用于生物成像的多功能纳米材料的制备与性质研究
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摘要
生物成像技术无论是在基础生物学研究领域，还是在涉及到人们日常医疗的实际应用领域都具有重要意义。但无论哪种成像技术，其发展的客观要求就是与其对应的成像材料的不断进步。一方面，成像技术在临床医疗诊断方面的应用对所需的成像材料提出了客观要求，即具有高灵敏性、高生物相容性、高的空间分辨率以及低毒性等等；另一方面，新型成像材料的不断发展又推动了既有成像技术的前进以及新型成像技术的诞生及演化。
荧光成像技术是众多生物成像技术当中的一个重要组成部分。其通常所用的成像材料包括稀土荧光材料，金属纳米簇材料以及最近发展起来的碳量子点。但是无论哪一种荧光成像材料的发展都不够完善，存在着包括生物毒性高、生物相容性不好、功能单一以及制备方法困难等诸多缺点。
为了提高上述荧光材料的生物相容性、我们在实验中引入了生物聚合物：壳聚糖。第一部分中，我们发展了一种简单的一步法制备chitosan/NaGdF_4:Eu~(3+)纳米复合物的合成方法。由于Eu~(3+)的掺杂以及纳米粒子中所带有的Gd(III)，所制备的纳米复合物具有很好的荧光性质以及顺磁性质。同时，我们研究了所制备的chitosan/NaGdF_4:Eu~(3+)纳米复合物的细胞毒性，并最终将其应用于成骨细胞的荧光成像。第二部分中，我们采用不良溶剂辅助静电络合法分别制备了具有荧光性质的壳聚糖-银纳米簇复合纳米微球以及具有荧光和磁性性质的壳聚糖-Eu~(3+),Gd~(3+)复合纳米微球。实验结果显示，复合纳米微球保留了银纳米簇以及Eu~(3+)的良好荧光性质以及Gd~(3+)的超顺磁性。实验中所得到的几种纳米复合物微球的尺寸约为100nm且尺寸分布均匀。实验发现，纳米复合物的荧光性质强烈的依赖于反应条件，包括反应温度以及反应物的添加量等等。壳聚糖的引入使得纳米复合物在水中具有很好的分散性以及生物相容性。最后，我们研究了所制备的纳米复合物的细胞毒性，并最终将其应用于成骨细胞以及人舌鳞癌细胞的荧光成像。第三部分中，我们首先应用稀土元素作为掺杂离子，采用高温注射法，成功合成了一系列铕掺杂比例不同的单分散硒化镉纳米晶。我们通过改变稀土掺杂比例及反应条件，体系发光可从黄绿光区到红光区再到蓝紫光区的转变，由此实现了对同一纳米晶合成体系在全部可见光区范围内的荧光调控。另外，我们以壳聚糖和聚乙二醇为起始原料，采用微波热解法来制备了具有荧光性质的碳量子点。荧光光谱显示所制备的样品具有很好的荧光性质，我们通过细胞毒性试验证明了所制备荧光碳量子点的生物安全性，在400μg/mL的高浓度下，细胞培养48h后，仍具有90%以上的存活率。并最终将其应用于海拉细胞的荧光成像。
Noninvasive imaging and minimally invasive in-vivo bioimaging techniquesare valuable tools in the arsenal of clinical diagnostics. Many types of bioimaging areavailable, spanning from techniques that enable whole-organism anatomical imaging(e.g., magnetic resonance imaging, MRI) to others that provide specific molecularimaging (e.g., optical fluorescence) at subcellular resolution. Such tools are expectedto be pivotal for advancing early-stage cancer diagnosis, guided stem cell therapies,drug delivery, pathogen detection, gene therapy, image-guided surgery, and cancerstaging, in addition to many other clinically relevant procedures, diagnostics, andtherapies.
