本文選題：熒光碳點(diǎn) + 光學(xué)影像探針��； 參考：《復(fù)旦大學(xué)》2014年博士論文

【摘要】：腫瘤是威脅人類生命和健康的重大疾病,尤其是腦腫瘤；導(dǎo)致腦腫瘤高死亡率的一個(gè)重要原因在于其診斷的滯后性。傳統(tǒng)的醫(yī)學(xué)影像雖然可以在解剖學(xué)基礎(chǔ)上發(fā)現(xiàn)腫瘤,但肉眼可以辨別的腫瘤往往已經(jīng)處于中、晚期,或者發(fā)生轉(zhuǎn)移,失去了最佳的治療時(shí)機(jī)。因此,腫瘤尤其是腦腫瘤的影像診斷具有極為重要的臨床意義。目前,常規(guī)的醫(yī)學(xué)影像在臨床上發(fā)揮著重要作用,但仍存在諸多不足。其中磁共振影像(MRI)的空間分辨率高,但靈敏度偏低；單光子發(fā)射計(jì)算機(jī)斷層成像(SPECT)和正電子發(fā)射計(jì)算機(jī)斷層掃描(PET)的靈敏度高,但空間分辨率較低。光學(xué)影像是一種新興的影像技術(shù),其在靈敏度和分辨率方面顯示出極大的優(yōu)勢(shì)和發(fā)展前景；尤其是基于納米技術(shù)的影像探針的應(yīng)用,使其在腫瘤光學(xué)影像,特別是早期腫瘤的影像診斷領(lǐng)域具有良好的發(fā)展前景和科學(xué)研究?jī)r(jià)值。近年,納米技術(shù)廣泛應(yīng)用于生物醫(yī)藥領(lǐng)域。熒光碳點(diǎn)(CDs)是繼量子點(diǎn)之后的又一種新型熒光納米材料。其具備諸多優(yōu)點(diǎn)：抗光漂白性強(qiáng)、無“光閃爍”現(xiàn)象,光穩(wěn)定性高；生物相容性好,毒性低；表面易于功能化修飾等。目前,國(guó)際上CDs作為光學(xué)影像探針的研究尚處于初期階段,主要在體外細(xì)胞的熒光影像方面有一些報(bào)道,而在體內(nèi)腫瘤光學(xué)影像中的應(yīng)用仍少有研究。關(guān)鍵問題主要是：①大多數(shù)CDs的發(fā)射光波長(zhǎng)處于400nm-500 nm的較短波長(zhǎng)可見光區(qū),對(duì)機(jī)體組織的穿透性差,限制其體內(nèi)深層組織的光學(xué)影像；同時(shí)還存在生物體自發(fā)熒光的干擾。因此,希望研制在紅光區(qū)或近紅外光區(qū)發(fā)光的CDs.②CDs在生物體內(nèi)的分布沒有選擇性,無法濃集于病灶如腫瘤部位,致使腫瘤組織與正常組織邊界的對(duì)比度弱,影像效果差。因此,希望設(shè)計(jì)具有腫瘤靶向性的CDs以提高其腫瘤信噪比。③CDs的粒徑尺度和表面性質(zhì)致使其體內(nèi)生物半衰期較短,進(jìn)一步削弱了影像效果。為了解決上述關(guān)鍵問題,本文擬針對(duì)腦腫瘤,首先通過選擇不同的碳源材料,逐步優(yōu)化CDs的光學(xué)性質(zhì),使之發(fā)射波長(zhǎng)趨于紅光區(qū)。其次在CDs表面進(jìn)行功能化的PEG修飾,以延長(zhǎng)生物半衰期。最后修飾兩類靶向分子：①腦毛細(xì)血管內(nèi)皮細(xì)胞和腫瘤細(xì)胞均高表達(dá)受體——低密度脂蛋白受體相關(guān)蛋白(LRP)的高親和配體Angiopep-2(ANG),通過主動(dòng)雙級(jí)腦靶向作用首先透過血腦屏障,進(jìn)而靶向進(jìn)入腦腫瘤組織和腫瘤細(xì)胞；或者②腫瘤新生血管和腫瘤細(xì)胞均高表達(dá)受體——整合素αvβ3的高親和配體c[RGDyK]環(huán)肽(RGD),通過主動(dòng)雙級(jí)腫瘤靶向作用首先透過腫瘤新生血管,進(jìn)而靶向進(jìn)入腫瘤細(xì)胞。從而增加CDs在腫瘤部位的富集和信噪比,提高腦腫瘤光學(xué)影像效果。本文的研究?jī)?nèi)容分為三章：第一章以蜘蛛絲為新型的有機(jī)碳源材料,采用水熱法制備了粒徑約5 nm的蜘蛛絲熒光碳點(diǎn)CDs-1,其最大發(fā)射峰波長(zhǎng)為447 nm。考察了碳點(diǎn)濃度和孵育時(shí)間對(duì)其細(xì)胞攝取的影響,結(jié)果表明CDs-1能夠被人源腦膠質(zhì)瘤U87細(xì)胞攝取,且呈濃度依賴性；觀察了在腦腫瘤細(xì)胞中的共定位情況,溶酶體和線粒體是其細(xì)胞內(nèi)運(yùn)行的可能途徑。CDs-1具有良好的生物相容性和較低的細(xì)胞毒性,有利其作為體外腫瘤細(xì)胞光學(xué)影像探針。CDs-1的缺點(diǎn)是發(fā)射波長(zhǎng)處于短波長(zhǎng)可見光區(qū),組織穿透性差、生物體本底熒光干擾大,尚不能用于體內(nèi)腫瘤的光學(xué)影像,有待進(jìn)一步提高和完善。第二章以甘氨酸為碳源材料,采用高溫?zé)峤夥ㄖ苽淞肆郊s5 nm的甘氨酸熒光碳點(diǎn)CDs-2,其最大發(fā)射峰波長(zhǎng)紅移至500 nm�？疾炝颂键c(diǎn)濃度和孵育時(shí)間對(duì)其細(xì)胞攝取的影響,結(jié)果表明鼠源腦膠質(zhì)瘤C6細(xì)胞攝取CDs-2具有顯著的時(shí)間和濃度依賴性；腦腫瘤細(xì)胞中的共定位結(jié)果表明,CDs-2細(xì)胞攝取后能夠迅速進(jìn)入溶酶體和線粒體,并且快速?gòu)募?xì)胞器中逸出。荷腦腫瘤裸鼠模型的體內(nèi)腫瘤光學(xué)影像觀察表明,CDs-2能夠迅速濃集于腦部,并且主要聚集于腦腫瘤接種部位。此外還證實(shí)了其良好的生物相容性和細(xì)胞低毒性。綜上所述,CDs-2能夠用于腫瘤的體外細(xì)胞影像,也具有一定的體內(nèi)腫瘤影像效果。但因其發(fā)射波長(zhǎng)仍處于較短波長(zhǎng)的可見光區(qū),致使體內(nèi)腫瘤影像信噪比不高,仍需進(jìn)一步提高和完善。第三章通過氨基酸和糖種類的選擇以及兩者配比的優(yōu)化,使熒光碳點(diǎn)的發(fā)射波長(zhǎng)進(jìn)一步紅移。優(yōu)化處方以谷氨酸和蔗糖按10：1復(fù)配為碳源材料,采用高溫?zé)峤夥ㄖ苽淞肆郊s5 nm的谷氨酸-蔗糖復(fù)合熒光碳點(diǎn)CDs-3,其有效發(fā)射波長(zhǎng)達(dá)600 nm。為了延長(zhǎng)生物半衰期,制備了PEG-CDs-3;并進(jìn)一步設(shè)計(jì)了兩種雙級(jí)腦腫瘤靶向的熒光碳點(diǎn)：ANG-PEG-CDs-3和RGD-PEG-CDs-3�？疾焯键c(diǎn)濃度和孵育時(shí)間對(duì)其細(xì)胞攝取的影響,結(jié)果顯示修飾后的CDs-3被腫瘤細(xì)胞攝取具有顯著的時(shí)間和濃度依賴性,且ANG或RGD修飾有利于其細(xì)胞攝取,提高了腫瘤細(xì)胞靶向性；其在腫瘤細(xì)胞中的共定位結(jié)果表明,修飾后的CDs-3通過溶酶體途徑進(jìn)入細(xì)胞。碳點(diǎn)的血漿蛋白穩(wěn)定性實(shí)驗(yàn)結(jié)果表明PEG修飾可以明顯降低血漿蛋白的吸附,有助于延長(zhǎng)其體內(nèi)長(zhǎng)循環(huán)。血液相容性和細(xì)胞毒性評(píng)價(jià)結(jié)果表明,ANG-PEG-CDs-3和RGD-PEG-CDs-3具有良好的生物相容性和低細(xì)胞毒性,適合于體內(nèi)影像應(yīng)用。