基于功能化纳米材料和Aptamer的细胞、活体层面肿瘤成像研究
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摘要
目前，恶性肿瘤发病率呈现逐年上升趋势，且死亡率居高不下，严重威胁人类健康和生命。探索肿瘤早期诊断及预后监测新方法，特别是通过细胞和活体水平上的非侵入式、原位、实时成像与表征揭示其发生发展过程中的规律和机制，对于患者治愈率和生存率的提高具有十分重要的意义。近年来，纳米技术和分子工程技术等的快速发展及其与生命科学的不断交叉融合，为开发新型肿瘤成像与表征方法提供了契机。一方面，多种多样的功能化纳米材料由于具有独特的信号发生和增强效应，为实现在克服背景干扰的前提下生物标记“靶子”的放大提供了可能；另一方面，逐渐兴起和不断丰富的肿瘤特异性Aptamer（核酸适配体）探针则由于具有传统生物抗体所不具备的诸多优越性能，为满足临床应用体系的选择性需求提供了一类理想的靶向识别分子。基于此，本论文即瞄准新型肿瘤成像与表征技术发展所面临的信号放大、靶向识别、信号激活和多功能化等问题与挑战，利用功能化纳米材料和Aptamer的优势，设计和构建了一系列新型探针，并从亚细胞、细胞水平到活体层面系统开展了灵敏、特异、原位、实时的肿瘤荧光成像与表征研究。具体包括以下工作：
一、基于二氧化硅荧光纳米颗粒新型标记物的肿瘤细胞溶酶体定位与示踪成像研究
为克服传统染料标记物所存在的信号强度低、易光漂白、标记寿命短等缺陷，满足细胞水平生命活动的实时、原位、动态和长时间成像与表征要求，利用二氧化硅荧光纳米颗粒（DSiNPs）的优越性能，在系统开展不同表面电荷DSiNPs在肿瘤细胞内的被动靶向定位与成像研究基础上，发展了一种新型肿瘤细胞溶酶体表征与示踪方法。首先采用反向微乳液法以四甲基罗丹明（TAMRA）为核，成功制备了粒径相当而表面电荷完全相反的两种DSiNPs。通过颗粒的荧光信号同步指示作用，结合细胞器常规标记物进行荧光共定位成像研究结果表明，表面带正电荷的TAMRA-NH_2-DSiNPs由于缓冲容量较大，在进入溶酶体后呈现出部分逃逸和被内质网捕获的特点；而表面带负电荷的TAMRA-DSiNPs则可长时间滞留于溶酶体中。随后以Hela宫颈癌细胞为模型，经条件优化后发现，TAMRA-DSiNPs能特异性定位于溶酶体内，且标记性能不受核材料限制，有望用于亚细胞结构的多色荧光成像研究。尤其与常规标记物（包括大分子Alexa488-dextran和小分子LysoTracker Green）相比，其不仅具有光稳定性强、循环寿命长且生物相容性好等一系列优点，而且还显示出优越的对细胞固定化和透化处理过程的耐受性能。最后，TAMRA-DSiNPs即被成功用于经氯喹处理的Hela细胞内溶酶体示踪以及多种不同细胞系的溶酶体表征研究，为其进一步在肿瘤发生发展过程中溶酶体相关生命活动的成像与表征中的应用提供了有力支持。
二、基于Annexin V功能化二氧化硅荧光纳米颗粒新型标记方法的早期凋亡肿瘤细胞成像研究
利用DSiNPs所具备的荧光信号强、光稳定性好、细胞毒性低等优点，结合“配体-受体”相互作用，基于Annexin V功能化DSiNPs与凋亡早期细胞膜上外翻磷脂酰丝氨酸（PS）的特异性结合，发展了一种灵敏、特异、稳定而简便的新型早期凋亡肿瘤细胞成像与表征方法。首先采用反向微乳液法以异硫氰酸罗丹明B（RBITC）为核，成功制备了包裹RBITC的二氧化硅纳米颗粒（RBITC-DSiNPs）。经TEM表征显示，该颗粒粒径为50±5nm、分散性好。通过进一步在其表面共价交联Annexin V，成功构建了一种主动靶向型早期凋亡肿瘤细胞识别与标记探针。结合激光共聚焦荧光成像考察发现，该探针不仅可有效区分紫杉醇诱导的早期凋亡MCF-7乳腺癌细胞和未经处理的MCF-7细胞，还能实现经药物诱导不同时间的早期凋亡细胞表面PS外翻程度的原位表征与示踪。尤其与Cy3染料标记方法相比，该探针显示出更为优越的光稳定性，在长达20分钟的激光连续照射后，仍能保持清晰而明亮的荧光标记信号，有望在肿瘤细胞凋亡相关研究以及抗癌药物的筛选等方面发挥重要作用。
三、基于cell-SELEX技术筛选的Aptamer探针的肿瘤活体荧光成像研究
为克服传统生物抗体的局限并开发新型肿瘤活体成像分子探针，采用基于cell-SELEX技术筛选的肿瘤细胞特异性Aptamer，通过直接修饰近红外荧光染料Cy5构建了一系列肿瘤靶向识别与标记探针，并系统开展了包括血液瘤和实体瘤在内的多种肿瘤的活体荧光成像研究。首先以Ramos淋巴瘤及其Aptamer TD05为模型，经流式细胞分析证实，Cy5-TD05在小鼠血清中能较好地保持对体外培养和原代培养Ramos细胞的结合性能。活体荧光成像结果也表明，其不仅具有对小鼠体内Ramos肿瘤的靶向成像能力，而且还显示出优越的序列依赖性和肿瘤特异性。在此基础上，为进一步拓展Aptamer在肺癌、肝癌等其他类型肿瘤荧光成像中的应用，利用其相应Cy5标记Aptamer探针成功实现了同一只小鼠体内不同类型肿瘤的选择性成像效果，为Aptamer作为一类新型肿瘤靶向识别分子今后在活体成像中的应用奠定了基础。
四、基于锁核酸修饰Aptamer探针的血液稳定性改善与肿瘤活体荧光成像研究
为解决Aptamer在用于复杂生物体系时仍然存在的稳定性不足、肿瘤部位滞留时间短、成像时间窗口窄等问题，以TD05为模型，采用锁核酸（LNA）和反转T碱基（3’-3’-T）修饰方法，成功构建了一种同时具有靶肿瘤细胞识别能力和血液稳定性的肿瘤成像探针。通过考察一系列不同取代组合、位置和数量的修饰策略对TD05抗酶切性能、亲和力和特异性的影响发现，LNA和3’-3’-T的共同修饰对其血清稳定性具有明显的协同改善效果，且一定程度上LNA取代数越多则对Aptamer半衰期的延长效果越明显。尤其经7对LNA取代和3’-3’-T修饰的TD05.6探针在37度血清的生理条件下显示出长达5-6小时的识别能力半衰期，提高至修饰前的10倍以上，并成功将活体肿瘤成像时间窗口从修饰前的105分钟延长到10小时以上。这一探针修饰策略将有望发展成为一种通用的方法为肿瘤活体成像研究提供更多具有临床实用价值的Aptamer探针。