No matter which bioimaging technique, its continuous development reliesgreatly on the improvement of corresponding contrast agents. On the one hand, thepractical application in clinical diagnostics requires the contrast agents to be of highsensitivity, biocompatibility, non-toxicity and can show images with high spacialresolution. On the other hand, the preparation of novel contrast agents is not onlyhelpful to the advancement of currently existed bioimaging techniques, but also theemergency and evolution of new techniques.
Currently, any single imaging technique has its own advantages anddisadvantages, whereas novel multifunctional contrast agents can joint differenttechniques together to achieve much better performance. In that case, one can obtain awealth of very useful information in single-dose rather than many tediouslyprocedures, which not only saves time but also palliate the patient's suffering. As aresult, the preparation and modification of contrast agents are of great significance.
Optical fluorescence imaging is one critical component of modern bioimagingtechniques. Contrast agents used in this technique include rare earth materials, metalclusters, carbon dots and so on. But these contrast agents have the disadvantages suchas high toxicity, low biocompatibility and functional singleness.
To improve the biocompatibility of contrast agent, chitosan (a kind ofbio-maromolecule) is introduced in the preparation system. The as-prepared chitosan-fluorescent hybrid contrast agents have low toxicity and much improvedbiocompatibility. On the other hand, chitosan can be used as a cost-effective moleculeprecursor to fabricate contrast agent such as carbon dots. In the end, magneticmaterials can be added to obtain multifunctional contrast agents.
In chapter2, we reported a novel and mild method for one-step synthesis ofchitosan/NaGdF_4:Eu~(3+)nanocomposites. The luminescent Eu~(3+)ions and magneticresonance imaging (MRI) contrast agent Gd~(3+)ions were incorporated to thesebiocompatible nanocomposites. The resultant nanocomposites exhibited strongfluorescence and attractive magnetic features. The nanocomposites also have purehexagonal phase with uniform size of about65nm. FT-IR spectra revealed that thesenanocomposites were successfully coated by hydrophilic chitosan, whose aminegroups conferred the nanocomposites excellent dispensability in aqueous solution.Besides, the MTT assay and laser confocal microscopy images have confirmed thegood biocompatibility of the nanocomposites. These results indicated that theas-prepared nanocomposites could be used as an excellent targeted imaging agent inbiological fields.
In chapter3, a facile approach to prepare Ag cluster-encapsulated chitosanhybrid nanospheres and Eu~(3+), Gd~(3+)-encapsulated chitosan hybrid nanospheres isdeveloped by utilizing ethanol-aided counterion complexation in aqueous solution.The obtained hybrid nanospheres have not only the loading space provided by thechitosan spherical matrix for loading multiply materials but also unique fluorescentproperties provided by the encapsulated Ag clusters and Eu~(3+), even more attractivemagnetic features from Gd~(3+). Besides, these hybrid nanospheres possess goodbiocompatibility and optical stability in physiological environment. It is demonstratedthat hybrid nanospheres can be internalized by MC3T3cells and Cal-27cells, andhence act as labeling agent in cell imaging by optical microscopy.
In chapter4, high quality CdSe/EuxSey hybrid nanocrystals were preparedthrough two methods. The photoluminescence of hybrid nanocrystals was tunablefrom blue to deep red by varying the different amount of rare earth in quantum dots(QDs) as well as the synthesis method. The resultant hybrid nanocrystals weremonodisperse of which sizewas controllable from2.55nmto5.86nm. Furthermore, ithas been proved that the hybrid nanocrystals were of cubic crystal structure with thehost of CdSe QDs. And the lanthanide ions were incorporated into the CdSe crystallattice successfully. Finally we have developed a general and simple microwave synthesis method to produce photoluminescent carbon dots with chitosan as acost-effective molecule precursor. Through TEM, XPS, XRD and FT-IR, wecharacterized the size、crystal structure and chemical composition of as-preparedcarbon dots. The as-prepared carbon dots present very good fluorescence property. Itis found that fluorescent emission peaks of samples shifted to longer wavelengthswith increasing excitation wavelength. Besides, the photoluminescence intensity ofthe carbon dots synthesized here is pH dependent, its intensity increases as theenvironmental pH decrease in the range of4-9, and this change is reversible. Thisphenomenon indicates the as-prepared carbon dots can be used as pH sensors. In theend the MTT assay confirmed the good biocompatibility and low toxicity of theas-prepared carbon dots.
