磁性碳量子點(diǎn)探針的構(gòu)建及其在腫瘤診療中的應(yīng)用研究
發(fā)布時間：2019-06-17 20:19
【摘要】：癌癥的早期診斷已引起人們的高度重視,分子影像學(xué)檢測作為癌癥早期診斷的重要工具,為腫瘤診斷提供更準(zhǔn)確和完整的信息,以達(dá)到早期診斷、早期治療的目的。基于多功能納米粒子的分子成像技術(shù)已在腫瘤檢測的靈敏度和特異性方面取得了一定進(jìn)展,量子點(diǎn)、熒光染料分子和磁性納米粒子等已經(jīng)作為成像探針運(yùn)用到了活體熒光成像和磁共振成像中。然而,這些探針有毒而且水溶液穩(wěn)定性差,靈敏度欠佳,極大的限制了其在體內(nèi)的應(yīng)用。因此,構(gòu)建一種體系簡單、生物友好、成像靈敏的多功能靶向探針是分子成像技術(shù)亟待解決的問題。基于此,本課題通過微波輔助法將氯化釓(GdCl3)以釓離子Gd3+的形式摻雜到碳點(diǎn)CDs的里面,同時PEI對CDs表面進(jìn)行氨基化修飾,我們得到了高量子產(chǎn)率的摻釓碳量子點(diǎn)Gd-CDs,即能夠在可見光照射下發(fā)出明亮的綠光,它具有熒光強(qiáng)度高、光學(xué)穩(wěn)定性和生物相容性好。以去鐵蛋白納米籠為載體,去鐵蛋白籠AFn獨(dú)特的籠狀結(jié)構(gòu)特性即在pH=2蛋白籠支解成多個亞甲基結(jié)構(gòu)使得籠打開,在pH=7蛋白籠重新自組裝從而實(shí)現(xiàn)封裝藥物阿霉素。Gd-CDs作為一個熒光兼磁性的探針偶聯(lián)載藥體系A(chǔ)Fn包裹DOX。在EDC和NHS催化劑的協(xié)同下將靶頭葉酸FA連接到AFn上賦予其腫瘤靶向能力。Gd-CDs/AFn(DOX)/FA在葉酸介導(dǎo)下特異性靶向腫瘤細(xì)胞以及腫瘤病變組織。實(shí)現(xiàn)體內(nèi)和體外的雙模式(熒光/核磁共振)成像及細(xì)胞追蹤,用于腫瘤的早期診斷及治療。采用X射線光電子能譜技術(shù)(XPS)對制備的Gd-CDs進(jìn)行元素分析,Gd元素軌道的變化表明Gd離子被摻進(jìn)去CDs中,通過透射電鏡觀察Gd-CDs的粒徑約6 nm,多分散系數(shù)PDI為0.201,Zeta電位測其電位約9.05 mV。Gd-CDs的正電性表明其表面修飾上氨基。通過掃描Gd-CDs光譜圖,制備的Gd-CDs具有明顯的上、下轉(zhuǎn)換發(fā)光性質(zhì)。通過對制備的Gd-CDs/AFn(DOX)/FA進(jìn)行物理化學(xué)性質(zhì)等表征,其平均粒徑為112±3.5 nm,電位約-23.7±2.1 mV。采用紫外可見分光光度計(jì)測定載藥系統(tǒng)中DOX的包封率,結(jié)果表明其包封率達(dá)86.6%。采用透析法分別測定了制劑在pH 5.0和pH 7.4時DOX的體外釋放,分析結(jié)果顯示通過調(diào)節(jié)pH值能較好的控制藥物釋放。在偏酸性環(huán)境pH 5.0時累計(jì)釋藥率為79.2%。Gd-CDs/AFn(DOX)/FA因含有釓離子具有T1成像能力,MRI體外實(shí)驗(yàn)表明Gd-CDs/AFn(DOX)/FA在T1磁場下,具有明顯的濃度依賴性,而且隨著濃度的增大呈現(xiàn)白化效應(yīng)。本研究選擇人源的乳腺癌細(xì)胞(MCF-7)作為其體外抗腫瘤活性評價的模型,考察了Gd-CDs/AFn(DOX)/FA細(xì)胞毒作用機(jī)制,包括細(xì)胞內(nèi)攝取實(shí)驗(yàn)、抑制率等。采用MTT法分別考察了Gd-CDs、Gd-CDs/AFn的細(xì)胞毒性,結(jié)果顯示,設(shè)定濃度為0~200μg/mL,Gd-CDs和Gd-CDs/AFn兩者均表現(xiàn)出低毒性,細(xì)胞存活率均為86%以上。且Gd-CDs鍵合到AFn表面后,相比Gd-CDs組Gd-CDs/AFn組的細(xì)胞存活率明顯增強(qiáng)。MCF-7細(xì)胞攝取實(shí)驗(yàn)中通過觀察Gd-CDs自身的綠色熒光,Gd-CDs能夠較好的標(biāo)記載藥系統(tǒng)的細(xì)胞攝取情況,與Gd-CDs/AFn(DOX)相比,Gd-CDs/AFn(DOX)/FA有較高的細(xì)胞攝取量,表明連有葉酸靶頭的去鐵蛋白給藥系統(tǒng)更有利于包裹藥物快速進(jìn)入腫瘤細(xì)胞。體外抗腫瘤活性實(shí)驗(yàn)表明:Gd-CDs/AFn(DOX)/FA可以有效的靶向MCF-7細(xì)胞,并高效的穿透細(xì)胞膜,攜帶更多的DOX進(jìn)入細(xì)胞內(nèi)部。去鐵蛋白籠在弱酸環(huán)境下籠漸漸離解打開,從而緩慢釋放出藥物DOX,從而誘導(dǎo)MCF-7細(xì)胞凋亡,最終抑制MCF-7細(xì)胞的生長和增殖。采用鼠源腹水瘤S180細(xì)胞株,建立KM雌性小白鼠荷瘤作為其體內(nèi)抗腫瘤活性評價的模型。體內(nèi)的抗腫瘤活性實(shí)驗(yàn)表明:Gd-CDs/AFn(DOX)/FA具有較低的系統(tǒng)毒性。在葉酸介導(dǎo)下,能將更多的Gd-CDs/AFn(DOX)/FA納米粒子滯留在腫瘤部位,在腫瘤組織偏酸性環(huán)境下,去鐵蛋白籠逐漸打開使藥物DOX緩慢從Gd-CDs/AFn(DOX)/FA釋放出來,從而大量的藥物更易于滯留在腫瘤部位。通過DOX嵌入DNA抑制核酸的合成從而誘導(dǎo)腫瘤細(xì)胞的凋亡,最終抑制腫瘤的生長。DOX在小鼠血漿的藥代動力學(xué)實(shí)驗(yàn)結(jié)果表明,納米復(fù)合物Gd-CDs/AFn(DOX)/FA能夠延長DOX在體內(nèi)的循環(huán)停留時間。具有葉酸靶向的Gd-CDs/AFn(DOX)/FA使藥物更多的聚集在腫瘤部位,進(jìn)一步證明構(gòu)建的納米給藥系統(tǒng)在體內(nèi)的靶向能力。此外,MRI體內(nèi)實(shí)驗(yàn)顯示注射Gd-CDs/AFn(DOX)/FA后,觀察到核磁成像中小鼠腫瘤部位T1場信號強(qiáng)度得到增強(qiáng),說明Gd-CDs/AFn(DOX)/FA可作為腫瘤核磁成像診斷的對比劑。
