Preparation of magnetic carbon nanotubes and their application in tumor cell photo-thermal therapy and magnetic resonance imaging
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摘要: 肿瘤是目前最主要的致死原因之一，实现对肿瘤的精准和非侵入性高效诊疗具有重要意义。以具有极高长径比、易于穿透细胞膜并具有优异生物相容性的碳纳米管(CNTs)作为载体，以乙酰丙酮铁为铁源，通过溶剂热法在其表面原位生长具有超顺磁特性的四氧化三铁纳米粒子(Fe3O4 NPs)，制备了具有优异水分散稳定性的磁性碳纳米管复合纳米材料。结果表明该磁性碳纳米管具有较高的近红外光热转换性能，在50 μg·mL−1浓度下808 nm激光照射10 min即可升温至48.6℃，且具有良好的光热稳定性。细胞及成像实验结果表明该复合纳米材料具有较好的生物相容性并对人宫颈癌细胞(HeLa)具有优异的光热杀伤效果，在体外模拟肿瘤微环境中磁共振成像(MRI) T2弛豫率r2可达215.61 mmol−1·L·s−1，表明制备的磁性碳纳米管具有出色的生物安全性、磁性和光热特性，有望用于磁靶向的肿瘤光热疗与磁共振成像的一体化诊疗。
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关键词:
- 碳纳米管 /
- 磁性Fe3O4纳米粒子 /
- 光热治疗 /
- 磁共振成像 /
- 肿瘤细胞
Abstract: Tumors are one of the leading causes of death in the world, and achieving precise and non-invasive efficient diagnosis and treatment of tumors is of great significance. We used carbon nanotubes (CNTs) with extremely high aspect ratio, easy to penetrate cell membrane and excellent biocompatibility as carriers, and acetylacetone iron as iron source, to synthesize magnetic carbon nanotube composite nanomaterials with excellent water dispersion stability by in situ growing superparamagnetic ferric oxide nanoparticles (Fe3O4 NPs) on their surface through solvothermal method. The results showed that the magnetic carbon nanotubes had high near-infrared photothermal conversion performance, and could reach 48.6℃ in 10 min under 808 nm laser irradiation at a concentration of 50 μg·mL−1, and had good photothermal stability. Cell and imaging experiments showed that the composite nanomaterials had good biocompatibility and excellent photothermal killing effect on human cervical cancer cells (HeLa). In vitro simulated tumor microenvironment, the magnetic resonance imaging (MRI) T2 relaxation rate r2 of the magnetic carbon nanotubes was up to 215.61 mmol−1·L·s−1, indicating that the prepared magnetic carbon nanotubes had outstanding biosafety, magnetism and photothermal properties, and were expected to be applied to the integration of magnetic targeted tumor photothermal therapy and magnetic resonance imaging. -
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图 1 (a) 羧化截短的多壁碳纳米管(cut-MWNTs)的TEM图像；(b) Fe3O4-MWNTs复合纳米材料(M@Fe3O4)的TEM图像；(c) 局部放大的M@Fe3O4 TEM图像；(d) M@Fe3O4表面Fe3O4纳米粒子粒径统计(200个颗粒)；(e) cut-MWNTs和M@Fe3O4的表面zeta电势；(f) M@Fe3O4水分散液在磁场作用下聚集；(g) M@Fe3O4水分散液放置5天前后的光学照片
