荧光磁性纳米微球的制备、表征及其初步应用
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摘要
本论文系统地开展了荧光磁性纳米微球的制备、表征以及初步应用等方面的工作,内容涉及亲水性Fe3O4磁流体的制备与表征；稀土配位的荧光磁性纳米微球Fe3O4@PHEMA-RE(RE=Sm, Eu, Tb, Dy)的制备与表征；合成了核中掺杂Dy的核壳结构的荧光磁性纳米微球Dy:Fe3O4@PHEMA-Tb;并就荧光磁性微球Dy:Fe3O4@PHEMA-Tb在蛋白标记和作为药物载体方面的初步应用进行了研究。具体说来,主要取得以下几方面的创新性研究成果：
首先,本实验通过改进化学共沉淀法,采用将Fe3+和Fe2+溶液按n(Fe3+)/n(Fe2+)摩尔比例为1.5：1混合,为防止Fe3O4被包覆时会有Na+被同时包覆,所以不用NaOH而用NH3·H20作为沉淀剂,亲水性很强的具有良好生物相容性的PEG-4000作为表面活性剂,形成稳定磁流体；实验的反应温度控制在35～40℃,pH=6～8等温和条件下可以得到最佳产物；经单因素预实验和三水平四因素正交实验确定了影响制备磁流体性能的主要参数,讨论了n(Fe3+)/n(Fe2+)摩尔比例,沉淀剂种类和用量,表面活性剂种类和用量,反应温度,搅拌速度,超声波分散等因素对形成磁流体影响,简化了制备过程,优化了制备条件；并对制备产物进行了表征,磁性纳米微球的磁含量为22.3%,微球的平均粒径为22.3 nrn,多分散系数为0.26,比饱和磁化强度为60.2 emu/g,无矫顽力和剩磁,产物具有超顺磁性。
其次,在亲水性Fe3O4磁流体中,以甲基丙烯酸-2-羟基乙酯(HEMA)为单体,N,N-亚甲基双丙烯酰胺(MBA)为交联剂,不加任何引发剂和乳化剂,采用光化学原位聚合合成了磁性聚甲基丙烯酸-2-羟乙酯(PHEMA)微球,并设计三水平四因素正交实验确定了Fe3O4@PHEMA的最佳工艺条件,讨论了磁流体与单体摩尔比,交联剂与单体摩尔比,光照时间,反应温度,搅拌速度等对磁性聚合物微球的影响。利用稀土离子易发光特性,将稀土离子与聚合单体进行配合作用,得到粒子的平均粒径在20～30nm之间,大小均匀,分散性好,具有超顺磁性的荧光磁性双功能纳米微球,对光化学原位聚合包覆过程中产生自由基引起链增长和稀土离子与聚合单体的配合作用可能机理进行了深入探讨。
为了提高荧光磁性微球在实际应用中磁响应性,在磁核Fe3O4铁氧体的制备过程中掺杂磁矩高于铁的Dy3+,成功制备镝铁氧体磁流体,由于镝的加入改变了磁性阳离子在晶体内部的分布状况,提高了铁氧体磁性,进而获得粒度更小、且分布更均匀,比饱和磁化强度更高、矫顽力较低的超顺磁性荧光磁性纳米微球,与没有加Dy3+相应的磁性微球,荧光磁性微球进行了对比讨论,得到了令人满意的结果,拓展了其在生物医药领域的应用空间。
同时探讨了荧光磁性微球在荧光标记和作为靶向药物载体方面的初步应用。由于在医学领域临床诊断和治疗是两个必要步骤,因此说荧光磁性微球对BSA的标记,是为临床诊断提供了理论依据,荧光磁性微球的靶向载药性,是为临床治疗提供方法参考。
利用荧光磁性微球中稀土离子具有多种荧光特性、发光效率高、荧光寿命长、Stock's位移大之特点,在温和条件下,用一定量的磁性微球Dy:Fe3O4@PHEMA,稀土离子Tb3+溶液,BSA混合合成了一种荧光磁性蛋白微球,通过红外光谱讨论了此微球表面高分子聚合物结构；荧光光谱测试发现,与Dy:Fe3O4@PHEMA-Tb相比,BSA的加入显著增强磁性微球的荧光性,荧光强度的增加量与BSA浓度在一定范围内成线性关系；在一定pH下,实现了磁性微球对BSA的荧光标记。因此,荧光磁性纳米微球Dy:Fe3O4@PHEMA-Tb作为荧光探针,是具有较高的灵敏度和选择性标记BSA的新方法,该法简单、快捷,且测定过程无毒和无放射性之优点,所以它必将成为临床BSA检测的新型探针。
利用荧光磁性微球掺杂Dy3+的铁氧体磁核具有很好磁响应性之特点,按照最佳配比将磁性微球Dy:Fe3O4@PHEMA,稀土离子Tb3+,抗肿瘤广谱药物丝裂霉素C(MMC)在一定条件下反应,制备了具有较高载药量和包封率的荧光磁性药物微球；实验发现,pH值对荧光磁性药物微球制备影响显著,荧光磁性药物微球在体外不同的pH值下释放率差别很大,并具有明显的磁响应性；荧光磁性药物微球制备方法简单,载药性好,缓释性好,可用作靶向给药载体,是胃肠道肿瘤治疗的好方法。
对制得每一个实验样品,均用光子相关光谱仪、红外光谱仪、紫外光谱仪、扫描电子显微镜、荧光光谱仪、振动样品磁强计、热失重分析仪等进行了全面表征,了解荧光磁性纳米微球的结构、组成、形态、磁学特性和荧光特性,为荧光磁性纳米微球作为蛋白标记和药物载体以及更为广泛的应用奠定了方法上的基础。
In the present paper, we systematically study the preparation, characterization and primary application of the fluorescent magnetic nano-microspheres, which involved the preparation and characterization of hydrophilic Fe3O4 magnetic fluid, the preparation and characterization of rare earth coordinated fluorescent magnetic nano-microspheres Fe3O4@PHEMA-RE(RE=Sm,Eu,Tb,Dy), the synthesis of Dy:Fe3O4@PHEMA-Tb doped with Dy core-shell structures and its primary application in protein labeling and as drug carriers. Concretely, we have achieved several innovative achievements as follows:
Firstly, we improved the chemical co-precipitation method and mixed Fe3+ with Fe2+ in proportion as 1.5:1 (n(Fe3+)/n(Fe2+)). We used NH3·H2O as precipitator rather than NaOH and PEG-4000 with high hydrophility and compatibility as surfactant to form stable magnetic fluid to avoid Na+ being coated with Fe3O4. As the reaction temperature was controlled at 35-40℃and PH at 6-8, we got the best product. Depending on the single element preliminary experiment and three levels-four elements orthogonal experiment, we decided the main parameters that affected the performance of the magnetic fluid in preparation, discussed the influences that the mole ratio of Fe3+ and Fe2+(n(Fe3+)/n(Fe2+)), the kinds and concentrations of precipitant and surfactant, reaction temperature, stirring velocity, ultrasonic dispersing had on formation of the magnetic fluid respectively. Besides, we simplified the preparation process, optimized the preparation conditions and conducted the characterization work of the product. As a result, we got magnetic nano-microspheres with a magnetism content to be 22.3%, average size 22.3 nm, polydispersity index 0.26, specific saturation magnetization 60.2 emu/g and without coercive force and remanent magnetism. The product had superparamagnetism.
Secondly, we synthesized the magnetic PHEME microspheres using the photochemical in situ polymerization method. In the experiment, we used HEMA as monomer, MBA as crosslinking agent and carried it out without any initiator and emulsifier in the hydrophilic Fe3O4 magnetic fluid. Meanwhile, we devised a three levels-four elements orthogonal experiment to decide the best process conditions for Fe3O4@PHEMA and discussed the influences of the mole ratio of magnetic fluid and monomer, the mole ratio of crosslinking agent and monomer, illumination time, reaction temperature and stirring velocity had on the magnetic microspheres respectively. Based on the luminescence-prone property of rare earth ion, after its coordination reaction with monomer we got the dual functional superparamagnetic fluorescent magnetic nano-microspheres with an average size between 20nm and 30nm and a high dispersivity. The possible mechanisms for the chain increase caused by the generation of free radicals and the coordination reaction between rare earth ions and monomers were deeply studied.
To improve the magnetic responsibility of this fluorescent magnetic microspheres in application, we doped Dy3+ whose magnetic moment is higher than iron and then prepared the dysprosium-ferrite magnetic fluid successfully. The addition of Dy changed the distribution of the positive charged magnetic ion, enhanced the magnetism of the ferrite and thus guaranteed the acquisition of the superparamagnetic fluorescent magnetic nano-microspheres with smaller range of size, more even distribution, higher specific saturation magnetization and lower coercive force. Through the comparison and discussion about the fluorescent magnetic microspheres with and without Dy3+, we got satisfying results and thus expanded its application space in biological and medical areas.
