功能化核壳型复合纳米颗粒的制备及其在生物医学研究中的应用
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摘要
复合纳米颗粒,尤其是核壳型复合纳米颗粒,克服了普通单组分纳米颗粒物质组成单一的不足,将不同物质所拥有的多种功能有机地结合在一起,显示出普通单组分纳米颗粒无可比拟的优越的理化性能,成为目前研究的热点。而且核壳型复合纳米颗粒在生化检测、医学成像、生物物质分离等生物医学领域显示出了广阔的应用前景。本论文瞄准这一重要的研究方向,在对当前迅速发展的复合纳米颗粒进行简要综述的基础上,以几种复合纳米颗粒的制备、性能表征及其在生物医学领域中的应用为主线,主要开展了以下几个方面的研究工作:
一、生物功能化硅壳复合纳米颗粒结合PCR技术用于SARS病毒基因检测。在本研究小组硅壳复合纳米颗粒技术平台的基础上,发展了一种将生物功能化硅壳磁性复合纳米颗粒(SMNPs)和硅壳荧光复合纳米颗粒(SFNPs)与PCR技术相结合来检测SARS病毒基因的新方法。我们首先利用修饰了捕获探针的硅壳磁性复合纳米颗粒对目标cDNA进行纯化和富集,然后对纯化的目标进行对称PCR扩增,接着再次利用修饰了捕获探针的硅壳磁性复合纳米颗粒将PCR扩增中产生的目标cDNA的互补链除去,最后以修饰了报告探针的硅壳荧光复合纳米颗粒通过三明治核酸杂交方式对扩增的目标cDNA进行定量检测。研究结果表明,该方法能成功地检测到目标cDNA,检测限达到2×103 copy/ mL,整个检测程序可以在6个小时内完成。该方法利用硅壳磁性复合纳米颗粒来纯化和富集DNA目标链,降低了普通PCR方法在检测SARS病毒基因时所存在的假阳性和假阴性问题;同时,这种以硅壳荧光复合纳米颗粒为检测信号采用三明治杂交方式检测PCR产物的方法同时具备荧光纳米颗粒的高灵敏度和核酸杂交技术的特异性,有效解决了电泳和同位素标记等方法带来的安全隐患问题,结果直观、特异性强、灵敏度高,是一种具有潜力的核酸检测方法。
二、Fe3O4@SiO2@Au核壳型复合纳米颗粒的制备及其在基因转染和细胞识别中的应用。
在硅壳磁性纳米颗粒的基础上,以Fe3O4@SiO2纳米颗粒为内核材料,采用自组装和化学还原法进一步对其进行包壳,构建了一种新型的Fe3O4@SiO2@Au核壳型复合纳米颗粒。对这种新型的核壳型复合纳米颗粒进行透射电镜、动态光散射、能谱表征的结果表明,该颗粒兼具磁性和金的表面特性及光谱特性,粒径为120±11 nm,并且具有较好的分散性。细胞毒性测定的研究结果也表明这种纳米颗粒具有很好的生物亲和性。在进一步探讨Fe3O4@SiO2@Au核壳型复合纳米颗粒在基因转染和细胞识别方面的应用中发现,该种纳米颗粒在多聚赖氨酸的协助下可以成功介导基因转染,并且偶联上RGD肽后还可以对乳腺癌细胞进行很好的识别。这种金包覆的核壳型复合磁性纳米颗粒可望在生物医药、细胞分离、DNA检测等领域具有较好的应用前景。
三、Fe3O4@Ag复合纳米颗粒的制备及其抑菌效果研究。
利用反相微乳液法制备了一种既具有磁性又具有抑菌效果的双功能Fe3O4@Ag复合纳米颗粒。这种纳米颗粒由具有超顺磁性的Fe3O4内核和单质银外壳组成,大小均匀,尺寸在60 nm左右,分散性好,在pH中性的溶液中Zata电势为20.5 mV。Fe3O4@Ag复合纳米颗粒的抑菌性能测定结果表明,该种纳米颗粒对大肠杆菌、葡萄球菌、枯草杆菌等革兰氏阴性菌、革兰氏阳性菌和孢子菌的代表性细菌都具有很好的抑菌功能。同时,利用其超顺磁性,可以非常容易地将发挥杀菌作用后的纳米颗粒从水溶液中回收,使处理水达到银残留标准。而且这种已经发挥过杀菌功能的回收颗粒依旧具有一定的抑菌功能,可以回收再利用。这种Fe3O4@Ag复合纳米颗粒制备方法相对简单、理化性能稳定,抑菌效果好,有望发展成为一种水的抑菌剂。
四、紫杉醇脂质体复合纳米颗粒的制备及其细胞靶向药效研究。
通过超声薄膜法制备了一种可连接靶向配体的紫杉醇脂质体复合纳米颗粒。这种载药脂质体纳米颗粒主体是由磷脂双层膜构成的大单室中空小球,不溶于水的紫杉醇药物被包裹在磷脂双分子疏水层中间,其中脂双层中参杂有PEG化的磷脂和二棕榈酰磷脂酰乙醇胺-聚乙二醇-对硝基苯碳酸酯(DPPE-PEG-pNP)。部分磷脂的PEG化使得脂质体在体内将具有长循环特性,增加了药物的肿瘤内选择性滞留(EPR)效应。DPPE-PEG-pNP在相对温和的条件下进行水解将生物配体分子修饰到纳米颗粒表面从而使脂质体具有生物靶向功能。该脂质体纳米颗粒的粒径均一,分散性和稳定性都比较好,对药物的包封率也比较高。对体外细胞的靶向识别和靶向药效试验证实连接了靶向配体的紫杉醇复合脂质体纳米颗粒能对目标细胞进行靶向识别并具有一定的靶向药效。这种靶向的紫杉醇脂质体复合纳米颗粒有望发展成为一种优良的靶向纳米药物。
Composite nanoparticles, especially core-shell composite nanoparticles, have more unique chemical and physical properties due to the combination of binary or multi-component nanocomposites, compared with conventional single component nanoparticles. In recent years, various forms of core-shell composite nanoparticles have been synthesized and studied, and their applications in the fields of biology and medicine have gained increasing attention and shown broad prospect in medical imaging, molecular diagnosis, biomaterial separation, and so on. Aiming at this important research direction, this thesis mainly focused on the synthesis and biomedical applications of several core-shell composite nanoparticles. 1. The method combinated functionalized silica composite nanoparticles with PCR for SARS associated coronavirus gene detection.
