本文選題：腫瘤微環(huán)境 + 含硅羅丹明　； 參考：《鄭州大學》2017年碩士論文

【摘要】：酸性腫瘤環(huán)境與控制腫瘤生長和轉(zhuǎn)移的許多因素密切相關,腫瘤部位的pH檢測和pH值變化的監(jiān)測已在納米醫(yī)學領域引起人們廣泛關注。本課題通過在含硅元素的羅丹明結構中引入硼酸基團合成pH敏感的近紅外(NIR)熒光探針SiRB。將吲哚青綠(ICG)和SiRB荷載到具有良好生物相容性的高聚物聚乳酸-羥基乙酸共聚物(PLGA)中形成PLGA-SiRB-ICG pH響應型近紅外比率熒光探針。結果表明,該熒光探針通過增強的滲透性和保留效應可有效地在腫瘤部位蓄積。PLGA在弱酸性環(huán)境下加速降解有利于探針的釋放,SiRB在pH 5.8~7.6范圍內(nèi)具有良好的pH響應性,而ICG的熒光強度幾乎不隨pH的變化而改變,兩種探針的熒光比值可定量測量出腫瘤部位的pH值及pH值的變化情況,對腫瘤的發(fā)生、發(fā)展和轉(zhuǎn)移有重要參考價值。本課題主要研究內(nèi)容包括以下四個部分:一、“自開關”型近紅外熒光探針SiRB的合成及其性質(zhì)考察本文以3-溴-N,N-二甲基苯胺和2-溴苯硼酸為原料,采用四步法成功合成“自開關”型近紅外熒光探針SiRB;通過柱層析法對中間體和終產(chǎn)物進行了純化;利用核磁共振氫譜,紫外-可見光譜和熒光光譜表征了SiRB的分子結構和激發(fā)/發(fā)射波長,結果證明,SiRB分子結構與預期結構相符,其激發(fā)波長和發(fā)射波長均位于近紅外區(qū);SiRB探針在pH 5.8~7.6范圍內(nèi)具有靈敏的pH響應性,且研究發(fā)現(xiàn)該熒光探針的pH響應性具有良好的可逆性,有利于監(jiān)測腫瘤微環(huán)境的pH值變化情況。二、基于SiRB的pH響應型近紅外比率熒光探針的制備及其性質(zhì)考察以SiRB為熒光pH指示劑;以吲哚菁綠(ICG)作為熒光參比試劑(其熒光強度在一定酸堿范圍內(nèi)不受pH值影響),高聚物PLGA為載體,通過溶劑乳化揮發(fā)法成功制備pH響應型近紅外比率熒光納米探針PLGA-SiRB-ICG,同法制備了對比試劑PLGA-SiRB和PLGA-ICG。通過透射電鏡圖和紫外-可見光譜圖證實了PLGA將SiRB與ICG荷載,形成了核殼式的結構;激光納米粒度儀測定結果表明,PLGA-SiRB-ICG的平均粒徑和電位分別為95.13±0.21 nm和-8.14±0.13 mV,粒徑大小均勻,穩(wěn)定;通過測定不同pH下的紫外-可見光譜可知:在pH 5.8~7.6之間,PLGA-SiRB-ICG的熒光比值隨pH變化而變化。實驗排除了人體常見鹽類對PLGA-SiRB-ICG探針的干擾,體外釋放實驗表明PLGA-SiRB-ICG在微酸性環(huán)境下具有更高的釋放速率,有利于在腫瘤微環(huán)境下的釋放。三、pH響應型近紅外比率熒光探針PLGA-SiRB-ICG的細胞實驗以人乳腺癌細胞MCF-7為模型細胞,考察了PLGA-SiRB-ICG的細胞毒性。SRB結果顯示,在較低濃度時,PLGA-SiRB-ICG對MCF-7細胞沒有明顯毒性,當探針濃度大于80μg/mL時,探針對MCF-7細胞具有一定的毒性,且游離的SiRB和ICG的毒性明顯高于探針組,說明PLGA的包裹降低了探針對細胞的毒性;激光共聚焦熒光顯微鏡實驗結果表明:探針SiRB可以進入腫瘤細胞內(nèi)部,并對溶酶體具有特殊靶向性,有望應用于細胞器定位研究。四、pH響應型近紅外比率熒光探針PLGA-SiRB-ICG的熒光成像和pH檢測研究首先,通過多模式活體成像系統(tǒng)(KODAK)記錄PLGA-SiRB-ICG的熒光比值與pH值之間的關系,通過線性回歸計算考察兩者之間的線性關系并得到線性回歸方程。然后,以KM雌性小鼠為模型動物,腹水瘤S180細胞為模型細胞,構建荷瘤鼠模型。通過多模式活體成像系統(tǒng),成功的對小鼠的腫瘤組織及主要器官進行了pH值檢測。通過實驗結果發(fā)現(xiàn):PLGA-SiRB-ICG對腫瘤組織具有一定的被動靶向作用,且腫瘤部位的pH數(shù)值明顯低于其他器官,通過對不同生長天數(shù)的腫瘤觀察發(fā)現(xiàn):腫瘤生長天數(shù)越長,尺寸越大,pH越低,此結果對腫瘤的發(fā)展階段界定與微環(huán)境研究具有重要參考價值。
[Abstract]:The acidic tumor environment is closely related to many factors controlling the growth and metastasis of tumor. The pH detection of the tumor site and the monitoring of the change of pH value have aroused widespread concern in the field of nanomedicine. This topic is based on the introduction of the boric acid group of the pH sensitive near infrared (NIR) fluorescence probe SiRB. to the indole in the Luo Danming structure containing silicon elements. Green (ICG) and SiRB loads form a PLGA-SiRB-ICG pH responsive near infrared fluorescence probe into a polymer of polymer polylactic acid copolymer (PLGA) with good biocompatibility. The results show that the fluorescence probe can effectively accumulate.PLGA in the tumor site to accelerate in the weak acid environment through enhanced permeability and retention effect. Degradation is beneficial to the release of the probe, SiRB has good pH responsiveness in the range of pH 5.8~7.6, and the fluorescence intensity of ICG almost does not change with the change of pH. The fluorescence ratio of the two probes can quantitatively measure the changes of the pH value and pH value of the tumor site, and have important reference value for the occurrence, development and metastasis of the tumor. The research contents include the following four parts: first, the synthesis and properties of "self switched" near infrared fluorescence probe SiRB and its properties, the "self switching" near infrared fluorescence probe SiRB is successfully synthesized by four step method using 3- bromine -N, N- two methyl aniline and 2- bromine boric acid as raw materials. The intermediate and final products are purified by column chromatography. The molecular structure and excitation / emission wavelengths of SiRB are characterized by NMR, UV visible and fluorescence spectra. The results show that the