進(jìn)一步評(píng)價(jià)了ANG-PEG-CDs-3、RGD-PEG-CDs-3和PEG-CDs-3在荷腦膠質(zhì)瘤裸鼠模型和荷乳腺癌皮下瘤裸鼠模型中的組織分布及腫瘤主動(dòng)靶向性,評(píng)估了各碳點(diǎn)的體內(nèi)腫瘤光學(xué)影像效果。結(jié)果表明,ANG-PEG-CDs-3能夠用于體內(nèi)腦腫瘤的光學(xué)影像,效果優(yōu)于RGD-PEG-CDs-3; RGD-PEG-CDs-3能夠用于乳腺癌皮下瘤的光學(xué)影像,效果優(yōu)于其腦腫瘤影像。組織器官和細(xì)胞水平評(píng)價(jià)結(jié)果進(jìn)一步驗(yàn)證了碳點(diǎn)經(jīng)ANG或RGD修飾有利于提高其腫瘤主動(dòng)靶向性,增加碳點(diǎn)在腫瘤部位的濃集和信噪比,提高腦腫瘤和皮下瘤的光學(xué)影像效果。綜上所述,本文創(chuàng)新性地通過復(fù)合碳源材料優(yōu)選及多種策略的協(xié)同設(shè)計(jì),研制了兩種腦腫瘤靶向的熒光碳點(diǎn)ANG-PEG-CDs和RGD-PEG-CDs-3,增加其在腫瘤部位的富集和信噪比,提高腦腫瘤和皮下瘤的光學(xué)影像效果,有望使該領(lǐng)域的研究由體外拓展至體內(nèi)；同時(shí)也為腫瘤的光學(xué)影像,尤其是早期影像診斷提供有價(jià)值的影像探針和研究資料。
[Abstract]:Cancer is a major disease that threatens human life and health, especially brain tumors. One of the important reasons for the high mortality of brain tumors is the hysteresis of the diagnosis. Although traditional medical images can find tumors on the basis of anatomy, the tumor that can be identified by the naked eye is already in the middle, late, or metastasize. Therefore, the imaging diagnosis of tumor, especially the brain tumor, has a very important clinical significance. At present, the conventional medical imaging plays an important role in clinical, but there are still many shortcomings. The spatial resolution of magnetic resonance imaging (MRI) is high, but the sensitivity is low; the single photon emission computer fault is formed. SPECT and positron emission computed tomography (PET) have high sensitivity, but low spatial resolution. Optical imaging is a new imaging technique, which shows great advantages and prospects in sensitivity and resolution, especially the application of nanotechnology based imaging probes to make it special in tumor optical images. In recent years, nanotechnology has been widely used in the field of biomedicine. CDs is another new kind of nanomaterial after quantum dots. It has many advantages: strong photobleaching, no "light scintillation", and high photostability. The biocompatibility is good, the toxicity is low, and the surface is easy to function modification. At present, the research of CDs as an optical image probe is still in the early stage. There are some reports on the fluorescence image of the cells in vitro, but the application of the optical imaging in the body is still less. The key issues are: (1) most of the CDs hair. The light wavelength in the shorter wavelength of the 400nm-500 nm is visible, the penetration of the body is poor and the optical images of the deep tissue in the body are limited. At the same time, there are also the interference of the spontaneous fluorescence of the organism. Therefore, the distribution of CDs. CDs in the red and near infrared light regions is not selective and cannot be concentrated. The contrast between the tumor tissue and the normal tissue boundary is weak and the image effect is poor. Therefore, it is hoped that the tumor targeting CDs can be designed to improve the tumor signal-to-noise ratio. (3) the size and surface properties of CDs make the biological half-life in the body shorter, and further weaken the image effect. The key problem is to optimize the optical properties