Rapid and sensitive detection of SARS associated coronavirus is critical for early diagnosis and control of severe acute respiratory syndrome. This study describes a method for the detection of SARS associated coronavirus gene by the combination of functionalized silica composite nanoparticles and PCR-based assay.
In this method, the target cDNA of SARS associated coronavirus was firstly captured and enriched from the mixture of target cDNA and non-target cDNA by the use of the functionalized superparamagnetic silica composite nanoparticles. Additionally, the enriched target cDNA was amplified through a general symmetry PCR and then was selectively isolated from the double strands PCR products by applying the superparamagnetic silica composite nanoparticles again. Finally, we detected the amplified target cDNA by employing the functionalized silica coated fluorescent dyes composite nanoparticles as signal probes with a sandwich hybridization format. The results show that the target cDNA can be assayed successfully with a detection limit of 2×103 copy/mL and the nonspecific amplification can be inhibited. In addition, the detection procedure is rapid and can be completed in less than 6 h. Our results suggest that the approache would provide promising prospects for other pathogen detection.
2. Preparation of Fe3O4@SiO2@Au core-shell composite nanoparticles and their potential applications in gene delivery and cells labeling.
Bifunctional Fe3O4@SiO2@Au composite nanoparticles with core-shell structures were prepared by self-assembling and reducing Au nanoparticles onto the surface of Fe3O4@SiO2 nanoparticles. Transmission electron microscopy (TEM), energy dispersive X-ray spectrometer (EDS), dynamic light scattering (DLS), UV-vis spectrophotometer and vibrating sample magnetometer were employed to investigate the physical and chemical properties of Fe3O4@SiO2@Au core-shell composite nanoparticles. The characterization results demonstrated that the nanoparticles with average diameter of 120±11 nm showed both the magnetic properties come from Fe3O4 nanoparticles and the optical properties due to the introduce of Au nanoparticles, and could be well dispersed in aqueous solution. Experimental data about the toxicity of Fe3O4@SiO2@Au core-shell composite nanoparticles to cells have also confirmed that the nanocomposites have good biocompatibility for cells. These novol nanoparticles were used as vehicles for gene delivery or label for cells. The results suggested that the Fe3O4@SiO2@Au core-shell composite nanoparticles could mediate gene transfection and the cells labeled by the nanoparticles could be detected by flow cytometer (FCM).
