肿瘤归巢穿膜肽介导靶向载药相变纳米粒用于乳腺癌体外超声分子成像与治疗研究

发布时间：2018-03-05 14:41

本文选题：肿瘤归巢穿膜肽　切入点：固相合成　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：第一部分肿瘤归巢穿膜肽合成及体外活性研究目的采用固相法人工合成异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的新型肿瘤归巢穿膜肽t Ly P-1(CGNKRTR),并于体外检测其肿瘤靶向性及穿膜特性。方法采用Na-芴甲氧羰基(Fmoc)法固相合成t Ly P-1,并用FITC标记N端。高效液相色谱及质谱仪检测其纯度及分子量;激光共聚焦显微镜及流式细胞仪观察其肿瘤靶向性及穿膜能力;建立三维肿瘤球模型检测其靶向穿膜性;CCK8(cell counting kit-8)评价其细胞毒性。结果合成的FITC-t Ly P-1纯度为99.14%,分子量为1334.7;激光共聚焦显微镜可见FITC-t Ly P-1能靶向聚集到MDA-MB-231细胞膜周围并部分穿膜进入到细胞内,而在HUVEC细胞膜周围未见明显的聚集和穿膜现象;流式细胞仪检测MDA-MB-231细胞内荧光强度值高于HUVEC;FITC-t Ly P-1可穿入MDA-MB-231三维肿瘤球模型内部约120μm;CCK8检测不同浓度的FITC-t Ly P-1对细胞活性没有明显影响(P0.05)。结论采用固相法成功合成FITC标记的新型肿瘤归巢穿膜肽t Ly P-1,具有乳腺癌MDA-MB-231肿瘤靶向性和穿膜性,能作为一种潜在的肿瘤靶向药物运输载体。第二部分肿瘤归巢穿膜肽介导的靶向载药相变纳米粒制备及体外靶向性、穿膜性研究目的制备一种新型超声分子探针--肿瘤归巢穿膜肽t Ly P-1介导的靶向载10-羟基喜树碱(10-HCPT)的液-气相变型氟碳(PFP)纳米粒(t Ly P-1-10-HCPT-PFP NPs),检测其基本表征,并评价其体外靶向性及穿膜性。方法薄膜水化-声振法制备新型超声分子探针,检测其形态、分布、粒径及表面电位;激光共聚焦显微镜定性观察、流式细胞仪定量分析其体外靶向性和细胞穿膜性;建立三维肿瘤球模型检测纳米粒的靶向穿膜性。结果t Ly P-1-10-HCPT-PFP NPs大小均一、分散性好,粒径为(366.30±11.50)nm,表面电位为(5.70±3.70)m V;激光共聚焦显微镜结果显示t Ly P-1-10-HCPT-PFP NPs可靶向聚集到MDA-MB-231细胞膜周围且部分穿膜进入到细胞质内,而HUVEC细胞膜周围未见明显聚集和穿膜现象;无t Ly P-1肽介导的非靶向载药相变纳米粒(10-HCPT-PFP NPs)在两种细胞周围也未见明显聚集和穿膜现象;流式细胞仪结果显示MDA-MB-231细胞内t Ly P-1-10-HCPT-PFP NPs的荧光强度明显高于其余各组(P0.05);MDA-MB-231三维肿瘤球模型的激光共聚焦显微镜结果显示t Ly P-1-10-HCPT-PFP NPs可穿入MDA-MB-231三维肿瘤球内部约80μm,而10-HCPT-PFP NPs在肿瘤球模型表面未见明显聚集,同时仅见极少量的纳米粒进入到肿瘤球内部,且距离仅约25μm。结论成功制备了肿瘤归巢穿膜肽t Ly P-1介导的靶向载10-羟基喜树碱(10-HCPT)的液-气相变型氟碳(PFP)纳米粒(t Ly P-1-10-HCPT-PFP NPs),其能有效地靶向乳腺癌MDA-MB-231细胞并穿膜进入细胞质内及肿瘤球内部,为后续实验奠定了基础。第三部分肿瘤归巢穿膜肽介导的靶向载药相变纳米粒体外超声成像及靶向治疗研究目的制备一种新型超声分子探针--肿瘤归巢穿膜肽t Ly P-1介导的靶向载10-羟基喜树碱(10-HCPT)的液-气相变型氟碳(PFP)纳米粒(t Ly P-1-10-HCPT-PFP NPs),评价其体外超声成像及对乳腺癌MDA-MB-231细胞的体外治疗效果。方法高效液相色谱仪检测所制备纳米粒10-HCPT的包封率及载药率;加热板加热和低强度聚焦超声仪(low intensity focused ultrasound,LIFU)辐照后观察纳米粒液气相变及体外超声成像效果;流式细胞仪检测纳米粒对MDA-MB-231细胞早晚期凋亡影响,CCK8检测纳米粒对MDA-MB-231细胞毒性作用。结果本实验制备的t Ly P-1-10-HCPT-PFP NPs中10HCPT的包封率为86.04±4.27%,载药率为7.82±0.38%。t Ly P-1-10-HCPT-PFP NPs加热到45℃可致相变,此外一定功率的LIFU辐照后也可致相变并能增强超声成像(P0.05);t Ly P-1-10-HCPT-PFP NPs联合LIFU辐照后,MDA-MB-231早晚期凋亡率明显增加,细胞毒性也明显增加,且高于其他各组(P0.05)。结论本实验所制备的t Ly P-1-10-HCPT-PFP NPs在一定功率的LIFU辐照后能增强超声成像,靶向破坏微泡后释放药物可实现更佳的治疗效果。
[Abstract]:The first part in tumor homing peptide to synthesis and in vitro activity of the solid phase synthesis of fluorescein isothiocyanate (fluorescein isothiocyanate FITC) new tumor marker homing penetrating peptide t Ly P-1 (CGNKRTR), and to detect the in vitro tumor targeting and membrane properties. Methods Na- fluorenylmethoxycarbonyl (Fmoc) t Ly P-1 solid synthesis method, and identified by FITC N. The high performance liquid chromatography and mass spectrometry to detect the purity and molecular weight; confocal laser scanning microscopy and flow cytometry were used to observe the tumor targeting and detection of its transmembrane ability; targeting transmembrane three-dimensional tumor sphere model; CCK8 (cell counting kit-8) to evaluate the cytotoxicity of FITC-t Ly P-1. The purity is 99.14%, molecular weight is 1334.7; laser scanning confocal microscopy showed FITC-t Ly P-1 targeting MDA-MB-231 gathered around the cell membrane and membrane into fine wear part Intracellular and around the membrane of HUVEC cells had no obvious aggregation and penetrating phenomenon; flow cytometry MDA-MB-231 intracellular fluorescence intensity was higher than that of HUVEC; FITC-t Ly P-1 MDA-MB-231 into the internal 3D tumor sphere model is about 120 m; FITC-t Ly P-1 CCK8 to detect different concentrations had no obvious effect on cell activity (P0.05). New tumor homing conclusion by solid phase synthesis of FITC successfully labeled membrane penetrating peptide t Ly P-1, with MDA-MB-231 breast cancer tumor targeting and transmembrane, can be used as a potential tumor to drug carriers. The second part tumor homing penetrating peptide mediated targeted drug loaded nanoparticles transformation preparation and in vitro targeting, targeting 10- hydroxycamptothecin through research