At the meantime, we discussed the primary application of the fluorescent magnetic microspheres in fluorescence labeling and as targeted drug carriers. As clinical diagnosis and treatment are two indispensable steps in medical field, labeling BSA with fluorescent magnetic microspheres offered a theoretical basis for clinical diagnosis and the ability to be targeted drugs carriers that the fluorescent magnetic microspheres had provided a reference in method for clinical treatment.
Rare earth ions in the fluorescent magnetic microspheres have some characteristics like containing several fluorescent properties, a high luminescence efficiency, a long life span of fluorescence and a long Stock's displacement. These characteristics were used to mix rare earth Tb3+ with a certain dose of magnetic microspheres Dy:Fe3O4@PHEMA and BSA to synthesize a kind of fluorescent magnetic protein microspheres. Depending on the Infrared Spectrum, we discussed the structure of macromolecular polymer on the surface of the microspheres. And the Fluorescence Spectrum indicated that compared with Dy:Fe3O4@PHEMA, the addition of BSA increased the magnetic microspheres'fluorescence significantly and the increase of fluorescence intensity was in linear relationship with the concentration of BSA in a certain range. Besides, we successfully conducted the fluorescent labeling to the magnetic microspheres under a certain PH. Therefore, it is a new way which has a high sensitivity and more alternative in labeling BSA that the fluorescent magnetic nano-microspheres Dy:Fe3O4@PHEMA-Tb is used as fluorescent probe. This simple and quick way with advantages of no toxicity and no radioactivity is sure to be a novel probe used in clinical BSA examination.
Because the fluorescent magnetic microspheres doped with Dy3+ had magnetic ferrite core with high magnetic responsibility, we prepared fluorescent magnetic microspheres with drugs which had a high drug loading capacity and embedding ratio when Dy:Fe3O4@PHEMA, Tb3+, and anti-tumor broad-spectrum medicine MMC reacted under a certain condition. The result show that PH has a great effect on the preparation of the fluorescent magnetic microspheres with drugs, as in vitro, they would have a great difference in release ratio under different PH and has a very remarkable magnetic responsibility. To sum up, the way we used here is simple, having a satisfying drug loading capacity as well as relaxation ability and therefore can be used as drug carrier. It is a good way in gastroenteric tumor treatment.
Every sample we prepared was overall characterized using photon correlation spectroscopy, infrared spectroscopy, ultraviolet spectroscopy, scanning electron microscope, fluorescence spectroscopy, vibration sample magnetometer, thermogravimetry analysis and so on. It is a must as well that understanding the fluorescent magnetic nano-microspheres' structure, composition, morphology, magnetic properties and fluorescent properties. So it will lay a foundation in method for the fluorescent nano-microspheres functioning as protein labels and drug carriers.
首先,本实验通过改进化学共沉淀法,采用将Fe3+和Fe2+溶液按n(Fe3+)/n(Fe2+)摩尔比例为1.5：1混合,为防止Fe3O4被包覆时会有Na+被同时包覆,所以不用NaOH而用NH3·H20作为沉淀剂,亲水性很强的具有良好生物相容性的PEG-4000作为表面活性剂,形成稳定磁流体；实验的反应温度控制在35～40℃,pH=6～8等温和条件下可以得到最佳产物；经单因素预实验和三水平四因素正交实验确定了影响制备磁流体性能的主要参数,讨论了n(Fe3+)/n(Fe2+)摩尔比例,沉淀剂种类和用量,表面活性剂种类和用量,反应温度,搅拌速度,超声波分散等因素对形成磁流体影响,简化了制备过程,优化了制备条件；并对制备产物进行了表征,磁性纳米微球的磁含量为22.3%,微球的平均粒径为22.3 nrn,多分散系数为0.26,比饱和磁化强度为60.2 emu/g,无矫顽力和剩磁,产物具有超顺磁性。