荧光成像技术是众多生物成像技术当中的一个重要组成部分。其通常所用的成像材料包括稀土荧光材料，金属纳米簇材料以及最近发展起来的碳量子点。但是无论哪一种荧光成像材料的发展都不够完善，存在着包括生物毒性高、生物相容性不好、功能单一以及制备方法困难等诸多缺点。
为了提高上述荧光材料的生物相容性、我们在实验中引入了生物聚合物：壳聚糖。第一部分中，我们发展了一种简单的一步法制备chitosan/NaGdF_4:Eu~(3+)纳米复合物的合成方法。由于Eu~(3+)的掺杂以及纳米粒子中所带有的Gd(III)，所制备的纳米复合物具有很好的荧光性质以及顺磁性质。同时，我们研究了所制备的chitosan/NaGdF_4:Eu~(3+)纳米复合物的细胞毒性，并最终将其应用于成骨细胞的荧光成像。第二部分中，我们采用不良溶剂辅助静电络合法分别制备了具有荧光性质的壳聚糖-银纳米簇复合纳米微球以及具有荧光和磁性性质的壳聚糖-Eu~(3+),Gd~(3+)复合纳米微球。实验结果显示，复合纳米微球保留了银纳米簇以及Eu~(3+)的良好荧光性质以及Gd~(3+)的超顺磁性。实验中所得到的几种纳米复合物微球的尺寸约为100nm且尺寸分布均匀。实验发现，纳米复合物的荧光性质强烈的依赖于反应条件，包括反应温度以及反应物的添加量等等。壳聚糖的引入使得纳米复合物在水中具有很好的分散性以及生物相容性。最后，我们研究了所制备的纳米复合物的细胞毒性，并最终将其应用于成骨细胞以及人舌鳞癌细胞的荧光成像。第三部分中，我们首先应用稀土元素作为掺杂离子，采用高温注射法，成功合成了一系列铕掺杂比例不同的单分散硒化镉纳米晶。我们通过改变稀土掺杂比例及反应条件，体系发光可从黄绿光区到红光区再到蓝紫光区的转变，由此实现了对同一纳米晶合成体系在全部可见光区范围内的荧光调控。另外，我们以壳聚糖和聚乙二醇为起始原料，采用微波热解法来制备了具有荧光性质的碳量子点。荧光光谱显示所制备的样品具有很好的荧光性质，我们通过细胞毒性试验证明了所制备荧光碳量子点的生物安全性，在400μg/mL的高浓度下，细胞培养48h后，仍具有90%以上的存活率。并最终将其应用于海拉细胞的荧光成像。
Noninvasive imaging and minimally invasive in-vivo bioimaging techniquesare valuable tools in the arsenal of clinical diagnostics. Many types of bioimaging areavailable, spanning from techniques that enable whole-organism anatomical imaging(e.g., magnetic resonance imaging, MRI) to others that provide specific molecularimaging (e.g., optical fluorescence) at subcellular resolution. Such tools are expectedto be pivotal for advancing early-stage cancer diagnosis, guided stem cell therapies,drug delivery, pathogen detection, gene therapy, image-guided surgery, and cancerstaging, in addition to many other clinically relevant procedures, diagnostics, andtherapies.
No matter which bioimaging technique, its continuous development reliesgreatly on the improvement of corresponding contrast agents. On the one hand, thepractical application in clinical diagnostics requires the contrast agents to be of highsensitivity, biocompatibility, non-toxicity and can show images with high spacialresolution. On the other hand, the preparation of novel contrast agents is not onlyhelpful to the advancement of currently existed bioimaging techniques, but also theemergency and evolution of new techniques.
