牛血清白蛋白—高分子结合体的制备及在肿瘤诊疗中的应用
发布时间：2018-12-14 08:37
【摘要】：近年来，血清白蛋白作为一种新型的生物材料，因其良好的生物相容性、精确的几何结构，且易于功能化，在纳米医药领域受到越来越多的关注。本文选用牛血清白蛋白(BSA)作为基材，从分子设计角度，制备出一种新型的蛋白质-高分子结合体，并对其自组装行为及在癌症化疗、核素治疗和荧光成像中的应用进行了研究。具体研究内容如下： （1）双亲性BSA-聚己内酯(PCL)结合体的合成与结构性能表征。本文通过开环聚合(ROP)，合成了端基马来酰亚胺化的PCL，并利用马来酰亚胺与BSA的自由巯基之间的迈克尔加成反应，制备出新型BSA-PCL结合体。通过核磁共振仪、基质辅助激光解吸附串联时间飞行质谱仪(MALDI-TOF MS)和SDS-PAGE凝胶电泳等对合成产物进行表征，并利用透射电子显微镜(TEM)和动态光散射(DLS)等进一步研究了该结合体的组装性能。结果显示，BSA-PCL结合体被成功制备，且具有双亲性，在水中可以组装得到不同形貌的纳米组装体。 （2）基于BSA-PCL结合体的纳米靶向药物载体的构建。利用制备的双亲性BSA-PCL结合体，通过自组装技术，构建了包载抗癌药物阿霉素的纳米高分子脂质体，并利用表面含有的功能基团，对高分子脂质体进行表面靶向修饰。采用U251细胞和MAD-MB-231细胞对构建的纳米载体的细胞内吞和细胞毒性进行了评估。结果表明，相比于未修饰的高分子脂质体，经西妥昔单抗修饰的高分子脂质体具有更好的进胞效果。MTT结果表明，制备的BSA-PCL结合体具有很低的细胞毒性和较好的生物相容性。包载抗癌药物阿霉素后，西妥昔单抗修饰的载药BSA-PCL结合体纳米粒子对两种肿瘤细胞的生长都具有明显的抑制效果。 （3）基于BSA-PCL结合体的核素靶向纳米载体的构建。利用BSA含有的酪氨酸作为位点，在BSA-PCL结合体制备的纳米组装体上标记放射性131I，并对纳米载体进行靶向修饰，提高放射性131I标记的纳米粒子对细胞的靶向性。采用γ计数器和MTT实验对构建的核素靶向纳米载体的进胞效果和肿瘤抑制生长效果进行了表征。结果显示，相比于游离的131I离子，构建的核素靶向纳米载体增加了放射性131I被肿瘤细胞的摄取量，延长了放射性131I在肿瘤细胞内的存留时间，提高了对肿瘤细胞的放射性杀伤效果。 （4）基于BSA-PCL结合体的近红外荧光纳米探针的构建。利用双亲性BSA-PCL结合体对疏水性近红外CuInS2/ZnS量子点进行表面改性，制备出新型的近红外荧光纳米探针。利用TEM、DLS、荧光分光光度计和紫外分光光度计对构建的纳米探针的形貌、粒径和荧光性能进行了表征。该纳米探针具有优异的荧光性能和胶体稳定性，同时由于最外层BSA的保护，有效地降低了纳米探针的非特异性吸附，并赋予纳米探针良好的生物相容性。将靶向制剂cRGD修饰到纳米探针的表面，有效增加了纳米探针对v3整合素过表达的肿瘤细胞的靶向性。此外，在裸鼠体内初步验证了该纳米探针在体内成像的可行性。以上结果为进一步构建体内靶向的近红外荧光纳米探针打下了基础。 （5） pH响应性BSA-聚甲基丙烯酸二异丙基氨基乙酯(PDPA)结合体的制备。将刺激响应性高分子连接到蛋白质上，可赋予蛋白质-高分子结合体敏感性。本文首先设计合成一种ATRP小分子引发剂，通过ATRP聚合，制备端基马来酰亚胺化的pH敏感高分子PDPA。利用BSA中的巯基，将PDPA连接到BSA上，制备了BSA-PDPA结合体。利用SDS-PAGE凝胶电泳对结合体进行分子量表征，证明PDPA被成功结合到BSA上。通过ZetaPALS电位分析仪测定BSA-PDPA结合体在不同pH值溶液中的Zeta电位，初步证明结合体具有pH响应性。通过自组装技术制备了BSA-PDPA结合体纳米组装体，粒径在135nm左右。 总之，本文以生物安全性好的BSA为基材，结合合成的优异性能的高分子，制备了新型蛋白质-高分子结合体。利用该结合体，，通过自组装技术，构建了一种新型载体平台，并初步验证了其在靶向化疗、核素治疗和荧光成像等方面具有良好的应用前景。本研究为开发基于BSA的多功能纳米诊疗试剂提供了新思路和新方法。
[Abstract]:In recent years, serum albumin, as a new kind of biological material, has been more and more concerned in the field of nano medicine because of its good biocompatibility, precise geometry and easy functionalization. In this paper, bovine serum albumin (BSA) is used as a base material, and a new type of protein-polymer combination is prepared from the molecular design angle, and its self-assembly behavior and its application in cancer chemotherapy, nuclide therapy and fluorescence imaging are studied. The specific contents of the study are as follows: (1) Synthesis and structural properties of amphiphilic BSA-polycaprolactone (PCL) combination In this paper, a new type of BSA-PCL was prepared by ring-opening polymerization (ROP), the synthesis of the terminal-based maleimide-modified PCL, and the Michael addition reaction between the free base of the maleimide and BSA. The synthesis products were characterized by means of a nuclear magnetic resonance instrument, a matrix-assisted laser desorption/ adsorption series time-of-flight mass spectrometer (MALDI-TOF MS) and SDS-PAGE gel electrophoresis, and the assembly property of the combination was further studied by means of transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results show that the BSA-PCL combination is successfully prepared and has both parents and can be assembled in water to obtain nano-assembly with different shapes. (2) Nanometer-targeting drug carrier based on BSA-PCL combination The preparation method comprises the following steps of: using the prepared amphiphilic BSA-PCL combination body, carrying out self-assembly technology, constructing a nano-polymer liposome carrying the drug-loaded anti-cancer drug adriamycin, and carrying out surface target on the high-molecular liposome by using the functional group contained in the surface, The cell endocytosis and cytotoxicity of the constructed nanocarrier were performed using the U251 cells and the MAD-MB-231 cells. The results show that, compared with the unmodified high-molecular liposome, the polymer liposome modified by the cidotin has a better effect. The results showed that the prepared BSA-PCL combination had very low cytotoxicity and better biological activity. The compatibility of the drug-loaded anti-cancer drug with the drug-loaded BSA-PCL combination nanoparticles on the growth of both tumor cells has a significant effect on the growth of both tumor cells. Preparation effect. (3) Nuclide targeting nanometer based on BSA-PCL combination The carrier is constructed by using the tyrosine as a site contained in the BSA, labeling the radioactive 131I on the nano-assembly