【作者】 刘爱红；

【导师】 孙康宁； 赵冬梅；

【作者基本信息】 山东大学 ， 材料学， 2008， 博士

【摘要】 肿瘤热疗当前最迫切需要解决的问题是如何在所需加热的组织进行均匀的加热,使其达到43℃以上的有效治疗温度,但又不引起附近正常组织严重的和永久性的损伤,即如何实现肿瘤靶向定位治疗。纳米科技的飞速发展为肿瘤热疗开启了新的思路。研发一种吸波性能优异、高产热率、生物相容性好的新型肿瘤热疗用热种子材料,实现微波靶向定位热疗,将为肿瘤的医治开辟一条高效的新途径,具有十分重要的科学意义与实用价值。碳纳米管（CNTs）是纳米材料的典型代表之一,具有良好的吸波性能和热学性能,决定了它作为热种子材料在微波肿瘤靶向热疗中具有广阔的应用前景。本文围绕碳纳米管热种子材料的生物学效应、表面修饰、吸波性能以及其在微波的作用下是否具有明显的热效应,这一主题进行了一系列的实验工作和理论研究,为促进热种子材料的实用化,同时制备了具有可注射性的热疗用温敏凝胶。本论文主要研究内容如下:1、本研究以Wistar大白鼠为研究对象,主要对不同尺寸的多壁碳纳米管的生物学效应进行了系统的评价。采用气管注入和尾静脉注射两种方式将直径为40-60nm多壁碳纳米管（40-60MWNTs）和直径小于10nm的多壁碳纳米管（10MWNTs）暴露于大白鼠的体内,同时分别采用纳米二氧化硅（SiO₂）和乙炔碳黑（Cb）作为阳性和阴性对照。详细评估了MWNTs缓慢注入大白鼠气管后在肺部产生的毒性及MWNTs进入体内后对靶器官的影响。气管注入法实验结果表明:实验中纳米颗粒均不同程度的引起了肺部的损伤,引起肺部损伤严重程度的顺序为:Cb＞10nm MWNTs＞40-60nm MWNTs＞SiO₂。另外,MWNTs对肺部损伤的程度与剂量呈依赖关系,当在低剂量（1mg/kg）时,40-60nm MWNTs仅仅对肺部引起了轻微的炎症损伤,与空白对照组几乎没有区别。随着剂量的增大,MWNTs对肺部的损伤越来越显著。尾静脉注射法实验结果表明:纳米粒子进入大鼠体内后,大鼠心脏、肾脏、肝脏和胃结构正常,未见明显的病理损伤。但是各种纳米粒子却在大鼠肺部沉积,随着暴露时间的延长,纳米粒子一部分被肺巨噬细胞清除体外,但是残余的纳米粒子引起了肺部的病理学改变。与气管注入法相似,商品化MWNTs对肺部的损伤呈现正的剂量一效应关系。另外,不同的暴露方式对靶器官的影响程度不同。在相同的剂量下,采用气管注入法MWNTs引起的肺部损伤比采用尾静脉注射法MWNTs引起的肺部损伤要显著。综合上述数据和结果,我们可以看出,碳纳米管对人类的身体健康具有潜在的毒性。2、针对CNTs潜在的毒性,本文选取羟基磷灰石（HAp）、四氧化三铁（Fe₃O₄）和壳聚糖（CS）三种目前公认的生物相容性较好的材料,对CNTs进行了表面修饰,在保证CNTs优异性能的同时,提高CNTs的生物相容性,扩大CNTs在医学中的应用。采用化学沉淀结合水热法成功实现了HAp对CNTs的表面修饰。当CNTs质量分数为10%,160℃水热处理5小时后,在CNTs的表面可以获得具有一定微观形貌、高结晶度、组分单一的HAp包覆层。采用化学沉淀结合水热法成功实现了Fe₃O₄对CNTs的表面修饰,Fe₃O₄纳米小粒子在CNTs的表面包覆均匀,具有普遍性;包覆Fe₃O₄后的CNTs具有纯Fe₃O₄相似的超顺磁性,当CNTs的质量分数为10%时,其比饱和磁化强度为64.53emu/g,矫顽力为14.03Oe,可以实现CNTs在磁场中的定向,说明我们成功制备了一种新的肿瘤靶向热疗用材料。采用表面沉积交联法,实现了CS对CNTs的表面修饰。包覆CS后CNTs的管径尺寸从40-60 nm增加到100nm左右。CS均匀的覆盖在CNTs表面,界面结合良好。由于CS覆盖层的静电排斥作用,使被包裹后CNTs的团聚减少。3、采用矢量网络分析仪测试碳纳米管复合材料的电磁参数,并对实测的电磁参数数据进行计算得反射率R（dB）—f（GHz）曲线,结果表明:CNTs/HAp复合材料为典型的电损耗型吸波材料,在0-5GHz范围内具有较好的吸波性能,其中CNTs在其中起到主导作用。随着CNTs含量的增加,CNTs/HAp复合材料的吸波能力逐渐增强,吸收峰向着高频移动,频宽增大。当CNTs的质量分数为8%时,复合材料的最大吸收峰在3.0 GHz附近,反射率达-26dB。Fe₃O₄对CNTs包覆起到了调整CNTs电磁参数的作用,在原有的介电损耗的基础上又增加了Fe₃O₄的磁滞损耗和共振吸收等特性。CNTs/Fe₃O₄复合材料在0-5GHz范围内具有较好的吸波性能,当CNTs的质量分数为4%时,CNTs/Fe₃O₄复合材料的吸收峰在2.2 GHz附近,反射率达-19dB,频宽为1G左右,能够满足微波热疗用热种子材料对吸波性能的要求。4、通过进行纳米材料在微波场中的升温实验,我们表征了材料的热效应,实验结果表明:经过HAp修饰后的碳纳米管的热效应远没有原始碳纳米管的热效应明显。随着CNTs含量的增大,CNTs/HAp复合材料粉体热效应越来越显著。当CNTs质量分数＞8%时,其热效应基本上趋于稳定状态。CNTs/HAp生物复合材料优异的吸波性能和在微波场中具有良好的产热能力,加上HAp的优异的生物相容性,说明该复合材料可以作为微波热种子材料应用于肿瘤热疗。CNTs/Fe₃O₄复合材料在微波辐照下,热效应效果明显。另外,由于Fe₃O₄具有很好的磁性能和生物相容性,可以很好的解决热种子材料在生物体内的靶向定位问题。CNTs/Fe₃O₄复合材料是实现微波靶向热疗理想的热种子材料。5、在壳聚糖—甘油磷酸钠温敏凝胶的基础上成功制备出含有碳纳米管复合热种子材料的热疗用温敏凝胶。流变学测试表明:碳纳米管复合热种子材料（CNTs、CNTs/Fe₃O₄复合材料和CNTs/HAp复合材料）的加入在一定程度上破坏了凝胶的网络结构,使温敏凝胶的初始凝胶温度升高,但是仍然具有温敏特性。在10℃时G′＜G″呈现液体的性质,在37℃时,G＇＞G″呈现凝胶的性质,并且随着剪切频率的改变,G＇＜G″曲线平直,受频率的影响不大,说明37℃时已经形成了完美的稳定的凝胶结构。碳纳米管复合热种子材料在凝胶体系中分布均匀,在微波场中具有良好的产热能力。25w下辐照10min,当CNTs含量为6mg/mL时,体系温度在初始温度基础上,升高了34.6℃;当CNTs/Fe₃O₄复合材料的含量为20mg/mL时,体系温度在初始温度基础上,温度升高了38.7℃;当CNTs/HAp复合材料的含量为20mg/mL时,体系温度在初始温度基础上,温度升高了35℃,说明含有碳纳米管复合热种子材料的温敏凝胶可以满足微波热疗的要求。6、用MTT法检测细胞增殖活力和计算相对增殖率。结果表明:CS温敏凝胶、CNTs-CS温敏凝胶、CNTs-Fe₃O₄-CS温敏凝胶、CNTs-HAp-CS温敏凝胶各浓度浸提液的细胞毒性为0-1级;采用直接接触法观察细胞与材料共同培养若干时间的细胞形态,结果表明:CNTs-Fe₃O₄-CS温敏凝胶生物相容性良好。形态学观察表明细胞在凝胶薄膜上具有良好的形态,细胞能在薄膜上面粘附、增殖。因此,可以认为所制备的热疗用温敏凝胶无细胞毒性,生物相容性良好,为进一步研究打下基础。更多还原

