基于生物功能化纳米颗粒的药物载体、乳腺癌成像及生物分离研究
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摘要
纳米生物技术是一门新兴的交叉学科,它的发展为生物医学研究应用向纵深发展开辟了崭新的途径并提供了重要的物质基础—生物纳米材料,特别是具有特定物理化学特性及生物活性的生物功能化纳米颗粒在生物活性物质检测、生物分离、基因与药物的靶向输送、生物医学成像等领域显示出巨大的应用价值。近年来,本研究小组结合纳米技术、分析技术、生物技术以及材料制备技术,开展了以生物功能化二氧化硅纳米颗粒及生物功能化壳聚糖纳米颗粒为核心的系统研究工作。本论文在相关工作的基础上,继续瞄准这一前沿方向,拓展了生物功能化二氧化硅纳米颗粒在乳腺癌靶向成像及生物分离中的应用,并开展了生物功能化壳聚糖纳米颗粒在药物传输中的应用研究工作,主要研究工作如下:
1.基于生物功能化二氧化硅荧光纳米颗粒的乳腺癌细胞及其组织靶向成像研究
利用本研究小组的二氧化硅荧光纳米颗粒技术平台,发展了一种新型的多肽荧光纳米标记方法。通过溴化氰(CNBr)交联法在联钌吡啶(RuBpy)二氧化硅荧光纳米颗粒表面修饰含有精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp)的RGD多肽,制备RGD生物功能化二氧化硅荧光纳米颗粒(RGD-FSiNPs),并开展其在细胞水平及组织水平上的靶向识别及成像研究。结果表明,RGD-FSiNPs对α_vβ_3整合素阳性表达的人乳腺癌细胞系(MDA-MB-231)的固定细胞及活细胞均能实现高特异性识别;通过将聚乙二醇(PEG)化的RGD-FSiNPs导入荷瘤裸鼠体内,可有效地减弱肝、脾对纳米颗粒的吞噬作用,其对裸鼠体内的乳腺癌组织表现出高特异性靶向特性,在组织水平上成功实现其对乳腺癌组织的靶向示踪及荧光成像。这种RGD多肽荧光纳米标记方法在肿瘤细胞靶向成像以及癌细胞微转移等研究领域具有重要的应用价值。
2.基于生物功能化二氧化硅磁纳米颗粒的蛋白质靶向分离方法研究
利用本研究小组的二氧化硅磁纳米颗粒(MSiNPs)技术平台,发展了一种基于生物功能化MSiNPs的蛋白质靶向分离方法。采用CNBr交联法在MSiNPs表面固定人IgG,制备人IgG生物功能化二氧化硅磁纳米颗粒(IgG-MSiNPs)。以羊抗人IgG-FITC为模型目标蛋白,利用抗原-抗体特异性作用原理和MSiNPs的超顺磁性,对目标蛋白进行特异性富集及磁分离;选择合适的解离剂对IgG-MSiNPs所捕获的目标蛋白进行解离。通过对分离条件及解离条件的进一步优化获得了较好的分离效果,捕获效率高达86.92%,选择性好;并且成功实现目标蛋白从生物功能化二氧化硅磁纳米颗粒上的解离,获得纯化的目标蛋白。该方法简便、快速,可望推广到直接从原始样品中靶向分离蛋白质等生物活性物质的研究领域中。
3.生物功能化荧光磁性纳米颗粒的制备及其对乳腺癌细胞的分离研究
基于化学沉降法和油包水反相微乳液制备技术,发展了一种简便、温和的二氧化硅荧光磁性纳米颗粒(FMSiNPs)制备方法。通过优化制备条件,得到由Fe_3O_4与人IgG标记的FITC混合内核及二氧化硅外壳构成的具有超顺磁性和荧光特性的FMSiNPs。采用CNBr交联法在FMSiNPs纳米颗粒表面固定RGD多肽,制备生物功能化二氧化硅荧光磁性纳米颗粒(RGD-FMSiNPs)。基于RGD多肽与MDA-MB-231乳腺癌细胞表面α_vβ_3整合素分子的亲和作用对MDA-MB-231细胞进行特异性识别及富集,利用其超顺磁性分离出靶细胞并结合流式细胞分析技术对靶细胞上所结合的RGD-FMSiNPs进行荧光检测。结果显示,RGD-FMSiNPs对乳腺癌细胞呈现良好的靶向效应和较高的分离效率。本法制备的生物功能化荧光磁性纳米颗粒有望作为一种有应用前景的免疫磁性纳米分离介质用于肿瘤细胞的磁性分离与检测。
4.基于生物功能化壳聚糖纳米颗粒的蛋白类药物载体研究
以天然、生物亲和性好的壳聚糖(CS)为载体,以牛血清白蛋白(BSA)为药物模型,基于CS与多聚磷酸纳(TPP)间静电作用的离子凝胶化方法,发展了一种基于生物功能化CS纳米颗粒的蛋白类药物载体的制备方法。本法实验条件温和,在最优化条件下可制备得到平均粒径小于100 nm,分散均匀、包封率大于50%的包载BSA的生物功能化CS纳米颗粒。在此基础之上,将该药物载体初步应用于丙种球蛋白的包载,包封率达到55.0%,并具有显著的缓释作用。从而为基于生物功能化壳聚糖纳米颗粒的蛋白类药物载体的开发提供了应用基础。
5.基于生物功能化壳聚糖纳米颗粒的抗肿瘤药物载体研究
以壳聚糖(CS)为载体,基于共价交联和离子凝胶化方法,制备了一种新型的甲氨蝶呤(MTX)缓释载体。首先通过戊二醛的醛基分别与CS和MTX的氨基发生缩合反应,形成稳定的化学键将MTX固定在CS分子上,再借助带负电荷的多聚磷酸纳(TPP)与带正电荷的CS之间的静电作用形成包载MTX的生物功能化CS纳米颗粒(CS-MTX-TPP)。本法制备条件温和,简便易行,在最佳药载比条件下,可获得平均粒径小于200 nm的CS-MTX-TPP纳米颗粒,对MTX的包封率达到53.0%。体外释放实验汪实CS与MTX之间稳定的共价键有利于保持CS-MTX-TPP纳米颗粒的缓释特征。此外,体外抗肿瘤活性研究表明MTX被CS纳米颗粒包载后,依然保持了较好的抑瘤活性。本法可望拓宽生物功能化CS纳米颗粒在抗肿瘤药物传输领域中的应用。