其次,在亲水性Fe3O4磁流体中,以甲基丙烯酸-2-羟基乙酯(HEMA)为单体,N,N-亚甲基双丙烯酰胺(MBA)为交联剂,不加任何引发剂和乳化剂,采用光化学原位聚合合成了磁性聚甲基丙烯酸-2-羟乙酯(PHEMA)微球,并设计三水平四因素正交实验确定了Fe3O4@PHEMA的最佳工艺条件,讨论了磁流体与单体摩尔比,交联剂与单体摩尔比,光照时间,反应温度,搅拌速度等对磁性聚合物微球的影响。利用稀土离子易发光特性,将稀土离子与聚合单体进行配合作用,得到粒子的平均粒径在20～30nm之间,大小均匀,分散性好,具有超顺磁性的荧光磁性双功能纳米微球,对光化学原位聚合包覆过程中产生自由基引起链增长和稀土离子与聚合单体的配合作用可能机理进行了深入探讨。
为了提高荧光磁性微球在实际应用中磁响应性,在磁核Fe3O4铁氧体的制备过程中掺杂磁矩高于铁的Dy3+,成功制备镝铁氧体磁流体,由于镝的加入改变了磁性阳离子在晶体内部的分布状况,提高了铁氧体磁性,进而获得粒度更小、且分布更均匀,比饱和磁化强度更高、矫顽力较低的超顺磁性荧光磁性纳米微球,与没有加Dy3+相应的磁性微球,荧光磁性微球进行了对比讨论,得到了令人满意的结果,拓展了其在生物医药领域的应用空间。
同时探讨了荧光磁性微球在荧光标记和作为靶向药物载体方面的初步应用。由于在医学领域临床诊断和治疗是两个必要步骤,因此说荧光磁性微球对BSA的标记,是为临床诊断提供了理论依据,荧光磁性微球的靶向载药性,是为临床治疗提供方法参考。
利用荧光磁性微球中稀土离子具有多种荧光特性、发光效率高、荧光寿命长、Stock's位移大之特点,在温和条件下,用一定量的磁性微球Dy:Fe3O4@PHEMA,稀土离子Tb3+溶液,BSA混合合成了一种荧光磁性蛋白微球,通过红外光谱讨论了此微球表面高分子聚合物结构；荧光光谱测试发现,与Dy:Fe3O4@PHEMA-Tb相比,BSA的加入显著增强磁性微球的荧光性,荧光强度的增加量与BSA浓度在一定范围内成线性关系；在一定pH下,实现了磁性微球对BSA的荧光标记。因此,荧光磁性纳米微球Dy:Fe3O4@PHEMA-Tb作为荧光探针,是具有较高的灵敏度和选择性标记BSA的新方法,该法简单、快捷,且测定过程无毒和无放射性之优点,所以它必将成为临床BSA检测的新型探针。
利用荧光磁性微球掺杂Dy3+的铁氧体磁核具有很好磁响应性之特点,按照最佳配比将磁性微球Dy:Fe3O4@PHEMA,稀土离子Tb3+,抗肿瘤广谱药物丝裂霉素C(MMC)在一定条件下反应,制备了具有较高载药量和包封率的荧光磁性药物微球；实验发现,pH值对荧光磁性药物微球制备影响显著,荧光磁性药物微球在体外不同的pH值下释放率差别很大,并具有明显的磁响应性；荧光磁性药物微球制备方法简单,载药性好,缓释性好,可用作靶向给药载体,是胃肠道肿瘤治疗的好方法。
对制得每一个实验样品,均用光子相关光谱仪、红外光谱仪、紫外光谱仪、扫描电子显微镜、荧光光谱仪、振动样品磁强计、热失重分析仪等进行了全面表征,了解荧光磁性纳米微球的结构、组成、形态、磁学特性和荧光特性,为荧光磁性纳米微球作为蛋白标记和药物载体以及更为广泛的应用奠定了方法上的基础。
In the present paper, we systematically study the preparation, characterization and primary application of the fluorescent magnetic nano-microspheres, which involved the preparation and characterization of hydrophilic Fe3O4 magnetic fluid, the preparation and characterization of rare earth coordinated fluorescent magnetic nano-microspheres Fe3O4@PHEMA-RE(RE=Sm,Eu,Tb,Dy), the synthesis of Dy:Fe3O4@PHEMA-Tb doped with Dy core-shell structures and its primary application in protein labeling and as drug carriers. Concretely, we have achieved several innovative achievements as follows:
Firstly, we improved the chemical co-precipitation method and mixed Fe3+ with Fe2+ in proportion as 1.5:1 (n(Fe3+)/n(Fe2+)). We used NH3·H2O as precipitator rather than NaOH and PEG-4000 with high hydrophility and compatibility as surfactant to form stable magnetic fluid to avoid Na+ being coated with Fe3O4. As the reaction temperature was controlled at 35-40℃and PH at 6-8, we got the best product. Depending on the single element preliminary experiment and three levels-four elements orthogonal experiment, we decided the main parameters that affected the performance of the magnetic fluid in preparation, discussed the influences that the mole ratio of Fe3+ and Fe2+(n(Fe3+)/n(Fe2+)), the kinds and concentrations of precipitant and surfactant, reaction temperature, stirring velocity, ultrasonic dispersing had on formation of the magnetic fluid respectively. Besides, we simplified the preparation process, optimized the preparation conditions and conducted the characterization work of the product. As a result, we got magnetic nano-microspheres with a magnetism content to be 22.3%, average size 22.3 nm, polydispersity index 0.26, specific saturation magnetization 60.2 emu/g and without coercive force and remanent magnetism. The product had superparamagnetism.