[Abstract]:Early diagnosis of cancer has attracted the attention of people, the molecular imaging detection is an important tool for early diagnosis of cancer, and provides more accurate and complete information for tumor diagnosis, so as to achieve the purpose of early diagnosis and early treatment. Molecular imaging techniques based on multi-function nanoparticles have made some progress in the sensitivity and specificity of tumor detection, and quantum dots, fluorescent dye molecules and magnetic nanoparticles have been used as imaging probes in vivo fluorescence imaging and magnetic resonance imaging. However, these probes are toxic and the aqueous solution has poor stability and poor sensitivity, which greatly limits its application in vivo. Therefore, to construct a multi-function targeting probe with simple system, bio-friendly and sensitive imaging is a problem to be solved in molecular imaging. On the basis of this, by microwave-assisted method, the chlorinated carbon (GdCl3) is doped into the inner surface of the carbon point CDs in the form of a fluorescent ion Gd3 +, and the PEI is subjected to the amination modification on the surface of the CDs, so that the doped carbon quantum dot Gd-CDs with high quantum yield is obtained, That is, the bright green light can be emitted under the irradiation of visible light, and has the advantages of high fluorescence intensity, good optical stability and good biocompatibility. And taking the ferritin nano-cage as a carrier, the unique cage-like structure characteristic of the ferritin cage AFn is that the cage is opened when the pH = 2 protein cage is resolved into a plurality of methylene structures, and the packaging drug adriamycin is realized by the self-assembly of the pH = 7 protein cage. Gd-CDs is used as a fluorescent and magnetic probe to couple the drug-carrying system AFn to the DOX. The target-head folic acid FA is connected to the AFn for the tumor targeting ability under the coordination of the EDC and the NHS catalyst. Gd-CDs/ AFn (DOX)/ FA specifically targeted tumor cells and tumor lesion tissues with folate-mediated. The dual-mode (fluorescence/ nuclear magnetic resonance) imaging and cell tracking in vivo and in vitro are realized for early diagnosis and treatment of tumors. The elements of Gd-CDs were analyzed by X-ray photoelectron spectroscopy (XPS). The change of Gd-element orbit showed that the Gd-ions were doped in the CDs. The particle size of Gd-CDs was observed by transmission electron microscope. The particle size of Gd-CDs was about 6 nm, and the multi-dispersion coefficient PDI was 0.201. The potential of Zeta potential is about 9.05 mV. The positive electrical property of Gd-CDs indicates that its surface is modified with amino group. The Gd-CDs prepared by scanning the Gd-CDs have obvious superior and lower conversion luminescence properties. The average particle size of the prepared Gd-CDs/ AFn (DOX)/ FA was 112-3.5 nm and the potential was about-23.7-2.1mV. The encapsulation rate of DOX in the drug-loaded system was determined by an ultraviolet-visible spectrophotometer. The results showed that the encapsulation rate was 86.6%. The in vitro release of DOX was determined by dialysis method at pH 5.0 and pH 7.4, and the results showed that the release of the drug was controlled by adjusting the pH value. The results showed