of CDs by selecting different carbon source materials to make the emission wavelengths tend to be red area. Secondly, the functional PEG modification on the surface of CDs is used to prolong the biological half-life. Finally, two kinds of target molecules are modified: the high surface of the brain capillary endothelial cells and tumor cells The high affinity ligand Angiopep-2 (ANG) of the receptor, low density lipoprotein receptor related protein (LRP), first penetrates the blood brain barrier through the active two-stage brain targeting, and then targets the brain tumor tissue and tumor cells, or the high affinity ligand C of the neovascularization and tumor cells of the tumor, the high affinity ligand C of integrin alpha v beta 3 [RGDyK] cyclic peptide (RGD), through the targeted action of the active two-stage tumor, first penetrates the neovascularization of the tumor and then targets the tumor cells, thus increasing the enrichment and signal to noise ratio of CDs at the tumor site, and improving the optical image effect of the brain tumor. The research contents of this paper are divided into three chapters: the first chapter is a new type of organic carbon source material with spider silk, which is used as a new type of organic carbon source. The fluorescence carbon point CDs-1 of the spider silk with a particle size of about 5 nm was prepared by hydrothermal method. The maximum emission wavelength was 447 nm., and the effects of carbon point concentration and incubation time on the cell uptake were investigated. The results showed that CDs-1 was able to be absorbed by human glioma U87 cells and was concentration dependent. The co localization of the brain tumor cells and the lysase were observed. Body and mitochondria are the possible pathways of its intracellular operation..CDs-1 has good biocompatibility and low cytotoxicity. The disadvantage of.CDs-1 as an optical image probe for tumor cells in vitro is that the emission wavelength is in the short wavelength visible light area, the tissue penetration is poor, the biological background fluorescence interference is large, and it is not yet used in the body tumor. The second chapter uses glycine as carbon source material in the second chapter to prepare the glycine fluorescent carbon point CDs-2 with a particle size of about 5 nm by high temperature pyrolysis. The maximum emission peak wavelength is red to 500 nm., and the effect of carbon point concentration and incubation time on the cell uptake is investigated. The results show that rat brain glioma C6 cells The uptake of CDs-2 has a significant time and concentration dependence; the co localization results in brain tumor cells indicate that CDs-2 cells can quickly enter lysosomes and mitochondria and escape from the organelles quickly. The tumor optical image of the nude mice model of the brain tumor bearing tumor of the brain of the nude mice shows that CDs-2 can quickly concentrate in the brain. In addition to the good biocompatibility and low cytotoxicity, CDs-2 can be applied to the cell image of the tumor in vitro and the tumor imaging effect in vivo. However, the signal to noise ratio of the tumor image in the body