五、基于细胞膜表面蛋白触发构型变化的发夹型激活式Aptamer探针的肿瘤活体荧光成像研究
为进一步解决上述染料直接标记“always on”Aptamer探针存在的诊断时间长、成像对比度不高、灵敏度有限等不足，以CCRF-CEM白血病细胞的特异性Aptamer Sgc8c为模型，巧妙地设计了一种基于细胞膜表面蛋白触发构型变化的发夹型激活式Aptamer探针。该探针在游离状态下主要为发夹构型，当没有目标物存在时，探针本体两端的荧光分子和淬灭分子相互靠近而导致荧光熄灭；而在与靶细胞作用后，该探针可有效发生构型重组而导致荧光激活，从而指示靶肿瘤细胞的存在。经流式细胞分析证实，该探针具有对靶肿瘤细胞的高特异性信号激活性能，尤其与“always on”探针相比，可显著提高分析灵敏度，有效检测低至118个CCRF-CEM细胞数的样品。而在应用于肿瘤活体成像时，该探针则不仅可明显降低来自非靶组织中未结合探针的信号干扰从而使肿瘤成像对比度得到明显增强，并将诊断时间从“always on”模式的数小时缩短至15分钟；而且还具有优越的序列特异性和肿瘤靶向性。鉴于Aptamer筛选技术对靶标范围的不断拓展，这一探针设计将有望作为一种通用的方法用于发展高灵敏、高特异性的肿瘤活体成像探针。
六、基于细胞膜表面蛋白触发构型变化的裂开型激活式Aptamer探针的肿瘤细胞和活体成像与检测研究
以Sgc8c为模型，通过在适当位点将其分裂为两条核酸片段，成功构建了一种基于细胞膜表面蛋白触发构型变化的裂开型激活式Aptamer探针。该探针在没有靶标存在时，两条游离核酸片段之间不会发生明显的相互作用；而当体系中引入靶肿瘤细胞后，细胞膜表面的靶蛋白会诱导其形成与完整Sgc8c类似的识别构型从而与靶细胞结合。经流式细胞分析发现，该探针对靶细胞的特异性亲和力具有明显的温度敏感性。利用低温下该探针的靶向识别性能，结合富G序列对银纳米簇的邻近荧光增强效应，发展了一种简单、方便、免洗、灵敏而特异的CCRF-CEM细胞成像与检测技术。利用温度从冰上逐渐上升至37度时该探针与靶细胞的结合能力逐渐下降的特点，选取探针修饰的96孔板作为捕获容器，建立了一种肿瘤细胞选择性捕获与温控释放方法；并证实该方法不仅具有良好的细胞亲和性，而且可有效用于靶细胞的循环捕获与释放以及混合体系中不同肿瘤细胞的分离和回收。最后，通过进一步修饰PEG linker将该探针的两条游离核酸片段相连，使分子间作用转换为分子内作用，成功克服了其在生理条件下容易失活的难题，并结合Cy3-Cy5供受体对的FRET效应初步展示了一定的肿瘤细胞检测与成像可行性。
七、基于肿瘤酸性微环境刺激响应的pH激活式Aptamer探针的肿瘤细胞和活体荧光成像研究
针对肿瘤组织的弱酸性特点以及肿瘤细胞溶酶体内的酸性环境（pH4-6），同时结合Aptamer的靶向识别性能，以A549肺癌细胞的特异性Aptamer S6为模型，利用可在低pH条件下水解的ATU（3,9-双(3-氨丙基)-2,4,8,10-四氧杂螺[5.5]十一烷）小分子将Cy5标记Aptamer与荧光淬灭分子BHQ3相连，成功构建了一种同时具备检测特异性和“signal on”信号模式的pH激活式Aptamer探针。该探针在中性环境中的荧光淬灭效率高达约98%，而当置于pH4.5的缓冲液中24小时即可发生几乎100%信号恢复。通过与发夹型激活式Aptamer探针比较发现，该探针无论在小鼠血清中还是裸鼠体内，均显示出优越的荧光稳定性。激光共聚焦显微成像结果则表明，该探针不仅具备在活细胞溶酶体内的定点酸性激活性能，而且与“always on”探针相比，具有成像特异性高、对比度高、简单、免洗等一系列优点。进一步的活体肿瘤荧光成像结果也证实，该探针完全具备在裸鼠体内肿瘤组织中发生信号激活的成像性能，且该激活行为具有高度的序列特异性和肿瘤靶向性，有望发展成为一种通用探针设计平台用于肿瘤等酸性疾病区域的表征和成像研究。
八、基于Aptamer-碳纳米管自组装激活式探针的肿瘤活体荧光成像研究
结合功能化纳米材料和Aptamer分别作为信号转换器件和靶向识别分子的优势，以Sgc8c为模型，利用单壁碳纳米管（SWNTs）对荧光标记DNA的强吸附性能和高效淬灭作用，成功构建了一种基于Aptamer-碳纳米管自组装功能体的激活式肿瘤成像探针。当体系中不存在靶标时，Cy5-Sgc8c牢固附着于SWNTs表面，Cy5荧光被淬灭；而在加入靶肿瘤细胞后，由于Aptamer与细胞表面的靶蛋白结合使Cy5离开SWNTs表面，荧光得以恢复。通过流式细胞术、活体荧光成像技术等考察发现，无论是在缓冲液体系中还是裸鼠移植瘤模型内，CCRF-CEM细胞均可有效激活该探针的荧光信号，且激活性能具有高度的序列特异性和肿瘤靶向性。尤其通过与“always on”探针对比证实，SWNTs可有效降低非靶体系中未结合探针的非特异性信号背景干扰，从而明显改善靶肿瘤细胞的体外分析信倍比和活体成像对比度。这一探针设计简单方便，有望发展成为一种低背景、高对比、高特异且具有普遍适用性的肿瘤成像方法。
九、基于激活式Aptamer探针功能化Au@Au/Ag纳米颗粒的肿瘤活体荧光成像及其引导下的近红外光热治疗研究