Bionanotechnology is a new interdiscipline,which has provided new opportunities and scientific methods for the deep extensive development of biomedicine research.As the novel products of bionanotechnology,bionanomaterials, especially biofunctionalized nanoparticles with specific physical,chemical or bioactive properties,have shown great potential in the researches of cancer diagnosis and treatment,cells and proteins separation,gene therapy and controlled drug delivery.However,many of these researches are still in their infants.Further studies need to be extended.Recently,our research group has developed systemic researches based on the biofunctionalized silica nanoparticles and chitosan nanoparticles with the combination of nanotechnology,analytical chemistry,biotechnology and material science.On the basis of our correlative researches,this dissertation has focused on the target imaging of breast cancer and bioseparation with biofunctionalized silica nanoparticles,and the further studies of the drug delivery with biofunctionalized chitosan nanoparticles.
1.Imaging breast cancer cells and tissues using biofunctionalized fluorescent silica nanoparticles.
A method using RuBpy doped silica nanoparticles(FSiNPs) as novel fluorescent labels of peptide has been developed for in vitro and ex vivo imaging of breast cancer cells and tissues.The biofunctionalized FSiNPs(RGD-FSiNPs) were prepared by covalent immobilization of arginine-glycine-aspartic acid(RGD) peptide onto FSiNPs according to the cyanogens bromide(CNBr) method.In vitro cell imaging showed that RGD-FSiNPs exhibited high target binding to fixed and living cells ofα_vβ_3 integrin receptor(ABIR)-positive MDA-MB-231 breast cancers.Further study regarding the ex vivo imaging of tumor tissue samples was also carried out by intravenously injecting PEGylated-RGD-FSiNPs into athymic nude mice bearing the MDA-MB-231 tumors.Tissue images demonstrated that it could reduce the phagocytosis of nanoparticles by liver and spleen and the high integrinα_vβ_3 expression level of the MDA-MB-231 tumors was clearly visible due to the special targeting effects of the RGD-FSiNPs.This RGD peptide-oriented fluorescent labeling technique based on the biofunctionalized FSiNPs will be promising for tumor cell imaging and cancer metastasis,etc.