Secondly, we synthesized the magnetic PHEME microspheres using the photochemical in situ polymerization method. In the experiment, we used HEMA as monomer, MBA as crosslinking agent and carried it out without any initiator and emulsifier in the hydrophilic Fe3O4 magnetic fluid. Meanwhile, we devised a three levels-four elements orthogonal experiment to decide the best process conditions for Fe3O4@PHEMA and discussed the influences of the mole ratio of magnetic fluid and monomer, the mole ratio of crosslinking agent and monomer, illumination time, reaction temperature and stirring velocity had on the magnetic microspheres respectively. Based on the luminescence-prone property of rare earth ion, after its coordination reaction with monomer we got the dual functional superparamagnetic fluorescent magnetic nano-microspheres with an average size between 20nm and 30nm and a high dispersivity. The possible mechanisms for the chain increase caused by the generation of free radicals and the coordination reaction between rare earth ions and monomers were deeply studied.
To improve the magnetic responsibility of this fluorescent magnetic microspheres in application, we doped Dy3+ whose magnetic moment is higher than iron and then prepared the dysprosium-ferrite magnetic fluid successfully. The addition of Dy changed the distribution of the positive charged magnetic ion, enhanced the magnetism of the ferrite and thus guaranteed the acquisition of the superparamagnetic fluorescent magnetic nano-microspheres with smaller range of size, more even distribution, higher specific saturation magnetization and lower coercive force. Through the comparison and discussion about the fluorescent magnetic microspheres with and without Dy3+, we got satisfying results and thus expanded its application space in biological and medical areas.
At the meantime, we discussed the primary application of the fluorescent magnetic microspheres in fluorescence labeling and as targeted drug carriers. As clinical diagnosis and treatment are two indispensable steps in medical field, labeling BSA with fluorescent magnetic microspheres offered a theoretical basis for clinical diagnosis and the ability to be targeted drugs carriers that the fluorescent magnetic microspheres had provided a reference in method for clinical treatment.
Rare earth ions in the fluorescent magnetic microspheres have some characteristics like containing several fluorescent properties, a high luminescence efficiency, a long life span of fluorescence and a long Stock's displacement. These characteristics were used to mix rare earth Tb3+ with a certain dose of magnetic microspheres Dy:Fe3O4@PHEMA and BSA to synthesize a kind of fluorescent magnetic protein microspheres. Depending on the Infrared Spectrum, we discussed the structure of macromolecular polymer on the surface of the microspheres. And the Fluorescence Spectrum indicated that compared with Dy:Fe3O4@PHEMA, the addition of BSA increased the magnetic microspheres'fluorescence significantly and the increase of fluorescence intensity was in linear relationship with the concentration of BSA in a certain range. Besides, we successfully conducted the fluorescent labeling to the magnetic microspheres under a certain PH. Therefore, it is a new way which has a high sensitivity and more alternative in labeling BSA that the fluorescent magnetic nano-microspheres Dy:Fe3O4@PHEMA-Tb is used as fluorescent probe. This simple and quick way with advantages of no toxicity and no radioactivity is sure to be a novel probe used in clinical BSA examination.
Because the fluorescent magnetic microspheres doped with Dy3+ had magnetic ferrite core with high magnetic responsibility, we prepared fluorescent magnetic microspheres with drugs which had a high drug loading capacity and embedding ratio when Dy:Fe3O4@PHEMA, Tb3+, and anti-tumor broad-spectrum medicine MMC reacted under a certain condition. The result show that PH has a great effect on the preparation of the fluorescent magnetic microspheres with drugs, as in vitro, they would have a great difference in release ratio under different PH and has a very remarkable magnetic responsibility. To sum up, the way we used here is simple, having a satisfying drug loading capacity as well as relaxation ability and therefore can be used as drug carrier. It is a good way in gastroenteric tumor treatment.
Every sample we prepared was overall characterized using photon correlation spectroscopy, infrared spectroscopy, ultraviolet spectroscopy, scanning electron microscope, fluorescence spectroscopy, vibration sample magnetometer, thermogravimetry analysis and so on. It is a must as well that understanding the fluorescent magnetic nano-microspheres' structure, composition, morphology, magnetic properties and fluorescent properties. So it will lay a foundation in method for the fluorescent nano-microspheres functioning as protein labels and drug carriers.
引文
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