3. Preparation and antibacterial activity of Fe3O4@Ag composite nanoparticles.
Bifunctional Fe3O4@Ag composite nanoparticles with both superparamagnetic and antibacterial properties were prepared by reducing silver nitrate on the surface of Fe3O4 nanoparticles using water-in-oil microemulsion method. Formation of well-dispersed nanoparticles with size of 60±20 nm was confirmed by transmission electron microscopy and dynamic light scattering. X-ray diffraction patterns and UV-visible spectroscopy indicated that both Fe3O4 and silver were present in the same particle. The superparamagnetism of Fe3O4@Ag composite nanoparticles was confirmed by vibrating sample magnetometer. Their antibacterial activity was evaluated by means of minimum inhibitory concentration value, flow cytometry, and antibacterial rate assays. The results showed that Fe3O4@Ag composite nanoparticles presented good antibacterial performance against Escherichia coli (gram-negative bacteria), Staphylcococcus epidermidis (gram-positive bacteria) and Bacillus subtilis (spore bacteria). Furthermore, Fe3O4@Ag composite nanoparticles can be easily removed from water by using magnetic field to avoid contamination of surroundings. Reclaimed Fe3O4@Ag composite nanoparticles can still hold antibacterial capability and be reused.
4. Preparation and study on the effect of targeted drug delivery for paclitaxel-containing liposome composite nanoparticles.
Paclitaxel-containing liposome composite nanoparticles were prepared by using film-ultrasonic method. The main body of paclitaxel-containing liposome composite nanoparticles was single hollow globules that were composed of soy bean phospholipids, cholesterol and hydrophobic paclitaxel that was entrapped in the interspace of a phospholipid bilayer. Additionally, some of dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol (DPPE-PEG) and a small fraction of dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol-p-Nitrophenylcarbonyl (DPPE-PEG-pNP) were contained in phospholipid bilayer, DPPE-PEG could increase the circulation time of drug in blood and pNP groups in DPPE-PEG-pNP allowed for the fast and efficient attachment of amino group-containing ligands on the surface of nanoparticles via the formation of the ethyl urethane (carbamate) bond, in which ligands could be bound to the surface of liposome composite nanoparticles and formed immunoliposomes. This kind of paclitaxel-containing liposomes has good dispersion morphology as well as physical stability, and the entrapment efficiency of the liposomes for paclitaxel is high. Based on the above work, applications of liposomes in targeted drug delivery were further studied; the results showed that the target paclitaxel-containing immunoliposomes could selectively recognized the target cells and were significantly more effective for the growth inhibition of tumor cell compared with non-target paclitaxel-containing liposome. These results indicated that the paclitaxel-containing liposome composite nanoparticles could be developing into a kind of good nanocomposite drug carriers.