that Gd-CDs/ AFn (DOX)/ FA had a significant concentration dependence under the T1 magnetic field and the whitening effect with the increase of the concentration. In this study, human-derived breast cancer cells (MCF-7) were selected as the model of their in vitro anti-tumor activity evaluation, and the mechanism of Gd-CDs/ AFn (DOX)/ FA (DOX)/ FA was investigated. The cytotoxicity of Gd-CDs and Gd-CDs/ AFn was studied by MTT method. The results showed that the concentration of Gd-CDs and Gd-CDs and Gd-CDs/ AFn showed low toxicity and the cell survival rate was over 86%. And the cell survival rate of the Gd-CDs/ AFn group in the Gd-CDs group was obviously enhanced after the Gd-CDs were bonded to the surface of the AFn. Compared with Gd-CDs/ AFn (DOX), the cell uptake of Gd-CDs/ AFn (DOX)/ FA was higher than that of Gd-CDs/ AFn (DOX). It is shown that the drug delivery system with the folic acid target head is more beneficial to the rapid entry of the packaged drugs into the tumor cells. In vitro anti-tumor activity experiments show that Gd-CDs/ AFn (DOX)/ FA can effectively target MCF-7 cells and penetrate cell membranes efficiently and carry more DOX into the cells. The cage gradually dissociates and opens in a weak acid environment, so that the drug DOX is slowly released, thereby inducing the apoptosis of the MCF-7 cell, and finally inhibiting the growth and the proliferation of the MCF-7 cell. The model of tumor-bearing activity of KM female mice was established by using mouse-derived ascites S180 cell line. The anti-tumor activity in vivo showed that Gd-CDs/ AFn (DOX)/ FA had lower systemic toxicity. In the presence of folic acid, more of the Gd-CDs/ AFn (DOX)/ FA nanoparticles can be retained in the tumor site. In the acidic environment of the tumor tissue, the de-ferritin cage is gradually opened to release the drug DOX slowly from the Gd-CDs/ AFn (DOX)/ FA, so that a large amount of the drug can be more easily retained in the tumor site. The DNA is embedded into the DNA to inhibit the synthesis of the nucleic acid, thereby inducing the apoptosis of the tumor cells, and finally inhibiting the growth of the tumor. The results of the pharmacokinetics of DOX in mouse plasma show that the nano-complex Gd-CDs/ AFn (DOX)/ FA can prolong the cyclic residence time of DOX in the body. The addition of folic acid-targeted Gd-CDs/ AFn (DOX)/ FA to the tumor site further demonstrates the targeted ability of the constructed nano-drug delivery system in vivo. In addition, after the injection of Gd-CDs/ AFn (DOX)/ FA in the MRI, the signal intensity of the T1 field in the tumor site of the mouse was enhanced and the Gd-CDs/ AFn (DOX)/ FA could be used as the contrast agent for the diagnosis of tumor nuclear magnetic resonance imaging.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：O657.3;R730.4
