本文選題：納米載藥體系 + 腫瘤微酸環(huán)境��； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的光動力學(xué)療法近些年越來越受到臨床工作者的關(guān)注,已經(jīng)被用于各種癌癥的治療。然而,對腫瘤診斷的敏感性,治療的有效性和患者腫瘤的異質(zhì)性無疑是臨床上光動力學(xué)療法面臨的巨大挑戰(zhàn)。納米顆粒同時裝載光敏劑及診斷試劑已經(jīng)成為一種新方法用于成像引導(dǎo)下的光動力學(xué)療法。為此我們設(shè)計一種包載光敏劑Ce6和螯合Gd3+的腫瘤細(xì)胞外酸敏感TAT靶向納米顆粒(DATAT-NP)并探究磁共振引導(dǎo)下的光動力學(xué)療法對胰腺癌治療的效果,為臨床上光動力學(xué)療法精準(zhǔn)治療腫瘤提供一個新的有效參考。方法我們使用雙嵌段聚合物(DATAT-PEG77-b-PCL25和PCL45-b-PAEP35-Cya/DTPA)包載Ce6和螯合Gd3+,形成在腫瘤組織微酸環(huán)境下激活的TAT靶向納米載藥體系DATAT-NP,用于光敏劑和造影劑的輸送。通過核磁表征、粒徑的檢測和TEM的觀察等實驗方法分別檢測納米顆粒的化學(xué)結(jié)構(gòu)、尺寸大小及形狀;用動態(tài)光散射儀(DLS)檢測顆粒表面電荷在p H 7.4或p H 6.5下隨時間的變化情況;采用高效液相色譜法(HPLC)檢測顆粒在p H 7.4或p H 6.5下光敏劑Ce6的釋放行為。在細(xì)胞水平上,通過流式細(xì)胞術(shù)(FACS)、激光共聚焦顯微鏡(CLSM)、高效液相色譜法(HPLC)等不同的實驗方法分別檢測納米顆粒在p H7.4或6.5條件下被細(xì)胞的攝取情況;進(jìn)一步利用活性氧指示劑DCFH-DA檢測細(xì)胞在攝取顆粒后產(chǎn)生活性氧的情況;最后采用MTT和活死染色法等方法分別檢測了光動力學(xué)療法對細(xì)胞的殺傷效果。在動物水平上,首先采用HPLC及小動物成像儀分別檢測了納米顆粒在小鼠體內(nèi)的循環(huán)、代謝和在腫瘤部位的富集情況;通過對荷瘤小鼠的給藥,光照,測量并記錄腫瘤體積大小和相關(guān)參數(shù)來觀察不同組對腫瘤生長的抑制作用;最后對離體器官及腫瘤分別進(jìn)行HE,PCNA細(xì)胞增殖和TUNEL細(xì)胞凋亡染色的檢測分析。結(jié)果我們成功制備出了粒徑在100 nm左右,粒徑分散度(PDI)為0.22的致密球形納米顆粒DATAT-NP。在體外p H 6.5緩沖液下納米顆粒表面電荷顯著提高,顆粒被細(xì)胞攝取量增加,光照后產(chǎn)生活性氧增加,對細(xì)胞有更強(qiáng)的殺傷作用。通過DA(2,3-二甲基馬來酸苷)分子對TAT表面的修飾,有效地屏蔽了TAT上的正電荷靶向基團(tuán),從而避免被網(wǎng)狀內(nèi)皮系統(tǒng)(RES)快速清除,使納米顆粒在血液中達(dá)到長循環(huán),通過腫瘤部位特有的EPR效應(yīng)及腫瘤組織特殊的微酸環(huán)境(低p H值),納米顆粒在腫瘤部位大量富集,另外DA與TAT之間的酸敏感鍵斷裂,從而暴露TAT穿膜肽,增強(qiáng)腫瘤細(xì)胞與顆粒之間的相互作用,增加腫瘤細(xì)胞對顆粒的攝取,釋放出更多的造影劑和光敏劑,從而放大腫瘤部位的熒光/核磁診斷信號強(qiáng)度,提高了診斷的敏感性和光動力學(xué)療法的抗腫瘤效果。結(jié)論光敏劑Ce6及造影劑Gd3+在腫瘤細(xì)胞外酸敏感TAT靶向納米載藥體系的遞送下,其納米顆粒(DATAT-NP)在血液循環(huán)中穩(wěn)定性好,顯著增加在腫瘤部位的富集量及良好的生物安全性,光照后極大的抑制了腫瘤細(xì)胞的生長。該設(shè)計為腫瘤微酸環(huán)境下響應(yīng)的靶向納米顆粒用于成像引導(dǎo)下的癌癥治療提供新的方法指導(dǎo)。
[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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.9

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