2.Targeted separation of protein based on biofunctionalized magnetic silica nanoparticles.
A method of silica coated magnetic nanoparticles(MSiNPs)-based targeted separation of proteins has been developed based on the technology platform of our research group.The biofunctionalized MSiNPs(IgG-MSiNPs) were synthesized by modification of human IgG onto the surface of MSiNPs with CNBr method.Target protein(goat-anti-human IgG-FITC) was specifically recognized with IgG-MSiNPs and isolated under magnetic field.Then the target protein could be dissociated from the nanoparticles with suitable dissociation agent.The effects of separation and dissociation conditions on the separation efficiency,selectivity and dissociation effiency were studied.At the optimal conditions,the relatively high separation and dissociation efficiency and good selectivity were obtained.This study will further explore the application of functionalized magnetic silica nanoparticles in direct bio-separation of proteins from complex samples.
3.Synthesis of biofunctionalized fluorescent magnetic nanoparticles and their potential applications in separation of breast cancer cells.
We have developed a simple method to synthesize FITC dye doped silica fluorescent magnetic nanoparticles(FMSiNPs) with chemical precipitation method and reversed microemulsion technique under mild condition.FMSiNPs were formed by employing human IgG modified FITC dye and magnetic fluid as core and the silica as shell.The biofunctionalized FMSiNPs(RGD-FMSiNPs) were prepared by immobilizing of RGD peptide onto the surface of FMSiNPs.The specific enrichment ofα_vβ_3 integrin receptor-positive MDA-MB-231 breast cancer cells using RGD-FMSiNPs were carried out due to the specific recognition of RGD peptide andα_vβ_3 integrin.Then the target cells were isolated with RGD-FMSiNPs under magnetic field and detected by FCM based on the fluorescence properties of RGD-FMSiNPs. Results indicated that RGD-FMSiNPs exhibited high target binding to MDA-MB-231 breast cancer cells and high separation efficiency.This new kind of RGD based biofunctionalized FMSiNPs could be widely used for the magnetic separation and fluorescent detection of tumor cells.
4.Research on protein drug carriers based on biofunctionalized chitosan nanoparticles.
A new type of biocompatible chitosan(CS) nanoparticles encapsulated with bovine serum albumin(BSA)(CS-BSA-TPP NPs) was prepared through ionic cross-linking technique based on CS and sodium tripolyphosphate(TPP) at room temperature.The influence of formulation conditions on the properties of CS-BSA-TPP NPs was investigated.Results showed that CS-BSA-TPP NPs with diameter of below 100 nm and good dispersion were prepared at the optimal conditions,and the BSA encapsulation efficiency was more than 50%.This system was also applied in the preparation of CS nanoparticles encapsulated with Gamma Seroglobulin(CS-IG-TPP NPs),the CS-IG-TPP NPs presented the encapsulation efficiency of IG with 55.0%and displayed sustained-release property.Thus,this protein delivery system based on biofunctionalized CS nanoparticles will be promising for protein drug delivery.
5.A novel anticancer drug delivery system based on biofunctionalized chitosan nanoparticles.