[Abstract]:Early diagnosis of cancer has attracted the attention of people, the molecular imaging detection is an important tool for early diagnosis of cancer, and provides more accurate and complete information for tumor diagnosis, so as to achieve the purpose of early diagnosis and early treatment. Molecular imaging techniques based on multi-function nanoparticles have made some progress in the sensitivity and specificity of tumor detection, and quantum dots, fluorescent dye molecules and magnetic nanoparticles have been used as imaging probes in vivo fluorescence imaging and magnetic resonance imaging. However, these probes are toxic and the aqueous solution has poor stability and poor sensitivity, which greatly limits its application in vivo. Therefore, to construct a multi-function targeting probe with simple system, bio-friendly and sensitive imaging is a problem to be solved in molecular imaging. On the basis of this, by microwave-assisted method, the chlorinated carbon (GdCl3) is doped into the inner surface of the carbon point CDs in the form of a fluorescent ion Gd3 +, and the PEI is subjected to the amination modification on the surface of the CDs, so that the doped carbon quantum dot Gd-CDs with high quantum yield is obtained, That is, the bright green light can be emitted under the irradiation of visible light, and has the advantages of high fluorescence intensity, good optical stability and good biocompatibility. And taking the ferritin nano-cage as a carrier, the unique cage-like structure characteristic of the ferritin cage AFn is that the cage is opened when the pH = 2 protein cage is resolved into a plurality of methylene structures, and the packaging drug adriamycin is realized by the self-assembly of the pH = 7 protein cage. Gd-CDs is used as a fluorescent and magnetic probe to couple the drug-carrying system AFn to the DOX. The target-head folic acid FA is connected to the AFn for the tumor targeting ability under the coordination of the EDC and the NHS catalyst. Gd-CDs/ AFn (DOX)/ FA specifically targeted tumor cells and tumor lesion tissues with folate-mediated. The dual-mode (fluorescence/ nuclear magnetic resonance) imaging and cell tracking in vivo and in vitro are realized for early diagnosis and treatment of tumors. The elements of Gd-CDs were analyzed by X-ray photoelectron spectroscopy (XPS). The change of Gd-element orbit showed that the Gd-ions were doped in the CDs. The particle size of Gd-CDs was observed by transmission electron microscope. The particle size of Gd-CDs was about 6 nm, and the multi-dispersion coefficient PDI was 0.201. The potential of Zeta potential is about 9.05 mV. The positive electrical property of Gd-CDs indicates that its surface is modified with amino group. The Gd-CDs prepared by scanning the Gd-CDs have obvious superior and lower conversion luminescence properties. The average particle size of the prepared Gd-CDs/ AFn (DOX)/ FA was 112-3.5 nm and the potential was about-23.7-2.1mV. The encapsulation rate of DOX in the drug-loaded system was determined by an ultraviolet-visible spectrophotometer. The results showed that the encapsulation rate was 86.6%. The in vitro release of DOX was determined by dialysis