在上述基于激活式Aptamer探针（AAP）的肿瘤活体成像研究基础上，结合功能化纳米材料的肿瘤杀伤性能，初步探讨了基于Aptamer-纳米材料功能组装体的“诊疗一体化”探针构建与应用研究。首先以金纳米棒（Au NRs）为模板，采用先包银后刻蚀金的方法成功合成了Au@Au/Ag球形纳米颗粒（Au@Au/AgNPs）。该颗粒在400-1100nm范围内显示出强烈的光吸收性能，且在980nm光激发下具有较Au NRs高约4.5倍的产热效率。随后，利用该颗粒同时作为光热转换元件和荧光淬灭元件，并以S6为模型设计竞争型AAP序列，通过“Au-S”作用下颗粒与DNA的自组装行为，成功构建了基于AAP功能化Au@Au/Ag NPs的“诊疗一体化”多功能探针。经过一系列体内外考察证实，该探针不仅具有对靶肿瘤细胞的选择性光热治疗效果，而且可有效实现对体内外靶肿瘤细胞的激活式成像与检测。进一步以尾静脉注射方式将该探针导入植有不同肿瘤的裸鼠体内，成功实现了对A549阳性肿瘤的特异性激活式成像及其引导下的近红外光热治疗。鉴于Aptamer的靶标种类丰富而Au@Au/Ag NPs的吸收光谱宽且强，该探针设计将有望发展成为一种具有多目标同时成像与治疗潜能的技术平台。
Due to its rising incidence and high mortality, the malignant tumor is a seriousthreat to human health and life. To explore novel diagnosis and prognostic monitoringmethods for early-stage tumors, especially to reveal the laws and mechanisms intumor occurrence and development through non-invasive, in-situ and real-timeimaging and characterization at the cell level and in vivo, holds great significance forthe improvement of its cure and survival rate. In recent years, the rapid progress ofnanotechnology and molecular engineering technology plus their continuousintercrossing with life science provides an opportunity for developing novel tumorimaging and characterization methods. On the one hand, a variety of functionalizednanomaterials with unique signal generation and enhancement properties afford agreat potential to overcome the background interference and realize the amplificationof targets. On the other hand, the tumor specific aptamer, a newly emerging andgradually enriching probe, provides an ideal kind of targeted recognition molecule tomeet the selectivity demand in clinical applications, resulting from a lot of advantagesover traditional biological antibodies. In this thesis, aiming at the problems andchallenges confronted in the exploration of novel tumor cell and in vivo imaging andcharacteriazation techniques including signal amplification, targeted recognition,signal activation and multi-functionality, a series of probes have been designed andconstructed by utilizing advantages of functionalized nanomaterials and aptamers. Onthis basis, a sensitive, specific, in-situ and real-time study on fluorescence tumorimaging and characteriazation has been systematically performed from the subcellularand cellular level to in vivo level. The detailed description is listed as follows:
1. Fluorescent silica nanoparticles-based novel marker for lysosome location,tracking and imaging in tumor cells
