【作者】 张哲；

【导师】 郑杰；

【作者基本信息】 吉林大学 ， 物理电子学， 2019， 博士

【摘要】 活体生物成像技术能够提供关键性的生理和病理信息,被广泛应用于生命科学研究和临床医疗中。借由活体生物成像技术,可以无创的获取病人体内的各种的结构与功能信息,实现对疾病的诊断、病情的监控甚至是外科手术的引导,极大的促进了医疗水平的发展。近年来,荧光成像技术的发展为活体成像领域注入了新的活力。相较于传统的成像方法,荧光成像可以对图像进行实时的宽视域采集,大大提高了时间分辨率。无害的光辐射激发和光辐射信号,大大降低了对成像目标的损害。除此之外,众多种类的荧光探针也可以实现多样化的功能性成像。光子在生物组织中较弱的穿透能力是制约活体荧光成像发展的关键因素之一。相关的研究表明,生物组织对于光子的散射和吸收,包括生物组织本身的自体荧光,都与光波长有极大的相关性。光的波长在700 nm–1700 nm的近红外区域时,各种组织均表现出极低的散射系数,生物组织对于光子的吸收能力和自体荧光的荧光强度也都大大降低。其中,700 nm-900 nm被称为近红外一区（Near-infrared I,NIR-I）,1000 nm-1700 nm被称为近红外二区（Near-infrared II,NIR-II）,这两个波长范围也被称为生物组织光学窗口。虽然采用近红外区区域内的荧光信号可以实现更深的成像深度和更高的荧光成像分辨率,但是大多数荧光探针激发波长和荧光波长均在可见光区内,现有的近红外荧光探针的荧光量子产率也普遍较低。为了活体荧光成像的进一步发展,开发出高荧光量子产率的近红外荧光探针就显得尤为关键。荧光聚合物纳米粒子由于其光学吸收截面大,荧光亮度高,光稳定性好,生物相容性佳,易于功能化等特点,在细胞荧光成像、荧光传感检测、活体荧光成像等方向表现出广阔的发展前景。聚合物纳米粒子的一个显著的优点是可以通过设计不同的分子结构,对其光学性能进行调控。本文就从荧光共轭聚合物的设计出发,有针对性的设计并合成适用于制备荧光纳米粒子探针的近红外荧光聚合物,并应用于生物成像,主要的研究内容概括如下:1、通过采用给受体结构和强给受体单元,成功合成了三种具有较低能隙的荧光共轭聚合物。聚合物显示出很强的吸光能力(560 nm处的摩尔吸光系数为3×10⁷ L mol^-1cm^-1),较长的激发光与荧光波长。利用光谱交叠图进行筛选,我们选择了小分子荧光染料NIR775与聚合物一起制备掺杂纳米粒子。纳米粒子的光学性能在掺杂后得到了极大的提升:首先,保留了聚合物强大的吸光能力和激发波长,可用676 nm激发光激发纳米粒子;其次,荧光发生了显著窄化,半峰宽从¹40 nm窄化至²0 nm,最大荧光峰为780 nm;最后,荧光量子产率由未2%（掺杂前）大幅提升至21%（掺杂后）。流式细胞术显示纳米粒子标记的MCF-7细胞具有很强的荧光信号。通过将标记的MCF-7细胞注入小鼠尾静脉,可以模拟血液中癌细胞的转移过程。利用活体荧光成像,我们监测了到了癌细胞在肺部富集的过程,并实现了在深层器官中对癌细胞的长期追踪成像。2、聚合物在纳米粒子中处于聚集态,由于大部分荧光聚合物具有聚集荧光猝灭现象,在制备成纳米粒子后,荧光量子产率会出现明显的降低。针对这一点,我们通过引入聚集诱导发光荧光团吩噻嗪,在保证近红外荧光（⁷00 nm）的同时,赋予了聚合物聚集诱导发光的性质。聚合物在四氢呋喃溶液中的荧光量子产率为9%,而在制备成纳米粒子后,由于聚集诱导发光性质带来的荧光增强,荧光量子产率不但没有降低反而提高了2倍以上,达到了23%。以PS-PEG进行包覆以后,荧光纳米粒子探针通过EPR效应在肿瘤区域富集,肿瘤区域显示出很高的荧光强度,最高信噪比达到了3.2。3、进一步的,我们设计并合成了8种近红外一区和近红外二区的聚集诱导发光聚合物。在对受体单元进行调节后,这些聚合物的荧光覆盖了600 nm-1400nm的波长范围。此外,我们通过引入具有较大空间位阻的侧基,减弱了聚合物在制备成纳米粒子后链内或链间的相互作用,在聚集诱导发光的基础上,进一步增强纳米粒子的荧光。其中,荧光峰位在1100 nm左右的聚合物在双重的荧光增强下,荧光量子产率从0.08%提升至1.5%。荧光量子产率的大幅度提升证明了这种双重荧光增强策略的有效性。使用具有NIR-II荧光的纳米粒子对小鼠血管进行标记,活体荧光成像清楚的检测出直径只有0.11 mm的脑部血管,显示出非常高的成像分辨率。更多还原

