瘤组织,可以选择性地在肿瘤部位富集,对肿瘤具有被动靶向功能[7, 8]。③可以通过有机功能化的方法将抗体或配体与载药接枝在CNTs表面,利用抗原、抗体及细胞膜表面受体、配体之间的专一性相互识别,实现针对肿瘤细胞的主动识别和靶向递药。 ④CNTs在红外光 (NIR)窗口的光热响应,使其可以在NIR光照条件下迅速升温,快速杀死肿瘤细胞,即肿瘤光热治疗 (photothermal therapy)[9]。综上所述, CNTs可作为肿瘤光热治疗过程中的“纳米天线”(nano-antenna)[10]和靶向递药过程中的“纳米舟”(nano-boat)[11],对肿瘤细胞和肿瘤干细胞 (CSCs)起到双重杀灭作用[12,13]

CNTs分为两种:由单层石墨片卷绕而成的称为单壁碳纳米管 (SWNTs),直径介于0.4~2.0 nm之间,由多层 (数层到数十层)石墨片同轴卷绕而成的称为多壁碳纳米管 (MWNTs),直径介于几个纳米到上百纳米之间。SWNTs的能带结构和电学、光学特性由石墨点阵与纳米管轴之间的夹角决定,可以用手性参数 (n, m)来表征。当两个手性参数之差不是3的整数倍时 (n-m = 3p ± 1, p为整数),为半导体特性碳纳米管 (s-SWNTs);当两个手性参数之差为3的整数倍时 (n-m = 3p, p为整数),为金属特性碳纳米管 (m-SWNTs)。当前,大多数方法制备出的SWNTs是成分复杂的混合体,包含数十种不同手性的碳管,其中1/3为金属特性, 2/3为半导体特性。理论上, m-SWNTs能直接将光激发的能量通过非辐射途径迅速转化为热能,光热转化效率高、转化速率快,是光热疗法理想的媒介材料[13, 14];而s-SWNTs在光激发跃迁到激发态后,存在多种相互竞争的激发态弛豫途径 (即能量耗散通道),只有一部分转换为热能,而且转化速率相对较慢。

因此,如果能够对SWNTs进行有效筛选分离,获得高纯度m-SWNTs,将会大幅度提高光热疗法的疗效。作者的研究[15, 16]表明PmPV (poly[(m-pheny­lenevinylene)-alt-(p-phenylenevinylene)])可以有效地实现对SWNTs的筛选分离,在此基础上,采用PmPV对金属特性碳纳米管进行分离,得到纯度较高的金属特性碳纳米管,并探讨金属特性碳纳米管光热效应强度及对MCF-7乳腺癌细胞的杀灭效果。 材料与方法 材料

噻唑蓝 (MTT), DMEM购自美国Gibco公司, MCF-7乳腺癌细胞为本实验室留存,新生牛血清为杭州四季青生物制品公司产品;Annexin V/PI试剂盒,嘉美生物公司产品。SWNTs通过HiPCO方法合成,美国Akron大学合成。 SWNTs的纯化、剪裁

考虑到尽可能降低SWNTs进入生物体内的毒性,必须对其进行严格纯化以重点除去催化剂粒子,并进行严格化学剪裁,使之具有相对均一的管径和长度。HiPCO SWNTs (0.5~1.0 g)在硝酸 (2.6 mol·L-1, 300 mL)中回流 48 h, 0.45 μm滤膜过滤,滤过物超声0.25 h,过滤后,将HiPCO SWNTs置入150 ℃烤箱中处理3 h。干燥后的HiPCO在225 ℃管式炉中处理18 h,冷却后,置入50 mL盐酸中超声0.5 h,过滤,用蒸馏水和甲醛洗3遍,自然干燥。通过在硝酸回流和盐酸中超声等一系列严格纯化处理和化学剪裁,除去了催化剂粒子,得到管径和长度分布均匀的SWNTs[16]m-SWNTs的分离、包覆