In order to overcome limitations of traditional fluorescent dyes, such as low signalintensity, easy photobleaching and short labeling life, and to satisfy demands forreal-time, in-situ, dynamic and long-time imaging and characterization of lifeactivities at the cell level, advantages of fluorescent silica nanoparticles (DSiNPs)have been utilized. On the basis of a systematic study on the passive-targeted locationand imaging of DSiNPs with different surface charges in tumor cells, a novellysosome characterization and tracking method was developed. Firstly, two kinds of DSiNPs with comparative diameters and opposite surface charges were successfullyprepared through a reverse microemulsion method using TAMRA as the core material.Utilizing the synchronous indication effect of NPs’ fluorescence combined withtraditional markers of organelles, a colocalization fluorescence imaging investigationwas conducted. It was revealed that due to a higher buffer capacity,TAMRA-NH2-DSiNPs with positive surface charge could enter into and then partiallyescape from lysosomes followed by the capture of endoplasmic reticulum. In contrast,TAMRA-DSiNPs with negative surface charge could locate in lysosomes for a longtime. After a further condition optimization using Hela cervix cancer cells as themodel, results showed that TAMRA-DSiNPs could specifically locate in lysosomes ofHela cells and the labeling pattern would not be affected by core materials, whichdisplays a potential application in multi-color staining of subcellular structures. Inparticular, compared with traditional markers, including big molecule Alexa488-dextran and small molecule LysoTracker Green, TAMRA-DSiNPs exhibitedseveral advantages like high photostability, long circulating-life and goodbiocompatibility. Moreover, TAMRA-DSiNPs-based lysosome marker also presentedexcellent tolerance to the cell fixation and permeabilization treatments. Subsequently,TAMRA-DSiNPs have been successfully applied to track lysosomes inchloroquine-treated cells and label lysosomes in different cell lines, which stronglysupported its further application in imaging and characterization of lysosomes-relatedlife activities in the tumor occurrence and development process.
2. Annexin V-functionalized fluorescent silica nanoparticles-based novellabeling method for imaging of early-stage apoptotic tumor cells