【Abstract】 One of the major problems of tumor hyperthermia is how to make the temperature in tumor increase to 43℃and therefore to kill the tumor cells,without damaging the normal tissue nearby in recent years.A novel thermo-seed material with high thermal efficiency and good biocompatibility can carry out tumor targeted therapy in microwave hyperthermia.It will be a newly and highly effective way for the tumor hyperthermia. CNTs is a typical representative of nanomaterial.Due to outstanding microwave absorbing and thermal properties,carbon nanotubes could be used as thermo-seed material for microwave tumor targeted therapy.In this paper,the biological effects、the surface modification、microwave absorbing properties and the thermal effects of MWNTs were researched.Following are the main contents:1、We exposed different sizes mutil-wall carbon nanotubes（MWNTs）into Wistar rats in intratracheal instillation and intravenous injection two different administration modes.At the same time,we used silica（SiO₂）and acetylene carbon black（Cb）,as positive and negative control.The results showed that:In intratracheal instillation experiments,the nanoparticles caused different levels of lung damage.The order of lung injury degree is:Cb＞10nm MWNTs＞40-60nm MWNTs＞SiO₂.In addition,MWNTs showed dose-dependent relationship on the extent of lung injury.In the low-dose（1mg/kg）,40-60 nm MWNTs only caused minor damage to lung inflammation,there were no difference with the control group almost.As the dose increased,the lung injury degree became more and more severely.When carbon nanotubes was exposed into rats by the method of intravenous injection the structure of lung tissue can produce obvious histopathologic changes but other tissues are almost in order.When the same dose carbon nanotubes was poured into the rats by intravenous injection and intratracheal instillation respectively,the inflammation reflection is mild that arose by the former than the latter. Under the conditions of this test,pulmonary exposure to MWNTs in rats by intratracheal instillation produced a series of multiple lesion in a dose-dependent and time-dependent manner,evidence of a foreign tissue body reaction.Although there are anormous differences in lung between rodent animals and human beings,the results could call attention to the human beings at potential risk for exposure to MWNTs.2、In this paper,the suface of CNTs were modified by hydroxyapatite（HAp）,iron oxide（Fe₃O₄）and chitosan（CS）which are currently recognized to