on membrane to prepare a new ultrasound molecular probe: tumor homing penetrating peptide t Ly mediated P-1 (10-HCPT) of the liquid gas phase transition of fluorocarbon (PFP) nanoparticles (t Ly P- 1-10-HCPT-PFP NPs), to detect the basic characterization, and evaluate its in vitro targeting and membrane. Method of thin film hydration - Acoustic preparation of new ultrasound molecular probe, detection of its morphology, particle size distribution, and surface potential; laser scanning confocal microscope, flow cytometry, quantitative analysis of the target in vitro the film and to establish three-dimensional model of tumor cells; ball detection nanoparticles targeting transmembrane t Ly P-1-10-HCPT-PFP NPs. The results of uniform size, good dispersion, particle size (366.30 + 11.50) nm, the surface potential is (5.70 + 3.70) m V; laser confocal microscope showed that t Ly P-1-10-HCPT-PFP NPs target gathered around MDA-MB-231 cell membrane and some in membrane into the cytoplasm, while HUVEC cell membrane around no obvious aggregation and penetrating phenomenon; no non target T Ly P-1 peptide mediated drug loaded nanoparticles to phase (10-HCPT-PFP NPs) in around two cells No obvious aggregation and penetrating phenomenon; flow cytometry results showed that the fluorescence intensity of MDA-MB-231 cells in t Ly P-1-10-HCPT-PFP NPs was significantly higher than other groups (P0.05); MDA-MB-231 laser 3D tumor sphere model confocal microscopy showed that t Ly P-1-10-HCPT-PFP NPs MDA-MB-231 can be inserted into the three-dimensional tumor inside the ball about 80 m, and 10-HCPT-PFP NPs in the model of tumor sphere surface no obvious aggregation, and only a very small amount of nanoparticles into the tumor ball inside, and the distance is only about 25 M. conclusion the successful preparation of tumor homing peptide t Ly mediated P-1 targeting 10- Hydroxycamptothecin (10-HCPT) of the liquid gas phase transition of fluorocarbon (PFP) nanoparticles (t Ly P-1-10-HCPT-PFP NPs), which can effectively target the MDA-MB-231 breast cancer cells and penetrating into the internal cytoplasm and tumor sphere, laid the foundation for subsequent experiments. The third part in tumor homing Targeting hydroxycamptothecin loaded 10- peptide mediated targeting of drug loaded nanoparticles in vitro transformation of ultrasound imaging and targeted therapy of the purpose of the preparation of a new ultrasound molecular probe: tumor homing penetrating peptide t Ly mediated P-1 (10-HCPT) of the liquid gas phase transition of fluorocarbon (PFP) nanoparticles (t Ly P-1-10-HCPT-PFP NPs), to evaluate the ultrasound imaging in vitro and in vitro treatment of breast cancer MDA-MB-231 cells. Methods the HPLC detection of 10-HCPT nanoparticles prepared by encapsulation efficiency and drug loading rate; heating board and low intensity focused ultrasound (low intensity, focused ultrasound, LIFU) were observed in vitro ultrasound and liquid gas phase transition the imaging effect after irradiation; flow cytometry nanoparticles on MDA-MB-231 cells of early and late apoptosis, CCK8 were detected on MDA-MB-231 cells. The results of this experiment prepared t Ly P-1-10-HCPT-PFP NPs 10H The encapsulation rate of CPT was 86.04 + 4.27%, the drug loading rate of 7.82 + 0.38%.t Ly P-1-10-HCPT-PFP NPs is heated to 45 DEG C can induce phase transition, in addition to some power after LIFU irradiation can cause phase change and can enhance ultrasound imaging (P0.05); t Ly P-1-10-HCPT-PFP NPs combined with LIFU irradiation, MDA-MB-231 and delayed apoptosis, cell the toxicity also increased significantly, and higher than the other groups (P0.05). Conclusion the preparation of t Ly P-1-10-HCPT-PFP NPs in power LIFU irradiation can enhance ultrasound imaging, targeted microbubble destruction after the release of the drug can achieve better therapeutic effect.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9;R445.1

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