MRI引导下细胞外酸敏感TAT靶向纳米颗粒用于光动力学疗法治疗胰腺癌

发布时间：2018-04-10 23:30

本文选题：纳米载药体系 + 肿瘤微酸环境　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的光动力学疗法近些年越来越受到临床工作者的关注,已经被用于各种癌症的治疗。然而,对肿瘤诊断的敏感性,治疗的有效性和患者肿瘤的异质性无疑是临床上光动力学疗法面临的巨大挑战。纳米颗粒同时装载光敏剂及诊断试剂已经成为一种新方法用于成像引导下的光动力学疗法。为此我们设计一种包载光敏剂Ce6和螯合Gd3+的肿瘤细胞外酸敏感TAT靶向纳米颗粒(DATAT-NP)并探究磁共振引导下的光动力学疗法对胰腺癌治疗的效果,为临床上光动力学疗法精准治疗肿瘤提供一个新的有效参考。方法我们使用双嵌段聚合物(DATAT-PEG77-b-PCL25和PCL45-b-PAEP35-Cya/DTPA)包载Ce6和螯合Gd3+,形成在肿瘤组织微酸环境下激活的TAT靶向纳米载药体系DATAT-NP,用于光敏剂和造影剂的输送。通过核磁表征、粒径的检测和TEM的观察等实验方法分别检测纳米颗粒的化学结构、尺寸大小及形状;用动态光散射仪(DLS)检测颗粒表面电荷在p H 7.4或p H 6.5下随时间的变化情况;采用高效液相色谱法(HPLC)检测颗粒在p H 7.4或p H 6.5下光敏剂Ce6的释放行为。在细胞水平上,通过流式细胞术(FACS)、激光共聚焦显微镜(CLSM)、高效液相色谱法(HPLC)等不同的实验方法分别检测纳米颗粒在p H7.4或6.5条件下被细胞的摄取情况;进一步利用活性氧指示剂DCFH-DA检测细胞在摄取颗粒后产生活性氧的情况;最后采用MTT和活死染色法等方法分别检测了光动力学疗法对细胞的杀伤效果。在动物水平上,首先采用HPLC及小动物成像仪分别检测了纳米颗粒在小鼠体内的循环、代谢和在肿瘤部位的富集情况;通过对荷瘤小鼠的给药,光照,测量并记录肿瘤体积大小和相关参数来观察不同组对肿瘤生长的抑制作用;最后对离体器官及肿瘤分别进行HE,PCNA细胞增殖和TUNEL细胞凋亡染色的检测分析。结果我们成功制备出了粒径在100 nm左右,粒径分散度(PDI)为0.22的致密球形纳米颗粒DATAT-NP。在体外p H 6.5缓冲液下纳米颗粒表面电荷显著提高,颗粒被细胞摄取量增加,光照后产生活性氧增加,对细胞有更强的杀伤作用。通过DA(2,3-二甲基马来酸苷)分子对TAT表面的修饰,有效地屏蔽了TAT上的正电荷靶向基团,从而避免被网状内皮系统(RES)快速清除,使纳米颗粒在血液中达到长循环,通过肿瘤部位特有的EPR效应及肿瘤组织特殊的微酸环境(低p H值),纳米颗粒在肿瘤部位大量富集,另外DA与TAT之间的酸敏感键断裂,从而暴露TAT穿膜肽,增强肿瘤细胞与颗粒之间的相互作用,增加肿瘤细胞对颗粒的摄取,释放出更多的造影剂和光敏剂,从而放大肿瘤部位的荧光/核磁诊断信号强度,提高了诊断的敏感性和光动力学疗法的抗肿瘤效果。结论光敏剂Ce6及造影剂Gd3+在肿瘤细胞外酸敏感TAT靶向纳米载药体系的递送下,其纳米颗粒(DATAT-NP)在血液循环中稳定性好,显著增加在肿瘤部位的富集量及良好的生物安全性,光照后极大的抑制了肿瘤细胞的生长。该设计为肿瘤微酸环境下响应的靶向纳米颗粒用于成像引导下的癌症治疗提供新的方法指导。
[Abstract]:Objective: photodynamic therapy in recent years has attracted more and more attention from clinical workers, has been used for the treatment of various cancers. However, the sensitivity of tumor diagnosis, heterogeneity and effectiveness of treatment of cancer is undoubtedly a huge challenge on clinical therapy. At the same time kinetics of nanoparticles loading photosensitizer and diagnostic reagent has become a new method for photodynamic therapy and imaging guided. So we design a package containing photosensitizer Ce6 and Gd3+ chelating acid sensitive tumor cells targeting TAT nanoparticles (DATAT-NP) and to explore the magnetic resonance guided photodynamic therapy for pancreatic cancer treatment effect, for clinical treatment of tumors on photodynamic therapy and provide an effective precision reference. Methods we used two block copolymers (DATAT-PEG77-b-PCL25 and PCL45-b-PAEP35-Cya/DTPA) and Ce6 loaded chelating Gd3+, formed in the TAT target tumor tissue under low pH environment to activate nanoparticle drug delivery system for delivery of DATAT-NP, photosensitizer and contrast agents. By NMR, observation and experiment method of particle size detection and TEM were used to detect the chemical structure of the nanoparticles, size and shape; by dynamic light scattering (DLS) detection of particle surface charge in P H 7.4 or P H 6.5 changes with time; using high performance liquid chromatography (HPLC) detection of particles in P H 7.4 or P H 6.5 Ce6. The release behavior of photosensitizer at the cellular level, by flow cytometry (FACS), laser scanning confocal microscope (CLSM), high liquid chromatography (HPLC) of different methods were tested for nanoparticles uptake by cells in the P H7.4 or the 6.5 conditions; the further use of active oxygen indicator DCFH-DA were detected in the uptake of particles after the production of reactive oxygen species; finally using MTT and live Die staining were used to detect the killing effect of photodynamic therapy on animal cells. In the first level, using HPLC and small animal imaging were detected by nanoparticles in vivo circulation, metabolism and enrichment in parts of the tumor; light through the tumor bearing mice administered, observe inhibition the effect of different groups on tumor growth were measured and recorded the tumor volume and related parameters; finally on isolated organs and tumors were HE, detection and analysis of PCNA cell proliferation and apoptosis of TUNEL cells staining. Results we successfully prepared the size of about 100 nm particle size dispersion (PDI) compact spherical nanoparticles of DATAT-NP. 0.22 in vitro P H 6.5 buffer solution significantly increased the surface charge of nanoparticles, the particles ingested by the cells increased after irradiation, ROS increase of cells have stronger anti-tumor effect. By DA (2,3- two methyl maleic acid glycoside) molecules on the surface of TAT, effectively shielding TAT positive charge on the target group, so as to avoid by the reticuloendothelial system (RES) rapid clearance, the nanoparticles can circulate in the blood, the EPR effect and tumor tissue tumor specific special acidic environment (low P value, H) nanoparticles in the tumor accumulation and also between DA and TAT acid sensitive bond rupture, thereby exposing transcriptionalactivatorprotein TAT, enhance the interaction between tumor cells and particles, increase the particle uptake of tumor cells, release more contrast agent and a photosensitizer, amplifying fluorescence / tumor parts of the magnetic resonance signal intensity, improves the diagnostic sensitivity and the antitumor effect of photodynamic therapy. Conclusion Gd3+ delivery of photosensitizer Ce6 and contrast agent in tumor extracellular acid sensitive TAT targeting nano drug delivery system, nano M particles (DATAT-NP) in the blood circulation in good stability, a significant increase in the accumulation of tumor location and good biological safety, light has greatly inhibited the growth of tumor cells. The design for the acidic environment in response to tumor targeting nanoparticles for cancer treatment under the guidance of imaging provides a new method for guidance.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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