Currently, any single imaging technique has its own advantages anddisadvantages, whereas novel multifunctional contrast agents can joint differenttechniques together to achieve much better performance. In that case, one can obtain awealth of very useful information in single-dose rather than many tediouslyprocedures, which not only saves time but also palliate the patient's suffering. As aresult, the preparation and modification of contrast agents are of great significance.
Optical fluorescence imaging is one critical component of modern bioimagingtechniques. Contrast agents used in this technique include rare earth materials, metalclusters, carbon dots and so on. But these contrast agents have the disadvantages suchas high toxicity, low biocompatibility and functional singleness.
To improve the biocompatibility of contrast agent, chitosan (a kind ofbio-maromolecule) is introduced in the preparation system. The as-prepared chitosan-fluorescent hybrid contrast agents have low toxicity and much improvedbiocompatibility. On the other hand, chitosan can be used as a cost-effective moleculeprecursor to fabricate contrast agent such as carbon dots. In the end, magneticmaterials can be added to obtain multifunctional contrast agents.
In chapter2, we reported a novel and mild method for one-step synthesis ofchitosan/NaGdF_4:Eu~(3+)nanocomposites. The luminescent Eu~(3+)ions and magneticresonance imaging (MRI) contrast agent Gd~(3+)ions were incorporated to thesebiocompatible nanocomposites. The resultant nanocomposites exhibited strongfluorescence and attractive magnetic features. The nanocomposites also have purehexagonal phase with uniform size of about65nm. FT-IR spectra revealed that thesenanocomposites were successfully coated by hydrophilic chitosan, whose aminegroups conferred the nanocomposites excellent dispensability in aqueous solution.Besides, the MTT assay and laser confocal microscopy images have confirmed thegood biocompatibility of the nanocomposites. These results indicated that theas-prepared nanocomposites could be used as an excellent targeted imaging agent inbiological fields.
In chapter3, a facile approach to prepare Ag cluster-encapsulated chitosanhybrid nanospheres and Eu~(3+), Gd~(3+)-encapsulated chitosan hybrid nanospheres isdeveloped by utilizing ethanol-aided counterion complexation in aqueous solution.The obtained hybrid nanospheres have not only the loading space provided by thechitosan spherical matrix for loading multiply materials but also unique fluorescentproperties provided by the encapsulated Ag clusters and Eu~(3+), even more attractivemagnetic features from Gd~(3+). Besides, these hybrid nanospheres possess goodbiocompatibility and optical stability in physiological environment. It is demonstratedthat hybrid nanospheres can be internalized by MC3T3cells and Cal-27cells, andhence act as labeling agent in cell imaging by optical microscopy.
In chapter4, high quality CdSe/EuxSey hybrid nanocrystals were preparedthrough two methods. The photoluminescence of hybrid nanocrystals was tunablefrom blue to deep red by varying the different amount of rare earth in quantum dots(QDs) as well as the synthesis method. The resultant hybrid nanocrystals weremonodisperse of which sizewas controllable from2.55nmto5.86nm. Furthermore, ithas been proved that the hybrid nanocrystals were of cubic crystal structure with thehost of CdSe QDs. And the lanthanide ions were incorporated into the CdSe crystallattice successfully. Finally we have developed a general and simple microwave synthesis method to produce photoluminescent carbon dots with chitosan as acost-effective molecule precursor. Through TEM, XPS, XRD and FT-IR, wecharacterized the size、crystal structure and chemical composition of as-preparedcarbon dots. The as-prepared carbon dots present very good fluorescence property. Itis found that fluorescent emission peaks of samples shifted to longer wavelengthswith increasing excitation wavelength. Besides, the photoluminescence intensity ofthe carbon dots synthesized here is pH dependent, its intensity increases as theenvironmental pH decrease in the range of4-9, and this change is reversible. Thisphenomenon indicates the as-prepared carbon dots can be used as pH sensors. In theend the MTT assay confirmed the good biocompatibility and low toxicity of theas-prepared carbon dots.
引文
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