将纯化的SWNTs 0.3 mg加入PmPV (MV = 25 000, 0.2 mg·mL-1)溶液 20 mL中,冰浴中超声3 h。在7 000×g下离心6 h。收集沉淀再次悬浮于PmPV中,重复离心超声10次,得到m-SWNTs。将m-SWNTs溶液加入1% PEG溶液100 mL中,超声辅助混合2 h,然后在室温下搅拌24 h。8 000 r·min-1离心5 min,分离不溶组分,最后得到深色的均匀溶液,即包裹PEG的m-SWNTs溶液[15]

Figure 1 Structure of PmPV (poly[(m-phenylenevinylene)-alt- (p-phenylenevinylene)])
m-SWNTs的表征及光热效应测定

样品的光学性质通过拉曼光谱仪检测 (HORIBA JOBIN YUON HR 800),用透射电子显微镜 (JEM 2100)观察制备的m-SWNTs的形貌和尺寸。将SWNTs和m-SWNTs置入1% PBS,配成不同浓度,在808 nm波长下,用NIR以2 W·cm-2的强度照射3 min,用MT-29/3热电耦仪测定样品的光热效应。 MCF-7细胞培养及光热效应对MCF-7细胞的影响

用含100 mL·L-1胎牛血清的DMEM (含1×105 u·L-1青霉素和100 mg·L-1链霉素)培养MCF-7细胞,在37 ℃、5% CO2及饱和湿度条件下培养。24 h换液,然后每2~3天更换一次培养液。细胞长满后,用2.5 g·L-1胰酶消化后传代。

细胞增殖测定 (MTT实验):将MCF-7细胞悬浮液接种于96孔板 (每孔2×103),每孔100 μL,待细胞贴壁生长后,分别加入5、10和50 μg·mL-1的SWNTs和m-SWNTs的DMEM溶液。设置阴性对照和空白对照。温育0.5 h后,在808 nm波长下,用NIR以2 W·cm-2的强度照射细胞3 min。每孔加入MTT (5 g·L-1) 20 μL,继续孵育4 h。吸弃孔内上清液,每孔加入DMSO 200 μL,振荡10 min,在490 nm波长处检测各孔吸光度 (OD值)[17]

细胞凋亡测定:调整细胞密度为每毫升3×104,接种于培养板中,用含10% FCS的DMEM培养液培养24 h后,换成含2% FCS的培养液,饥饿24 h以达到同步化,然后分别加入50 μg·mL-1 SWNTs和m-SWNTs的DMEM溶液, 0.5 h后,在808 nm波长下,用NIR以2 W·cm-2的强度照射细胞3 min。收集细胞,将每毫升2×105~1×106细胞数,悬浮于200 μL结合缓冲液中,加入Annexin V-FITC 10 μL和PI 5 μL,轻轻混匀,避光静置15 min,流式细胞仪分析[17]统计学处理

实验数据以x±s表示,所有实验至少重复3次,采用SPSS统计软件进行方差分析 (one- way ANOVA)。 结果 1 m-SWNTs的形貌特征及拉曼光谱

图 2透射电镜图片是经过纯化、剪裁、分离和包覆的m-SWNTs形貌。图 2A可以看到m-SWNTs的尺寸均匀、分散良好、无聚集现象、PEG包覆均匀, 图 2B为单根m-SWNTs的局部放大图片,可以看到单根m-SWNTs表面光滑, PEG包覆完整、均匀。

Figure 2 TEM images of m-SWNTs. A: Low-magnification TEM image of the product, showing the large quantity of m-SWNTs; B: High-magnification image of a single m-SWNT

图 3为m-SWNTs的分离示意图,共轭高分子PmPV对m-SWNTs具有很强的亲合力,通过将SWNTs置于PmPV溶液中经过一系列超声-离心过程,可以分离m-SWNTs。图 4是SWNTs的拉曼光谱,从图中可看到采用PmPV分离方法, m-SWNTs含量可提高到80%。