一、生物功能化硅壳复合纳米颗粒结合PCR技术用于SARS病毒基因检测。在本研究小组硅壳复合纳米颗粒技术平台的基础上,发展了一种将生物功能化硅壳磁性复合纳米颗粒(SMNPs)和硅壳荧光复合纳米颗粒(SFNPs)与PCR技术相结合来检测SARS病毒基因的新方法。我们首先利用修饰了捕获探针的硅壳磁性复合纳米颗粒对目标cDNA进行纯化和富集,然后对纯化的目标进行对称PCR扩增,接着再次利用修饰了捕获探针的硅壳磁性复合纳米颗粒将PCR扩增中产生的目标cDNA的互补链除去,最后以修饰了报告探针的硅壳荧光复合纳米颗粒通过三明治核酸杂交方式对扩增的目标cDNA进行定量检测。研究结果表明,该方法能成功地检测到目标cDNA,检测限达到2×103 copy/ mL,整个检测程序可以在6个小时内完成。该方法利用硅壳磁性复合纳米颗粒来纯化和富集DNA目标链,降低了普通PCR方法在检测SARS病毒基因时所存在的假阳性和假阴性问题;同时,这种以硅壳荧光复合纳米颗粒为检测信号采用三明治杂交方式检测PCR产物的方法同时具备荧光纳米颗粒的高灵敏度和核酸杂交技术的特异性,有效解决了电泳和同位素标记等方法带来的安全隐患问题,结果直观、特异性强、灵敏度高,是一种具有潜力的核酸检测方法。
二、Fe3O4@SiO2@Au核壳型复合纳米颗粒的制备及其在基因转染和细胞识别中的应用。
在硅壳磁性纳米颗粒的基础上,以Fe3O4@SiO2纳米颗粒为内核材料,采用自组装和化学还原法进一步对其进行包壳,构建了一种新型的Fe3O4@SiO2@Au核壳型复合纳米颗粒。对这种新型的核壳型复合纳米颗粒进行透射电镜、动态光散射、能谱表征的结果表明,该颗粒兼具磁性和金的表面特性及光谱特性,粒径为120±11 nm,并且具有较好的分散性。细胞毒性测定的研究结果也表明这种纳米颗粒具有很好的生物亲和性。在进一步探讨Fe3O4@SiO2@Au核壳型复合纳米颗粒在基因转染和细胞识别方面的应用中发现,该种纳米颗粒在多聚赖氨酸的协助下可以成功介导基因转染,并且偶联上RGD肽后还可以对乳腺癌细胞进行很好的识别。这种金包覆的核壳型复合磁性纳米颗粒可望在生物医药、细胞分离、DNA检测等领域具有较好的应用前景。
三、Fe3O4@Ag复合纳米颗粒的制备及其抑菌效果研究。
利用反相微乳液法制备了一种既具有磁性又具有抑菌效果的双功能Fe3O4@Ag复合纳米颗粒。这种纳米颗粒由具有超顺磁性的Fe3O4内核和单质银外壳组成,大小均匀,尺寸在60 nm左右,分散性好,在pH中性的溶液中Zata电势为20.5 mV。Fe3O4@Ag复合纳米颗粒的抑菌性能测定结果表明,该种纳米颗粒对大肠杆菌、葡萄球菌、枯草杆菌等革兰氏阴性菌、革兰氏阳性菌和孢子菌的代表性细菌都具有很好的抑菌功能。同时,利用其超顺磁性,可以非常容易地将发挥杀菌作用后的纳米颗粒从水溶液中回收,使处理水达到银残留标准。而且这种已经发挥过杀菌功能的回收颗粒依旧具有一定的抑菌功能,可以回收再利用。这种Fe3O4@Ag复合纳米颗粒制备方法相对简单、理化性能稳定,抑菌效果好,有望发展成为一种水的抑菌剂。
四、紫杉醇脂质体复合纳米颗粒的制备及其细胞靶向药效研究。
通过超声薄膜法制备了一种可连接靶向配体的紫杉醇脂质体复合纳米颗粒。这种载药脂质体纳米颗粒主体是由磷脂双层膜构成的大单室中空小球,不溶于水的紫杉醇药物被包裹在磷脂双分子疏水层中间,其中脂双层中参杂有PEG化的磷脂和二棕榈酰磷脂酰乙醇胺-聚乙二醇-对硝基苯碳酸酯(DPPE-PEG-pNP)。部分磷脂的PEG化使得脂质体在体内将具有长循环特性,增加了药物的肿瘤内选择性滞留(EPR)效应。DPPE-PEG-pNP在相对温和的条件下进行水解将生物配体分子修饰到纳米颗粒表面从而使脂质体具有生物靶向功能。该脂质体纳米颗粒的粒径均一,分散性和稳定性都比较好,对药物的包封率也比较高。对体外细胞的靶向识别和靶向药效试验证实连接了靶向配体的紫杉醇复合脂质体纳米颗粒能对目标细胞进行靶向识别并具有一定的靶向药效。这种靶向的紫杉醇脂质体复合纳米颗粒有望发展成为一种优良的靶向纳米药物。
Composite nanoparticles, especially core-shell composite nanoparticles, have more unique chemical and physical properties due to the combination of binary or multi-component nanocomposites, compared with conventional single component nanoparticles. In recent years, various forms of core-shell composite nanoparticles have been synthesized and studied, and their applications in the fields of biology and medicine have gained increasing attention and shown broad prospect in medical imaging, molecular diagnosis, biomaterial separation, and so on. Aiming at this important research direction, this thesis mainly focused on the synthesis and biomedical applications of several core-shell composite nanoparticles. 1. The method combinated functionalized silica composite nanoparticles with PCR for SARS associated coronavirus gene detection.
Rapid and sensitive detection of SARS associated coronavirus is critical for early diagnosis and control of severe acute respiratory syndrome. This study describes a method for the detection of SARS associated coronavirus gene by the combination of functionalized silica composite nanoparticles and PCR-based assay.