molecular structure of SiRB is in accordance with the expected structure, the excitation wavelength and the emission wavelength are in the near infrared region, and the SiRB probe has a sensitive pH responsiveness in the pH 5.8~7.6 enclosure, and the research has found the fluorescein. The pH responsiveness of the light probe has good reversibility, which is beneficial to monitoring the pH value change of the tumor microenvironment. Two, the preparation and properties of the pH responsive near infrared fluorescence probe based on SiRB and its properties are SiRB as the fluorescent pH indicator, and using the indocyanine green (ICG) as the fluorescence reference reagent (its fluorescence intensity is not subject to a certain acid base range. " The effect of pH value), polymer PLGA as the carrier, pH responsive near infrared ratio fluorescent nano probe PLGA-SiRB-ICG was successfully prepared by solvent emulsification and volatilization, and the comparison reagent PLGA-SiRB and PLGA-ICG. were prepared by transmission electron microscopy and UV visible spectrogram to confirm PLGA with SiRB and ICG loads, forming a nuclear shell structure; laser nanoscale. The results showed that the average particle size and potential of PLGA-SiRB-ICG were 95.13 + 0.21 nm and -8.14 + 0.13 mV respectively, and the size of grain was uniform and stable. By measuring the UV visible spectrum under different pH, the fluorescence ratio of PLGA-SiRB-ICG changed with pH in pH 5.8~7.6. The experiment ruled out common salt of human body to PLGA-Si. The interference of RB-ICG probe and in vitro release experiments show that PLGA-SiRB-ICG has a higher release rate in the microacid environment and is beneficial to the release of the tumor microenvironment. Three, the cell experiment of the pH responsive near infrared ratio fluorescence probe PLGA-SiRB-ICG is based on the human breast cancer cell MCF-7 as the model cell, and the cytotoxic.S of PLGA-SiRB-ICG is investigated. RB results showed that PLGA-SiRB-ICG had no obvious toxicity to MCF-7 cells at a lower concentration. When the probe concentration was greater than 80 g/mL, the probe had a certain toxicity to MCF-7 cells, and the toxicity of free SiRB and ICG was significantly higher than that of the probe group. It indicated that the encapsulation of PLGA reduced the toxicity of the probe to the cells; the laser confocal fluorescence microscopy experiment was used. The results show that the probe SiRB can enter the tumor cells and have special targeting to the lysosomes and is expected to be applied to the study of organelles. Four, the fluorescence imaging and pH detection of the pH responsive near infrared ratio fluorescence probe PLGA-SiRB-ICG, first, records the fluorescence ratio of PLGA-SiRB-ICG by multimode living imaging system (KODAK). The relationship between pH values, linear regression and linear regression equation were obtained by linear regression calculation. Then, KM female mice were used as model animals and ascites tumor S180 cells as model cells to construct a tumor bearing rat model. The pH value of the tumor tissues and major organs of mice was successfully carried out by multi mode living imaging system. The test results show that PLGA-SiRB-ICG has a certain passive targeting effect on tumor tissue, and the pH value of the tumor site is obviously lower than that of other organs. Through the observation of the tumor growth days, the longer the growth days, the larger the size and the lower the pH, the result is the definition of the development stage of the tumor and the study of the microenvironment. It has important reference value.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R943

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