Figure 3 Molecular modeling of PmPV oligomer (cyan color) wrapped on a SWNT

Figure 4 Raman spectra of SWNTs at 633 nm laser excitation. The red spectrum is metallic SWNTs after the PmPV separation. The black spectrum is from the SWNTs dispersion before separation
2 m-SWNTs的光热效应

将SWNTs和m-SWNTs置入1% PBS,配成5、10、20、50、100、200、500和1 000 μg·mL-1等不同浓度,在808 nm波长下,用红外光照射SWNTs和m-SWNTs溶液,观察碳纳米管的光热效应。随着碳纳米管浓度的升高,溶液温度也逐渐升高。碳纳米管质量浓度为5 μg·mL-1时,二者升温作用都不明显。质量浓度为10 μg·mL-1时, m-SWNTs的升温效果开始高于SWNTs。当质量浓度为100 μg·mL-1时, m-SWNTs溶液温度为37 ℃, SWNTs溶液温度为30 ℃, m-SWNTs的升温效果明显高于SWNTs,表明通过PmPV分离得到的m-SWNTs有更高的光热转化效率。 3 m-SWNTs对MCF-7细胞活性和细胞凋亡的影响

图 5所示,对数生长的MCF-7细胞中,分别加入不同浓度的SWNTs和m-SWNTs,在808 nm波长下,用NIR照射细胞后,加入m-SWNTs的MCF-7细胞存活率下降,小剂量、中剂量和大剂量相对存活率分别为对照组的84.2% (P < 0.05)、35.6% (P < 0.01)和25% (P < 0.01),而加入SWNTs的MCF-7细胞相对存活率分别为对照组的88.1% (P < 0.05)、55.8% (P < 0.01)和43.5% (P < 0.01),实验结果表明,光照后, m-SWNTs对MCF-7细胞的增殖有明显抑制作用,抑制作用呈剂量依赖性,剂量超过10 μg·mL-1后,对MCF-7细胞的抑制作用明显强于SWNTs (P < 0.01)。

Figure 5 Effects of m-SWNTs and SWNTs on the viability of MCF-7 cells. The cells were suspended in DMEM and plated at a density of 2.0×103 cells/well into a 96-well culture dish. After 24 h, the medium was replaced with DMEM in the presence or absence of m-SWNTs and SWNTs, followed by Laser excitation (3 min, 808 nm, 2 W·cm-2). The cell viability normalized to 100% compared with the vehicle DMSO was measured by MTT. n = 3, x±s. P < 0.05, **P < 0.01 vs control group, ##P < 0.01 vs SWNTs group

图 6所示,在808 nm波长下,用NIR照射细胞后,加入50 μg·mL-1 m-SWNTs组的细胞出现大量的凋亡和坏死,效果强于等剂量的SWNTs组。

Figure 6 Effects of SWNTs/PEG on cell apoptosis detected by flow cytometry using Annexin V/PI staining. A: Control sample (standard cell culture conditions); B: Exposure to 50 μg·mL-1 SWNTs (0.5 h, 37 ℃), followed by Laser excitation (3 min, 808 nm, 2 W·cm-2). C: Exposure to 50 μg·mL-1 m-SWNTs (0.5 h, 37 ℃), followed by Laser excitation (3 min, 808 nm, 2 W·cm-2)
讨论