prepared by the BSA-PCL combination, carrying out targeted modification on the nano carrier, and improving the fine particle size of the radioactive 131I-labeled nano-carrier. Cell targeting. The effect of the tumor-targeting nano-carrier and the effect of the tumor-inhibiting growth on the constructed nuclide-targeting nano-carrier by the counter and MTT assay. The results show that, compared with the free 131I ion, the constructed nuclide-targeting nano-carrier increases the uptake of the radioactive 131I by the tumor cells, prolongs the retention time of the radioactive 131I in the tumor cells, and improves the radiation of the tumor cells. (4) Near-infrared fluorescence based on BSA-PCL combination The surface modification of the hydrophobic near-infrared CuInS2/ ZnS quantum dot is carried out by using the amphiphilic BSA-PCL combination, and a novel near-red is prepared. the morphology, the particle size and the fluorescence of the constructed nano-probe by using a TEM, DLS, a fluorescence spectrophotometer and an ultraviolet spectrophotometer The nano-probe has excellent fluorescence performance and colloidal stability, and at the same time, due to the protection of the outermost layer BSA, the non-specific adsorption of the nano-probe is effectively reduced, good biocompatibility. The target preparation cRGD is modified to the surface of the nano probe, and the swelling of the nano-probe to the overexpression of the v3 integrin is effectively increased. The targeting of the tumor cells. In addition, the nanoprobe was initially validated in the nude mice The above results are to further construct the in vivo target near-infrared fluorescent nanometer. The probe laid the foundation. (5) pH responsive BSA-poly (diisopropylamino) ethyl methacrylate (PD PA) preparation of a combination of a stimulus-responsive polymer to a protein, which can be imparted with a protein-In this paper, an ATRP small-molecular initiator is designed, and the pH of the terminal-based maleimide is prepared by ATRP polymerization. The sensitive polymer PDPA was prepared by connecting the PDPA to the BSA by using the base of the BSA in the BSA. A-PDPA combination. The molecular weight of the combination was characterized by SDS-PAGE gel electrophoresis, and the PDPA was proved to be The Zeta potential of BSA-PDPA combination in different pH value solutions was determined by the ZetaPALS potential analyzer. in that invention, a BSA-PDPA combination nano-assembly body and a particle are prepared by self-assembly technology, The diameter is about 135nm. In general, the new high-molecular polymer with good biological safety is used as the base material, and the new polymer with excellent properties is prepared. A new type of carrier platform is constructed by self-assembly technology, and its target chemotherapy, nuclide therapy and fluorescence imaging are also preliminarily verified. The purpose of this study is to develop a multi-functional nano-diagnosis and treatment trial based on BSA.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08;O629.73
[Abstract]:In recent years, serum albumin, as a new kind of biological material, has been more and more concerned in the field of nano medicine because of its good biocompatibility, precise geometry and easy functionalization. In this paper, bovine serum albumin (BSA) is used as a base material, and a new type of protein-polymer combination is prepared from the molecular design angle, and its self-assembly behavior and its application in cancer chemotherapy, nuclide therapy and fluorescence imaging are studied. The specific contents of the study are as follows: (1) Synthesis and structural properties of amphiphilic BSA-polycaprolactone (PCL) combination In this paper, a new type of BSA-PCL was prepared by ring-opening polymerization (ROP), the synthesis of the terminal-based maleimide-modified PCL, and the Michael addition reaction between the free base of the maleimide and BSA. The synthesis products were characterized by means of a nuclear magnetic resonance instrument, a matrix-assisted laser desorption/ adsorption series time-of-flight mass spectrometer (MALDI-TOF MS) and SDS-PAGE gel electrophoresis, and the assembly property of the combination was further studied by means of transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results show that the BSA-PCL combination is successfully prepared and has both parents and can be