牛血清白蛋白—高分子結(jié)合體的制備及在腫瘤診療中的應(yīng)用
發(fā)布時(shí)間：2018-12-14 08:37
【摘要】：近年來(lái)，血清白蛋白作為一種新型的生物材料，因其良好的生物相容性、精確的幾何結(jié)構(gòu)，且易于功能化，在納米醫(yī)藥領(lǐng)域受到越來(lái)越多的關(guān)注。本文選用牛血清白蛋白(BSA)作為基材，從分子設(shè)計(jì)角度，制備出一種新型的蛋白質(zhì)-高分子結(jié)合體，并對(duì)其自組裝行為及在癌癥化療、核素治療和熒光成像中的應(yīng)用進(jìn)行了研究。具體研究?jī)?nèi)容如下： （1）雙親性BSA-聚己內(nèi)酯(PCL)結(jié)合體的合成與結(jié)構(gòu)性能表征。本文通過(guò)開(kāi)環(huán)聚合(ROP)，合成了端基馬來(lái)酰亞胺化的PCL，并利用馬來(lái)酰亞胺與BSA的自由巰基之間的邁克爾加成反應(yīng)，制備出新型BSA-PCL結(jié)合體。通過(guò)核磁共振儀、基質(zhì)輔助激光解吸附串聯(lián)時(shí)間飛行質(zhì)譜儀(MALDI-TOF MS)和SDS-PAGE凝膠電泳等對(duì)合成產(chǎn)物進(jìn)行表征，并利用透射電子顯微鏡(TEM)和動(dòng)態(tài)光散射(DLS)等進(jìn)一步研究了該結(jié)合體的組裝性能。結(jié)果顯示，BSA-PCL結(jié)合體被成功制備，且具有雙親性，在水中可以組裝得到不同形貌的納米組裝體。 （2）基于BSA-PCL結(jié)合體的納米靶向藥物載體的構(gòu)建。利用制備的雙親性BSA-PCL結(jié)合體，通過(guò)自組裝技術(shù)，構(gòu)建了包載抗癌藥物阿霉素的納米高分子脂質(zhì)體，并利用表面含有的功能基團(tuán)，對(duì)高分子脂質(zhì)體進(jìn)行表面靶向修飾。采用U251細(xì)胞和MAD-MB-231細(xì)胞對(duì)構(gòu)建的納米載體的細(xì)胞內(nèi)吞和細(xì)胞毒性進(jìn)行了評(píng)估。結(jié)果表明，相比于未修飾的高分子脂質(zhì)體，經(jīng)西妥昔單抗修飾的高分子脂質(zhì)體具有更好的進(jìn)胞效果。MTT結(jié)果表明，制備的BSA-PCL結(jié)合體具有很低的細(xì)胞毒性和較好的生物相容性。包載抗癌藥物阿霉素后，西妥昔單抗修飾的載藥BSA-PCL結(jié)合體納米粒子對(duì)兩種腫瘤細(xì)胞的生長(zhǎng)都具有明顯的抑制效果。 （3）基于BSA-PCL結(jié)合體的核素靶向納米載體的構(gòu)建。利用BSA含有的酪氨酸作為位點(diǎn)，在BSA-PCL結(jié)合體制備的納米組裝體上標(biāo)記放射性131I，并對(duì)納米載體進(jìn)行靶向修飾，提高放射性131I標(biāo)記的納米粒子對(duì)細(xì)胞的靶向性。采用γ計(jì)數(shù)器和MTT實(shí)驗(yàn)對(duì)構(gòu)建的核素靶向納米載體的進(jìn)胞效果和腫瘤抑制生長(zhǎng)效果進(jìn)行了表征。結(jié)果顯示，相比于游離的131I離子，構(gòu)建的核素靶向納米載體增加了放射性131I被腫瘤細(xì)胞的攝取量，延長(zhǎng)了放射性131I在腫瘤細(xì)胞內(nèi)的存留時(shí)間，提高了對(duì)腫瘤細(xì)胞的放射性殺傷效果。 （4）基于BSA-PCL結(jié)合體的近紅外熒光納米探針的構(gòu)建。利用雙親性BSA-PCL結(jié)合體對(duì)疏水性近紅外CuInS2/ZnS量子點(diǎn)進(jìn)行表面改性，制備出新型的近紅外熒光納米探針。利用TEM、DLS、熒光分光光度計(jì)和紫外分光光度計(jì)對(duì)構(gòu)建的納米探針的形貌、粒徑和熒光性能進(jìn)行了表征。該納米探針具有優(yōu)異的熒光性能和膠體穩(wěn)定性，同時(shí)由于最外層BSA的保護(hù)，有效地降低了納米探針的非特異性吸附，并賦予納米探針良好的生物相容性。將靶向制劑cRGD修飾到納米探針的表面，有效增加了納米探針對(duì)v3整合素過(guò)表達(dá)的腫瘤細(xì)胞的靶向性。此外，在裸鼠體內(nèi)初步驗(yàn)證了該納米探針在體內(nèi)成像的可行性。以上結(jié)果為進(jìn)一步構(gòu)建體內(nèi)靶向的近紅外熒光納米探針打下了基礎(chǔ)。 （5） pH響應(yīng)性BSA-聚甲基丙烯酸二異丙基氨基乙酯(PDPA)結(jié)合體的制備。將刺激響應(yīng)性高分子連接到蛋白質(zhì)上，可賦予蛋白質(zhì)-高分子結(jié)合體敏感性。本文首先設(shè)計(jì)合成一種ATRP小分子引發(fā)劑，通過(guò)ATRP聚合，制備端基馬來(lái)酰亞胺化的pH敏感高分子PDPA。利用BSA中的巰基，將PDPA連接到BSA上，制備了BSA-PDPA結(jié)合體。利用SDS-PAGE凝膠電泳對(duì)結(jié)合體進(jìn)行分子量表征，證明PDPA被成功結(jié)合到BSA上。通過(guò)ZetaPALS電位分析儀測(cè)定BSA-PDPA結(jié)合體在不同pH值溶液中的Zeta電位，初步證明結(jié)合體具有pH響應(yīng)性。通過(guò)自組裝技術(shù)制備了BSA-PDPA結(jié)合體納米組裝體，粒徑在135nm左右。 總之，本文以生物安全性好的BSA為基材，結(jié)合合成的優(yōu)異性能的高分子，制備了新型蛋白質(zhì)-高分子結(jié)合體。利用該結(jié)合體，，通過(guò)自組裝技術(shù)，構(gòu)建了一種新型載體平臺(tái)，并初步驗(yàn)證了其在靶向化療、核素治療和熒光成像等方面具有良好的應(yīng)用前景。本研究為開(kāi)發(fā)基于BSA的多功能納米診療試劑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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R318.08;O629.73
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