Figure 1. (a) TEM image of carboxylated truncated multi-walled carbon nanotubes (cut-MWNTs); (b) TEM image of Fe3O4-MWNTs (M@Fe3O4); (c) TEM image of locally magnified M@Fe3O4; (d) Particle size statistics of iron tetroxide nanoparticles on the surface of M@Fe3O4 (200 particles); (e) Surface zeta potential of cut-MWNTs and M@Fe3O4; (f) M@Fe3O4 aqueous dispersions aggregated under magnetic field; (g) Photographs of M@Fe3O4 aqueous dispersion at 0 day and 5 days
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图 2 (a) cut-MWNTs和M@Fe3O4的紫外可见吸收光谱；(b) M@Fe3O4的XRD图谱；(c) M@Fe3O4的XPS全谱；(d) M@Fe3O4的XPS Fe2p谱；(e) M@Fe3O4的磁滞回线
Figure 2. (a) UV-visible absorption spectrum of cut-MWNTs and M@Fe3O4; (b) XRD spectrum of M@Fe3O4; (c) XPS survey spectrum of M@Fe3O4; (d) XPS Fe2p spectrum of M@Fe3O4; (e) Magnetic hysteresis curves measured for M@Fe3O4
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图 3 光热性能：(a) M@Fe3O4在不同浓度和激光辐照功率下的光热升温曲线(A：150 μg·mL−1、1.5 W·cm−2；B：100 μg·mL−1、1.5 W·cm−2；C：50 μg·mL−1、1.5 W·cm−2；D：50 μg·mL−1、1.0 W·cm−2；E：20 μg·mL−1、0.5 W·cm−2)；(b) cut-MWNTs和M@Fe3O4在808 nm激光下的光热升温曲线，浓度为50 μg·mL−1，激光功率为1.5 W·cm−2；(c) M@Fe3O4在1.5 W·cm−2下的循环升温-降温曲线
Figure 3. Photothermal properties: (a) Photothermal heating curves of M@Fe3O4 at different concentrations and laser irradiation power (A: 150 μg·mL−1, 1.5 W·cm−2; B: 100 μg·mL−1, 1.5 W·cm−2; C: 50 μg·mL−1, 1.5 W·cm−2; D: 50 μg·mL−1, 1.0 W·cm−2; E: 20 μg·mL−1, 0.5 W·cm−2); (b) Photothermal heating curves of cut-MWNTs and M@Fe3O4 at 808 nm laser, 50 μg·mL−1, Laser power is 1.5 W·cm−2; (c) Cyclic heating-cooling curve of M@Fe3O4 at 1.5 W·cm−2
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图 4 不同共孵育时间和不同浓度条件下，cut-MWNTs和M@Fe3O4对HeLa宫颈癌细胞 (a) 和LO2正常肝细胞 (b) 的细胞毒性
Figure 4. HeLa cervical cancer cells (a) and LO2 normal hepatocytes (b) at different co-incubation time and at different concentrations of cytotoxicity of cut-MWNTs and M@Fe3O4
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图 5 在1.5 W·cm−2 的激光功率(808 nm)下，不同浓度的cut-MWNTs和M@Fe3O4对HeLa宫颈癌细胞的杀伤效果
Figure 5. Killing effect of HeLa cervical cancer cells by cut-MWNTs and M@Fe3O4 at a laser power of 1.5 W·cm−2 (808 nm)