method at pH 5.0 and pH 7.4, and the results showed that the release of the drug was controlled by adjusting the pH value. The results showed that Gd-CDs/ AFn (DOX)/ FA had a significant concentration dependence under the T1 magnetic field and the whitening effect with the increase of the concentration. In this study, human-derived breast cancer cells (MCF-7) were selected as the model of their in vitro anti-tumor activity evaluation, and the mechanism of Gd-CDs/ AFn (DOX)/ FA (DOX)/ FA was investigated. The cytotoxicity of Gd-CDs and Gd-CDs/ AFn was studied by MTT method. The results showed that the concentration of Gd-CDs and Gd-CDs and Gd-CDs/ AFn showed low toxicity and the cell survival rate was over 86%. And the cell survival rate of the Gd-CDs/ AFn group in the Gd-CDs group was obviously enhanced after the Gd-CDs were bonded to the surface of the AFn. Compared with Gd-CDs/ AFn (DOX), the cell uptake of Gd-CDs/ AFn (DOX)/ FA was higher than that of Gd-CDs/ AFn (DOX). It is shown that the drug delivery system with the folic acid target head is more beneficial to the rapid entry of the packaged drugs into the tumor cells. In vitro anti-tumor activity experiments show that Gd-CDs/ AFn (DOX)/ FA can effectively target MCF-7 cells and penetrate cell membranes efficiently and carry more DOX into the cells. The cage gradually dissociates and opens in a weak acid environment, so that the drug DOX is slowly released, thereby inducing the apoptosis of the MCF-7 cell, and finally inhibiting the growth and the proliferation of the MCF-7 cell. The model of tumor-bearing activity of KM female mice was established by using mouse-derived ascites S180 cell line. The anti-tumor activity in vivo showed that Gd-CDs/ AFn (DOX)/ FA had lower systemic toxicity. In the presence of folic acid, more of the Gd-CDs/ AFn (DOX)/ FA nanoparticles can be retained in the tumor site. In the acidic environment of the tumor tissue, the de-ferritin cage is gradually opened to release the drug DOX slowly from the Gd-CDs/ AFn (DOX)/ FA, so that a large amount of the drug can be more easily retained in the tumor site. The DNA is embedded into the DNA to inhibit the synthesis of the nucleic acid, thereby inducing the apoptosis of the tumor cells, and finally inhibiting the growth of the tumor. The results of the pharmacokinetics of DOX in mouse plasma show that the nano-complex Gd-CDs/ AFn (DOX)/ FA can prolong the cyclic residence time of DOX in the body. The addition of folic acid-targeted Gd-CDs/ AFn (DOX)/ FA to the tumor site further demonstrates the targeted ability of the constructed nano-drug delivery system in vivo. In addition, after the injection of Gd-CDs/ AFn (DOX)/ FA in the MRI, the signal intensity of the T1 field in the tumor site of the mouse was enhanced and the Gd-CDs/ AFn (DOX)/ FA could be used as the contrast agent for the diagnosis of tumor nuclear magnetic resonance imaging.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：O657.3;R730.4
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