【Abstract】 In vivo bio-imaging technology can provide critical physiological and pathological information and was widely used in biomedical research and clinical medicine.Through in vivo bio-imaging technology,various structural and functional information of patients can be non-invasively obtained,which is helpful for realizing the diagnosis and monitoring of diseases,even the guidance of surgery.In recent years,the development of fluorescence imaging technology has injected new vitality into the field of in vivo bio-imaging.Compared with the traditional imaging method,fluorescence imaging can capture images in a real-time and wide field of view,greatly improving the time resolution.Harmless optical radiation excitation and signal response greatly reduce damage to imaging targets.In addition,a wide variety of fluorescent probes enable a variety of functional imaging.The poor photon penetration depth in biological tissues is one of the key factors that restrict the development of in vivo fluorescence imaging.Related studies have shown that the scattering and absorption of photons by biological tissues,including the autofluorescence,is highly correlated with the wavelength of the photon.In the broad 700 nm–1700 nm NIR window,tissues exhibit extremely low scattering coefficients,absorption of photons and autofluorescence.Particularly,700 nm-900 nm is called Near-infrared I（NIR-I）window,and 1000 nm-1700 nm is called Near-infrared II（NIR-II）window.However,most organic fluorophores emit in the visible region,and current near-infrared fluorescent probes also suffer from low fluorescence quantum yield.Promising applications inspire the researchers to pursue new NIR fluorophores.Fluorescent conjugated polymer nanoparticles have attracted considerable attention in fluorescence imaging because of their tunable optical properties and superior biocompatibility.A significant advantage of polymer nanoparticles is their tunable optical properties.By designing the structure of the fluorescent conjugated polymer,adjustment of the excitation wavelength,the fluorescence wavelength,and the fluorescence quantum yield can be achieved.In this paper,we synthesized various fluorescent conjugated polymers which are suitable for in vivo biological imaging.The main research results are summarized as follows:1.Three fluorescent conjugated polymers with lower energy gaps were successfully synthesized by employ strong donor and acceptor structure.The polymers possessed a strong light absorption capacity(molecular absorption coefficient at 560 nm is 3×107 L mol^-1 cm^-1),deep red excitation and NIR fluorescence.Fluorescent dye NIR775 was selected to dope with polymers according to the spectral overlap.The optical properties of the nanoparticles are greatly improved after doping:firstly,the strong absorption capacity and deep red excitation（676 nm）are retained;secondly,the FWHM peak is narrowed from¹40 nm to²0 nm with an emission peak of 780 nm;and last,the fluorescence quantum yield is significantly improved from 2%to 21%.After labeled by the nanoparticles,MCF-7 cells show strong fluorescent signals in flow cytometry.Labeled MCF-7 cells were injected into the mice to simulate the cancer cells metastasis in blood.And the enrichment of cancer cells in the lungs was clearly detected by in vivo fluorescence imaging,which achieves long-term cell tracking in deep organs.2.In the nanoparticles,the polymer is normally in a state of aggregation.Due to the aggregation-caused quenching（ACQ）effects,the fluorescence quantum yield of the polymer would significantly reduce after preparing into nanoparticles.Accordingly,we employed the aggregation-induced emission（AIE）fluorophore phenothiazine to endow the AIE character to the polymer.The fluorescence quantum yield of polymer in tetrahydrofuran is around 9%,but substantially increase to 23%after preparing into nanoparticles.After coating with PS-PEG,the nanoparticles achieved targeted fluorescence imaging of the tumor by EPR effect,the tumor area demonstrated a high fluorescence signal with the signal-to-noise ratio of 3.2.3.Furthermore,we designed and synthesized 8 kinds of near-infrared AIE polymers.The fluorescence of these polymers covers from 600 nm to 1400 nm.In addition,the inter-and intra-chain interactions were successfully weakened by the introduction of steric hindrance side groups.As a result,nanoparticles fluorescence intensity was further enhanced based on AIE.Among these polymers,the fluorescence quantum yield of the NIR-II polymer（fluorescence peak at around 1100 nm）increased from 0.08%to 1.5%under such double fluorescence enhancement.This large increase demonstrates the effectiveness of such dual enhancement strategy.In addition,using NIR-II fluorescence polymer nanoparticle,the cerebrovascular structures were clearly visualized with an outstanding clarity to 0.11 mm by FWHM.Such excellent performance revels the potential of fluorescence dual-enhancement with AIE and anti-ACQ effect,which is a promising approach to achieve more superior NIR-II fluorophores.更多还原