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图 6 M@Fe3O4与HeLa细胞共孵育经过活死细胞双染后的共聚焦扫描图像：(a) 明场图像；(b) 活细胞图像；(c) 死细胞图像；(d) 明场和暗场叠加图像
Figure 6. Confocal scanning images of M@Fe3O4 coincubated with HeLa cells after double staining with live-dead cells: (a) Bright field image; (b) Live cell image; (c) Dead cell image; (d) Bright field and dark field superimposed image
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目的
肿瘤是目前最主要的致死原因之一，实现对肿瘤的精准和非侵入性高效诊疗具有重要意义。发展兼具生物安全性、靶向性和非侵入性治疗试剂是实现上述目标的主要途径之一。本文利用具有较高长径比的碳纳米管和具有磁靶向及MRI造影特性的四氧化三铁纳米粒子制备一类新型诊疗试剂，研究其抗肿瘤性能。
方法将表面疏水的碳纳米管利用混酸处理，将碳管截短的同时在其表面引入丰富的亲水基团，提升其生物相容性；将羧化截短的碳纳米管与铁源—乙酰丙酮铁在高沸点溶剂中混合加热，制备了碳纳米管表面原位负载超小四氧化三铁纳米粒子的复合材料MWNTs@FeO(记为M@FeO)，并对其结构和性能进行表征和测试。
结果(1)羧化截短后的多壁碳纳米管长度约为1 μm，表面较为粗糙，表面电势为-23.5±0.3 mV，表明混酸处理过程在碳纳米管表面引入了大量的亲水官能团，而修饰后的碳纳米管降至-53.8±2.6 mV，表明M@FeO表面同样富含亲水官能团，使其在水中放置5天仍具有优异的分散稳定性；(2)FeO NPs均匀地分布在碳纳米管表面，粒径主要分布在6-12 nm范围，其水分散液经过磁铁吸附时，15s即可完全聚集在靠近磁铁的一侧，表明其具有优异的顺磁性；(3)羧化截短的碳纳米管具有较宽的吸收，最大吸收波长在280 nm左右，而在原位生长FeO NPs后，其在近红外区域的吸收明显提高，最大吸收波长出现一定偏移，达到324 nm左右，表明碳纳米管表面成功负载上大量四氧化三铁纳米粒子。XRD中位于30.09°、35.42°、43.05°、53.39°、56.94°、62.52°处左右的尖锐峰可分别归属于四氧化三铁纳米粒子(220)、(311)、(400)、(422)、(511)、(440)晶面，XPS测试表明复合碳纳米管表面Fe主要有三种结合态，其磁饱和强度为31.905emu/g，表明磁性碳纳米管的成功制备；(4)不同浓度和激光功率下磁性碳纳米管的光热转换性能测试表明，在808 nm激光器辐照下，在50 μg/mL浓度下，经过10 min光照之后，羧化截短的碳纳米管升温至45.9 ℃，而M@FeO的升温幅度则为48.6 ℃，在150 μg/mL和1.5 W/cm的条件下温度更是达到55.3 ℃，表现出明显的激光功率和浓度依赖性；(5)在50 μg/mL的共孵育浓度下，经过24和48小时共孵育之后正常细胞存活率仍均超过85%，表明所制备的羧化截短的碳纳米管和磁性碳纳米管具有较好的生物相容性，利用1.5 W/cm的808nm激光进行照射，随着材料浓度的提高，细胞存活率有明显下降，在材料浓度为50 μg/mL时，细胞存活率低至46.2%，激光共聚焦测试进一步说明经过808nm激光照射后肿瘤细胞大量死亡，与细胞存活率实验结果一致；(6)在模拟肿瘤微环境的弱酸性条件(pH 5.0)下，其弛豫率r可达215.6 mmol·L·s，而在正常体液微环境中，弛豫率仅为118.9 mmol·L·s。
结论制备的磁性碳纳米管M@FeO具有优异的生物相容性、顺磁性和近红外光热转换性能，具有肿瘤微环境响应性的MRI-T造影性能，有望用做磁靶向的肿瘤诊疗一体化试剂。此外，材料在细胞内的分布以及在动物体内循环稳定性、各器官的分布及肿瘤靶向性仍有待进一步深入研究。
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本文选择碳纳米管作为载体和光热转换试剂，在其表面原位生长四氧化三铁纳米粒子，制得四氧化三铁-碳纳米管复合纳米材料(M@Fe3O4)，该磁性碳纳米管具有较高的近红外光热转换性能，在50 μg·mL-1浓度下808nm激光照射10分钟即可升温至48.6℃，且具有良好的光热稳定性。细胞实验表明，该复合纳米材料对人宫颈癌细胞具有优异的光热杀伤效果，肿瘤微环境中T2弛豫率r2可达215.61 mmol-1·L·s-1 。一方面能提高光热转化效果，另一方面利用四氧化三铁纳米粒子提供潜在的磁靶向和T2-MRI成像应用。并研究了该复合纳米材料对宫颈癌细胞的光热杀伤性能与在肿瘤微环境中的磁共振造影性能，为肿瘤诊疗一体化提供了新材料和新思路。
光热性能：(a) 羧化截短碳纳米管和磁性碳纳米管在808 nm激光下的光热升温曲线，浓度为50 μg·mL-1，激光功率为1.5 W·cm-2；W·cm-2; (b) 磁性碳纳米管在1.5 W·cm-2下的循环升温-降温曲线。
磁性碳纳米管在不同pH条件下的T2-MRI造影效果和弛豫率
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