is not high because its emission wavelength is still in the short wavelength visible light region. The third chapter, through the selection of amino acids and sugars and the optimization of their ratio, further redshift the emission wavelengths of the fluorescent carbon points. The optimized formulation was made of glutamic acid and sucrose as carbon source materials according to 10:1, and a high temperature pyrolysis method was used to prepare the glutamate sucrose compound fluorescent carbon point CDs-3 with a particle size of about 5 nm. The effective emission wavelength was 600 nm. in order to prolong the biological half-life, and PEG-CDs-3 was prepared, and the target fluorescence carbon points of two kinds of two-stage brain tumors were further designed. ANG-PEG-CDs-3 and RGD-PEG-CDs-3. were used to investigate the effects of carbon point concentration and incubation time on their cell uptake. The results showed that the modified CDs-3 was significantly absorbed by the tumor cells. The time and concentration dependence, and the ANG or RGD modification was beneficial to the cell uptake and enhanced the targeting of the tumor cells. The co localization results in the tumor cells showed that the modified CDs-3 entered the cells through the lysosome pathway. The experimental results of plasma protein stability in the carbon point showed that the PEG modification could significantly reduce the adsorption of plasma protein. The results of blood compatibility and cytotoxicity show that ANG-PEG-CDs-3 and RGD-PEG-CDs-3 have good biocompatibility and low cytotoxicity and are suitable for the application of imaging in vivo. Further evaluation of ANG-PEG-CDs-3, RGD-PEG-CDs-3 and PEG-CDs-3 in the nude mice model of glioma and the subcutaneous of breast cancer The tissue distribution and active targeting of tumor in nude mice model were used to evaluate the optical image effect of the tumor in vivo. The results showed that ANG-PEG-CDs-3 could be used in the optical image of brain tumor in vivo, and the effect was better than that of RGD-PEG-CDs-3; RGD-PEG-CDs-3 could be used in the optical image of the subcutaneous tumor of breast cancer, and the effect was better than that of the brain tumor image. The evaluation results of the weave and cell level further verify that the ANG or RGD modification can improve the active targeting of the tumor, increase the concentration and signal-to-noise ratio of the carbon point at the tumor site, and improve the optical image effect of the brain tumor and subcutaneous tumor. The fluorescent carbon points ANG-PEG-CDs and RGD-PEG-CDs-3 targeted by two kinds of brain tumors are designed and developed. The enrichment and signal to noise ratio of the tumor sites are increased and the optical image effects of brain tumors and subcutaneous tumors are improved. It is expected that the research in this field will be extended from in vitro to the body, and it is also provided for the optical image of the swelling tumor, especially the early imaging diagnosis. Valuable imaging probes and research materials.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943

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