be biocompatible in order to attain a kind of multi-wall nanotubes composites combining the biocompatibility and the unique properties of pristine multi-wall carbon nanotubes.This will enlarge the potential application of CNTs in tumor hyperthermia.HAp nanoparticles were decorated on CNTs successfully by a simple and effective coprecipitation and hydrothermal treatment method.When CNTs content was 10%, after hydrothermal treatment,a certain high degree of crystallinity,pure component single HAp layer were obtained on the surface of CNTs.Fe₃O₄ nanoparticles were densely decorated on CNTs successfully prepared by a simple and effective coprecipitation and hydrothermal treatment method.Fe₃O₄ nanoparticles in the surface of carbon nanotubes were uniform and universal;The hysteresis loops of the decorated MWNTs were measured by vibrating sample magnetometer（VSM）,and the results showed that the composite was ferromagnetism with the saturated magnetization of 64.53 emu/g,and the coercive of 14.03 Oe.This results showed that CNTs/Fe₃O₄ composite had superparamagnetic character so that it could be easily magnetized in a applied magnetic field and could be used as a new tumor-tageted material in hyperthermia.We have successfully decorated the CNTs with chitosan by a controlled surface deposition and crosslinking process.The results of XRD and IR all expressed that the surface of CNTs were fully coverd with chitosan.This condition resulted in the external diameter of CNTs became thicker from 40-60nm to about 100nm which was very clear in TEM micrograph.We expect that the decoration can improve the biocompatibility of carbon nanotubes and the wettability of CNTs surface.3、The complex permeability and permittivity of the carbon nanotube composite were measured.Based on the theoretical calculation of reflection loss,the microwave absorption property of carbon nanotube composite has been measured in the frequency range from 0.5-5.0 GHz.The results showed that: CNTs/HAp composite had better absorbing properties in the 0.5-5.0 GHz range. With increasing of carbon nanotubes content,the microwave absorbing capacity of CNTs/HAp composite has been gradually strengthened,the absorption peak towards high frequency,the bandwidth was increased.When CNTs content was 8%,a maximum reflection loss value of-26 dB had been obtained at 3.0 GHz.The results indicated that this composite had significant potential for electromagnetic wave absorption application in tumor hyperthermia.CNTs/Fe₃O₄ composite also had better absorbing properties in the 0-5 GHz range. when CNTs content was 4%,the reflection loss value of -19dB and the bandwidth of 1G had been obtained at 2.2GHz.The results indicated that this composite had significant potential for electromagnetic wave