In this method, the target cDNA of SARS associated coronavirus was firstly captured and enriched from the mixture of target cDNA and non-target cDNA by the use of the functionalized superparamagnetic silica composite nanoparticles. Additionally, the enriched target cDNA was amplified through a general symmetry PCR and then was selectively isolated from the double strands PCR products by applying the superparamagnetic silica composite nanoparticles again. Finally, we detected the amplified target cDNA by employing the functionalized silica coated fluorescent dyes composite nanoparticles as signal probes with a sandwich hybridization format. The results show that the target cDNA can be assayed successfully with a detection limit of 2×103 copy/mL and the nonspecific amplification can be inhibited. In addition, the detection procedure is rapid and can be completed in less than 6 h. Our results suggest that the approache would provide promising prospects for other pathogen detection.
2. Preparation of Fe3O4@SiO2@Au core-shell composite nanoparticles and their potential applications in gene delivery and cells labeling.
Bifunctional Fe3O4@SiO2@Au composite nanoparticles with core-shell structures were prepared by self-assembling and reducing Au nanoparticles onto the surface of Fe3O4@SiO2 nanoparticles. Transmission electron microscopy (TEM), energy dispersive X-ray spectrometer (EDS), dynamic light scattering (DLS), UV-vis spectrophotometer and vibrating sample magnetometer were employed to investigate the physical and chemical properties of Fe3O4@SiO2@Au core-shell composite nanoparticles. The characterization results demonstrated that the nanoparticles with average diameter of 120±11 nm showed both the magnetic properties come from Fe3O4 nanoparticles and the optical properties due to the introduce of Au nanoparticles, and could be well dispersed in aqueous solution. Experimental data about the toxicity of Fe3O4@SiO2@Au core-shell composite nanoparticles to cells have also confirmed that the nanocomposites have good biocompatibility for cells. These novol nanoparticles were used as vehicles for gene delivery or label for cells. The results suggested that the Fe3O4@SiO2@Au core-shell composite nanoparticles could mediate gene transfection and the cells labeled by the nanoparticles could be detected by flow cytometer (FCM).
3. Preparation and antibacterial activity of Fe3O4@Ag composite nanoparticles.
Bifunctional Fe3O4@Ag composite nanoparticles with both superparamagnetic and antibacterial properties were prepared by reducing silver nitrate on the surface of Fe3O4 nanoparticles using water-in-oil microemulsion method. Formation of well-dispersed nanoparticles with size of 60±20 nm was confirmed by transmission electron microscopy and dynamic light scattering. X-ray diffraction patterns and UV-visible spectroscopy indicated that both Fe3O4 and silver were present in the same particle. The superparamagnetism of Fe3O4@Ag composite nanoparticles was confirmed by vibrating sample magnetometer. Their antibacterial activity was evaluated by means of minimum inhibitory concentration value, flow cytometry, and antibacterial rate assays. The results showed that Fe3O4@Ag composite nanoparticles presented good antibacterial performance against Escherichia coli (gram-negative bacteria), Staphylcococcus epidermidis (gram-positive bacteria) and Bacillus subtilis (spore bacteria). Furthermore, Fe3O4@Ag composite nanoparticles can be easily removed from water by using magnetic field to avoid contamination of surroundings. Reclaimed Fe3O4@Ag composite nanoparticles can still hold antibacterial capability and be reused.
4. Preparation and study on the effect of targeted drug delivery for paclitaxel-containing liposome composite nanoparticles.
Paclitaxel-containing liposome composite nanoparticles were prepared by using film-ultrasonic method. The main body of paclitaxel-containing liposome composite nanoparticles was single hollow globules that were composed of soy bean phospholipids, cholesterol and hydrophobic paclitaxel that was entrapped in the interspace of a phospholipid bilayer. Additionally, some of dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol (DPPE-PEG) and a small fraction of dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol-p-Nitrophenylcarbonyl (DPPE-PEG-pNP) were contained in phospholipid bilayer, DPPE-PEG could increase the circulation time of drug in blood and pNP groups in DPPE-PEG-pNP allowed for the fast and efficient attachment of amino group-containing ligands on the surface of nanoparticles via the formation of the ethyl urethane (carbamate) bond, in which ligands could be bound to the surface of liposome composite nanoparticles and formed immunoliposomes. This kind of paclitaxel-containing liposomes has good dispersion morphology as well as physical stability, and the entrapment efficiency of the liposomes for paclitaxel is high. Based on the above work, applications of liposomes in targeted drug delivery were further studied; the results showed that the target paclitaxel-containing immunoliposomes could selectively recognized the target cells and were significantly more effective for the growth inhibition of tumor cell compared with non-target paclitaxel-containing liposome. These results indicated that the paclitaxel-containing liposome composite nanoparticles could be developing into a kind of good nanocomposite drug carriers.
引文
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