A chitosan-methotrexate covalently conjugated nanoparticles(CS-MTX-TPP NPs) has been developed as a potential delivery system for methotrexate(MTX). MTX was first conjugated to CS by using glutaraldehyde as cross-linked agent,and followed by the process of ionic gelation between MTX-conjugated CS and sodium tripolyphosphate(TPP) to form CS-MTX-TPP NPs at mild reaction conditions.At the optimal conditions,CS-MTX-TPP NPs with diameter of sub-200-nm and encapsulation efficiency of 53.0%were obtained.Additionally,in vitro release test revealed that the stable covalent bonding of CS and MTX was beneficial for providing slow release for MTX.Especially,cellular toxicity study in MCF-7 cancer cells further demonstrated the effective anticancer efficacy of this new type of delivery for MTX.In the future,this work could be expected to broaden the applications of biofunctionalized CS NPs in anticancer drug delivery.
1.基于生物功能化二氧化硅荧光纳米颗粒的乳腺癌细胞及其组织靶向成像研究
利用本研究小组的二氧化硅荧光纳米颗粒技术平台,发展了一种新型的多肽荧光纳米标记方法。通过溴化氰(CNBr)交联法在联钌吡啶(RuBpy)二氧化硅荧光纳米颗粒表面修饰含有精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp)的RGD多肽,制备RGD生物功能化二氧化硅荧光纳米颗粒(RGD-FSiNPs),并开展其在细胞水平及组织水平上的靶向识别及成像研究。结果表明,RGD-FSiNPs对α_vβ_3整合素阳性表达的人乳腺癌细胞系(MDA-MB-231)的固定细胞及活细胞均能实现高特异性识别;通过将聚乙二醇(PEG)化的RGD-FSiNPs导入荷瘤裸鼠体内,可有效地减弱肝、脾对纳米颗粒的吞噬作用,其对裸鼠体内的乳腺癌组织表现出高特异性靶向特性,在组织水平上成功实现其对乳腺癌组织的靶向示踪及荧光成像。这种RGD多肽荧光纳米标记方法在肿瘤细胞靶向成像以及癌细胞微转移等研究领域具有重要的应用价值。
2.基于生物功能化二氧化硅磁纳米颗粒的蛋白质靶向分离方法研究
利用本研究小组的二氧化硅磁纳米颗粒(MSiNPs)技术平台,发展了一种基于生物功能化MSiNPs的蛋白质靶向分离方法。采用CNBr交联法在MSiNPs表面固定人IgG,制备人IgG生物功能化二氧化硅磁纳米颗粒(IgG-MSiNPs)。以羊抗人IgG-FITC为模型目标蛋白,利用抗原-抗体特异性作用原理和MSiNPs的超顺磁性,对目标蛋白进行特异性富集及磁分离;选择合适的解离剂对IgG-MSiNPs所捕获的目标蛋白进行解离。通过对分离条件及解离条件的进一步优化获得了较好的分离效果,捕获效率高达86.92%,选择性好;并且成功实现目标蛋白从生物功能化二氧化硅磁纳米颗粒上的解离,获得纯化的目标蛋白。该方法简便、快速,可望推广到直接从原始样品中靶向分离蛋白质等生物活性物质的研究领域中。
3.生物功能化荧光磁性纳米颗粒的制备及其对乳腺癌细胞的分离研究
基于化学沉降法和油包水反相微乳液制备技术,发展了一种简便、温和的二氧化硅荧光磁性纳米颗粒(FMSiNPs)制备方法。通过优化制备条件,得到由Fe_3O_4与人IgG标记的FITC混合内核及二氧化硅外壳构成的具有超顺磁性和荧光特性的FMSiNPs。采用CNBr交联法在FMSiNPs纳米颗粒表面固定RGD多肽,制备生物功能化二氧化硅荧光磁性纳米颗粒(RGD-FMSiNPs)。基于RGD多肽与MDA-MB-231乳腺癌细胞表面α_vβ_3整合素分子的亲和作用对MDA-MB-231细胞进行特异性识别及富集,利用其超顺磁性分离出靶细胞并结合流式细胞分析技术对靶细胞上所结合的RGD-FMSiNPs进行荧光检测。结果显示,RGD-FMSiNPs对乳腺癌细胞呈现良好的靶向效应和较高的分离效率。本法制备的生物功能化荧光磁性纳米颗粒有望作为一种有应用前景的免疫磁性纳米分离介质用于肿瘤细胞的磁性分离与检测。
4.基于生物功能化壳聚糖纳米颗粒的蛋白类药物载体研究
以天然、生物亲和性好的壳聚糖(CS)为载体,以牛血清白蛋白(BSA)为药物模型,基于CS与多聚磷酸纳(TPP)间静电作用的离子凝胶化方法,发展了一种基于生物功能化CS纳米颗粒的蛋白类药物载体的制备方法。本法实验条件温和,在最优化条件下可制备得到平均粒径小于100 nm,分散均匀、包封率大于50%的包载BSA的生物功能化CS纳米颗粒。在此基础之上,将该药物载体初步应用于丙种球蛋白的包载,包封率达到55.0%,并具有显著的缓释作用。从而为基于生物功能化壳聚糖纳米颗粒的蛋白类药物载体的开发提供了应用基础。
5.基于生物功能化壳聚糖纳米颗粒的抗肿瘤药物载体研究