现有方法制备的SWNTs包含数十种不同手性参数 (n, m)的SWNTs,彼此混杂而且紧紧束缚在一起。因此,对SWNTs进行筛选分离,是实现其广泛应用的关键步骤。2002年以来,国际上在SWNTs的筛选分离方面开展大量研究工作,主要包括不同电子类型(M-SWNTs vs S-SWNTs)、不同手性 (chirality- specific)的筛选分离[18]。密度梯度超速离心工艺 (density gradient ultracentrifugation, DGU)可以实现对不同直径和不同电子特性SWNTs (m-SWNTs和s-SWNTs)的分离但是分离效率不高,往往需要多次重复分离过程,才能得到较高的纯度。而且对于各组分密度差别非常小的HiPCo SWNTs, DGU法难以实现各组分有效分离[19]。有机小分子 (如烷基胺和四羟酮醇等)、共轭高分子 (如poly(9,9-dioctylfluorenyl-2,7- diyl (POF)等)以及DNA对m-SWNTs与s-SWNTs具有不同的亲合力,通过非共价化学的方法可以对m-SWNTs与s-SWNTs进行有效筛选分离,同时保证SWNTs本征电子结构不受破坏[20]

研究发现,共轭高分子PmPV对m-SWNTs具有很强的亲合力[15],通过将SWNTs置于不同浓度PmPV溶液中经过一系列超声-离心过程萃取, m-SWNTs含量可提高到80%以上。如图 2所示,将SWNTs加入到PmPV溶液中进行十次超声-离心处理,在光斑聚焦区域m-SWNTs含量达到80%。m-SWNTs经过化学剪裁,其长度可控制在1 µm左右,通过PEG包裹,其具有良好的组织相容性。PEG为线型柔性水溶性高分子,在超声辅助下,能够缠绕在m-SWNTs表面, PEG不仅具有良好的生物相容性,而且能够抑制血液中巨噬细胞以及其它单核吞噬细胞的清洁功能,因此用PEG包裹m-SWNTs可以在血液循环系统中具有较长的半衰期。

SWNTs能将光激发的能量通过非辐射途径迅速转化为热能,通过光热效应,杀死肿瘤细胞。有报道将SWNTs注入到裸鼠肿瘤中,用3 W·cm-2的红外光照射3 min,肿瘤细胞被完全杀灭,并且在几个月内未见复发。但是,造成裸鼠严重烧伤。而在相对温和的实验条件下 (200 mW·cm-2, 10 min)进行类似实验,虽然观察到肿瘤生长减少,但有一部分肿瘤细胞能够依然生存下来,不能持久缓解病症,且与病灶相邻的正常组织有坏死迹象,表明热从靶点部位传递给周围正常组织并造成损伤[21]。因此,使用未经分离的SWNTs的光热效率不够高,升温速度不够快,导致杀灭肿瘤细胞需要的光照功率过强或光照时间过长,很容易造成周围组织损伤[22]

m-SWNTs当中还包含金属特性碳管 (pure metallic SWNTs, Eg = 0)和半金属特性碳管 (semi-metallic SWNTs, Eg < 0.1 eV)两部分。当两个手性参数相等时 (n = m),带隙为零 (Eg = 0),是严格意义上的m-SWNTs。其余的“m-SWNTs”则存在一个很小的“准带隙” (Eg < 0.1 eV),为半金属特性碳管,研究者[23]通过PmPV方法获得的m-SWNT浓度大约为80%,对肿瘤细胞已表现出很强的光热效应,大幅度提高了肿瘤光热疗法的效应,因此如果能够在m-SWNTs分离基础上,对m-SWNTs进行进一步筛选分离,获得严格意义上的零带隙 (Eg = 0) m-SWNTs,将会进一步提高光热疗法的疗效。

综上所述,作者采用PmPV方法分离了SWNTs,得到了纯度达80%以上的m-SWNTs,研究了在近红外光[3,4]照射下m-SWNTs对MCF-7细胞的光热治疗效应。实验结果表明,在细胞中加入m-SWNTs,光照后能明显抑制肿瘤细胞活性并促进其凋亡,作用明显强于未经分选的SWNTs; PmPV方法对SWNTs有很好的筛选效果,筛选得到的m-SWNTs对MCF-7细胞的光热效应更强,提示对乳腺癌的光热治疗有着潜在的应用前景。

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