assembled in water to obtain nano-assembly with different shapes. (2) Nanometer-targeting drug carrier based on BSA-PCL combination The preparation method comprises the following steps of: using the prepared amphiphilic BSA-PCL combination body, carrying out self-assembly technology, constructing a nano-polymer liposome carrying the drug-loaded anti-cancer drug adriamycin, and carrying out surface target on the high-molecular liposome by using the functional group contained in the surface, The cell endocytosis and cytotoxicity of the constructed nanocarrier were performed using the U251 cells and the MAD-MB-231 cells. The results show that, compared with the unmodified high-molecular liposome, the polymer liposome modified by the cidotin has a better effect. The results showed that the prepared BSA-PCL combination had very low cytotoxicity and better biological activity. The compatibility of the drug-loaded anti-cancer drug with the drug-loaded BSA-PCL combination nanoparticles on the growth of both tumor cells has a significant effect on the growth of both tumor cells. Preparation effect. (3) Nuclide targeting nanometer based on BSA-PCL combination The carrier is constructed by using the tyrosine as a site contained in the BSA, labeling the radioactive 131I on the nano-assembly prepared by the BSA-PCL combination, carrying out targeted modification on the nano carrier, and improving the fine particle size of the radioactive 131I-labeled nano-carrier. Cell targeting. The effect of the tumor-targeting nano-carrier and the effect of the tumor-inhibiting growth on the constructed nuclide-targeting nano-carrier by the counter and MTT assay. The results show that, compared with the free 131I ion, the constructed nuclide-targeting nano-carrier increases the uptake of the radioactive 131I by the tumor cells, prolongs the retention time of the radioactive 131I in the tumor cells, and improves the radiation of the tumor cells. (4) Near-infrared fluorescence based on BSA-PCL combination The surface modification of the hydrophobic near-infrared CuInS2/ ZnS quantum dot is carried out by using the amphiphilic BSA-PCL combination, and a novel near-red is prepared. the morphology, the particle size and the fluorescence of the constructed nano-probe by using a TEM, DLS, a fluorescence spectrophotometer and an ultraviolet spectrophotometer The nano-probe has excellent fluorescence performance and colloidal stability, and at the same time, due to the protection of the outermost layer BSA, the non-specific adsorption of the nano-probe is effectively reduced, good biocompatibility. The target preparation cRGD is modified to the surface of the nano probe, and the swelling of the nano-probe to the overexpression of the v3 integrin is effectively increased. The targeting of the tumor cells. In addition, the nanoprobe was initially validated in the nude mice The above results are to further construct the in vivo target near-infrared fluorescent nanometer. The probe laid the foundation. (5) pH responsive BSA-poly (diisopropylamino) ethyl methacrylate (PD PA) preparation of a combination of a stimulus-responsive polymer to a protein, which can be imparted with a protein-In this paper, an ATRP small-molecular initiator is designed, and the pH of the terminal-based maleimide is prepared by ATRP polymerization. The sensitive polymer PDPA was prepared by connecting the PDPA to the BSA by using the base of the BSA in the BSA. A-PDPA combination. The molecular weight of the combination was characterized by SDS-PAGE gel electrophoresis, and the PDPA was proved to be The Zeta potential of BSA-PDPA combination in different pH value solutions was determined by the ZetaPALS potential analyzer. in that invention, a BSA-PDPA combination nano-assembly body and a particle are prepared by self-assembly technology, The diameter is about 135nm. In general, the new high-molecular polymer with good biological safety is used as the base material, and the new polymer with excellent properties is prepared. A new type of carrier platform is constructed by self-assembly technology, and its target chemotherapy, nuclide therapy and fluorescence imaging are also preliminarily verified. The purpose of this study is to develop a multi-functional nano-diagnosis and treatment trial based on BSA.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08;O629.73
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