absorption application in tumor hyperthermia.4、The thermal effect of thermo-seed material were described by heating experiment in the microwave field.The results showed that:With the increasing of CNTs content,the thermal effect was more obvious.But when CNTs content exceeded 8%,the thermal effect was gradually stabilized.CNTs played a dominant role in the thermal effect of CNTs/HAp composite powders.The thermal effects of CNTs/Fe₃O₄ composite was evident in the microwave field.When CNTs content exceeded 8%,the thermal effects of CNTs/Fe₃O₄ composite was comparative with pure CNTs.5、Carbon nanotubes composite-chitosan thermosensitive gels were prepared, which were characterized by Scanning Electronic Microscope（SEM）and Fourier Transform Infrared Spectrometer（FTIR）.In this system chitosan thermosensitive gels were used as the carrier with CNTs composite dispersed in it.IR and SEM images indicated that CNTs composite were dispersed homogeneously in the carrier,and there were no chemical reactions between CNTs composite and gels components.The initial gelation temperature rised because the network structure of gel were damaged by adding nano thermal-seed material,Meanwhile,they still had thermo-sensitive properties.The viscoelastic properties of thermo-sensitive gel for hyperthermia,as investigated rheologically,indicated that if the region was below 37℃, where G′was lower than G″,showed the common viscoelastic behavior of a liquid;in the second region,G′and G″increased with the increasing of temperature and G′was higher than G″,indicated that an elastic gel network had been formed.Nano thermal-seed materials had a good heat production capacity in the microwave field,and were distributed uniform in the gel system.Under this condition（the power, 25w;irradiated temperature,10 minutes）,when the content of CNTs was 6 mg/ml,the temperature of the system could increased by 34.6℃.when the content of CNTs/Fe₃O₄ was 20 mg/ml,the temperature of the system could increased by 34.6℃,when the content of CNTs/HAp was 20 mg/ml,the temperature could increased by 35℃.6、Cytotoxicity evaluation using L-929 cells as model system showed that the CNTs-CS thermo-sensitive gels,CNTs-Fe₃O₄-CS thermo-sensitive gels and CNTs-HAp- CS thermo-sensitive gels were nontoxic in proper composition.Option and SEM micrographs showed good morphology of L-929 cells in these hydrogels.MTT assay confirmed that there were no signicant difference between the hydrogels and control in cell avtivity,the toxicity level were 0-1 level.The results would be useful for the further research of developing these thermo-sensitive gels for tumor hyperthermia.This paper was supported by the Natural Science Foundation of the People’s Republic of China（Grant No.30540061 and 50672051）更多还原