以壳聚糖(CS)为载体,基于共价交联和离子凝胶化方法,制备了一种新型的甲氨蝶呤(MTX)缓释载体。首先通过戊二醛的醛基分别与CS和MTX的氨基发生缩合反应,形成稳定的化学键将MTX固定在CS分子上,再借助带负电荷的多聚磷酸纳(TPP)与带正电荷的CS之间的静电作用形成包载MTX的生物功能化CS纳米颗粒(CS-MTX-TPP)。本法制备条件温和,简便易行,在最佳药载比条件下,可获得平均粒径小于200 nm的CS-MTX-TPP纳米颗粒,对MTX的包封率达到53.0%。体外释放实验汪实CS与MTX之间稳定的共价键有利于保持CS-MTX-TPP纳米颗粒的缓释特征。此外,体外抗肿瘤活性研究表明MTX被CS纳米颗粒包载后,依然保持了较好的抑瘤活性。本法可望拓宽生物功能化CS纳米颗粒在抗肿瘤药物传输领域中的应用。
Bionanotechnology is a new interdiscipline,which has provided new opportunities and scientific methods for the deep extensive development of biomedicine research.As the novel products of bionanotechnology,bionanomaterials, especially biofunctionalized nanoparticles with specific physical,chemical or bioactive properties,have shown great potential in the researches of cancer diagnosis and treatment,cells and proteins separation,gene therapy and controlled drug delivery.However,many of these researches are still in their infants.Further studies need to be extended.Recently,our research group has developed systemic researches based on the biofunctionalized silica nanoparticles and chitosan nanoparticles with the combination of nanotechnology,analytical chemistry,biotechnology and material science.On the basis of our correlative researches,this dissertation has focused on the target imaging of breast cancer and bioseparation with biofunctionalized silica nanoparticles,and the further studies of the drug delivery with biofunctionalized chitosan nanoparticles.
1.Imaging breast cancer cells and tissues using biofunctionalized fluorescent silica nanoparticles.
A method using RuBpy doped silica nanoparticles(FSiNPs) as novel fluorescent labels of peptide has been developed for in vitro and ex vivo imaging of breast cancer cells and tissues.The biofunctionalized FSiNPs(RGD-FSiNPs) were prepared by covalent immobilization of arginine-glycine-aspartic acid(RGD) peptide onto FSiNPs according to the cyanogens bromide(CNBr) method.In vitro cell imaging showed that RGD-FSiNPs exhibited high target binding to fixed and living cells ofα_vβ_3 integrin receptor(ABIR)-positive MDA-MB-231 breast cancers.Further study regarding the ex vivo imaging of tumor tissue samples was also carried out by intravenously injecting PEGylated-RGD-FSiNPs into athymic nude mice bearing the MDA-MB-231 tumors.Tissue images demonstrated that it could reduce the phagocytosis of nanoparticles by liver and spleen and the high integrinα_vβ_3 expression level of the MDA-MB-231 tumors was clearly visible due to the special targeting effects of the RGD-FSiNPs.This RGD peptide-oriented fluorescent labeling technique based on the biofunctionalized FSiNPs will be promising for tumor cell imaging and cancer metastasis,etc.
2.Targeted separation of protein based on biofunctionalized magnetic silica nanoparticles.
A method of silica coated magnetic nanoparticles(MSiNPs)-based targeted separation of proteins has been developed based on the technology platform of our research group.The biofunctionalized MSiNPs(IgG-MSiNPs) were synthesized by modification of human IgG onto the surface of MSiNPs with CNBr method.Target protein(goat-anti-human IgG-FITC) was specifically recognized with IgG-MSiNPs and isolated under magnetic field.Then the target protein could be dissociated from the nanoparticles with suitable dissociation agent.The effects of separation and dissociation conditions on the separation efficiency,selectivity and dissociation effiency were studied.At the optimal conditions,the relatively high separation and dissociation efficiency and good selectivity were obtained.This study will further explore the application of functionalized magnetic silica nanoparticles in direct bio-separation of proteins from complex samples.
3.Synthesis of biofunctionalized fluorescent magnetic nanoparticles and their potential applications in separation of breast cancer cells.
We have developed a simple method to synthesize FITC dye doped silica fluorescent magnetic nanoparticles(FMSiNPs) with chemical precipitation method and reversed microemulsion technique under mild condition.FMSiNPs were formed by employing human IgG modified FITC dye and magnetic fluid as core and the silica as shell.The biofunctionalized FMSiNPs(RGD-FMSiNPs) were prepared by immobilizing of RGD peptide onto the surface of FMSiNPs.The specific enrichment ofα_vβ_3 integrin receptor-positive MDA-MB-231 breast cancer cells using RGD-FMSiNPs were carried out due to the specific recognition of RGD peptide andα_vβ_3 integrin.Then the target cells were isolated with RGD-FMSiNPs under magnetic field and detected by FCM based on the fluorescence properties of RGD-FMSiNPs. Results indicated that RGD-FMSiNPs exhibited high target binding to MDA-MB-231 breast cancer cells and high separation efficiency.This new kind of RGD based biofunctionalized FMSiNPs could be widely used for the magnetic separation and fluorescent detection of tumor cells.
4.Research on protein drug carriers based on biofunctionalized chitosan nanoparticles.
A new type of biocompatible chitosan(CS) nanoparticles encapsulated with bovine serum albumin(BSA)(CS-BSA-TPP NPs) was prepared through ionic cross-linking technique based on CS and sodium tripolyphosphate(TPP) at room temperature.The influence of formulation conditions on the properties of CS-BSA-TPP NPs was investigated.Results showed that CS-BSA-TPP NPs with diameter of below 100 nm and good dispersion were prepared at the optimal conditions,and the BSA encapsulation efficiency was more than 50%.This system was also applied in the preparation of CS nanoparticles encapsulated with Gamma Seroglobulin(CS-IG-TPP NPs),the CS-IG-TPP NPs presented the encapsulation efficiency of IG with 55.0%and displayed sustained-release property.Thus,this protein delivery system based on biofunctionalized CS nanoparticles will be promising for protein drug delivery.
5.A novel anticancer drug delivery system based on biofunctionalized chitosan nanoparticles.
A chitosan-methotrexate covalently conjugated nanoparticles(CS-MTX-TPP NPs) has been developed as a potential delivery system for methotrexate(MTX). MTX was first conjugated to CS by using glutaraldehyde as cross-linked agent,and followed by the process of ionic gelation between MTX-conjugated CS and sodium tripolyphosphate(TPP) to form CS-MTX-TPP NPs at mild reaction conditions.At the optimal conditions,CS-MTX-TPP NPs with diameter of sub-200-nm and encapsulation efficiency of 53.0%were obtained.Additionally,in vitro release test revealed that the stable covalent bonding of CS and MTX was beneficial for providing slow release for MTX.Especially,cellular toxicity study in MCF-7 cancer cells further demonstrated the effective anticancer efficacy of this new type of delivery for MTX.In the future,this work could be expected to broaden the applications of biofunctionalized CS NPs in anticancer drug delivery.
引文
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