Taking advantages of DSiNPs like strong fluorescence intensity, goodphotostability and low cell cytoxicity, and combining the “ligand-receptor”interaction, a novel, sensitive, specific, stable and facile method for early-stageapoptotic tumor cell imaging and characterization has been developed based on thespecific binding between the Annexin V-modified DSiNPs and the externalizedphosphatidylserine (PS) on the membrane of early-stage apoptotic tumor cells. Firstly,rhodamine B isothiocyanate (RBITC)-doped silica nanoparticles (RBITC-DSiNPs)were successfully prepared through a reverse microemulsion method using RBITC asthe core material. TEM characterization showed that RBITC-DSiNPs were uniformand well-dispersed with a diameter of50±5nm. By further modifying Annexin V ontoNP’s surface, an active-targeted recognition and labeling probe for early-stage apoptotic cells was successfully constructed. Through confocal fluorescence imaging,it was found that this probe not only effectively identified the paclitaxel-inducedearly-stage apoptotic MCF-7breast cancer cells from untreated MCF-7cells, but alsoachieved the in-situ characterization and tracking of PS’s externalization progress atthe apoptotic cell surface along with the extended induction time. In particular,compared with the pure Cy3-based labeling method, this probe exhibited much betterphotostability. Its fluorescent labeling signal could still be clear and bright after acontinuous laser irradiation as long as20minutes. This probe might play an importantrole in tumor cell apoptosis-related researches and the screening of anti-tumor drugs.
3. Aptamers generated by cell-SELEX for in vivo fluorescence tumor imaging
To address the limitations of traditional biological antibodies and explore novelmolecular probes for in vivo tumor imaging, a series of tumor-targeted recognitionand labeling probes were constructed by adopting tumor specific aptamers evoledfrom cell-SELEX and directly modifying the near-infrared fluorescent dye Cy5. Then,a systematic in vivo fluorescence imaging study was performed for several tumorsincluding blood cancer and solid cancer. Ramos lymphoma and its aptamer TD05waschosen as the model firstly. Flow cytometry assays demonstrated that Cy5-TD05inmouse serum could well retain its recognition ability to both in vitro and ex vivocultured Ramos cells. In vivo fluorescence imaging results also showed thatCy5-TD05could achieve the targeted imaging of Ramos tumor inside mice withexcellent sequence dependence and tumor selectivity. On this basis, to further expandaptamers’ application in fluorescence imaging of other tumor types, both lung cancerand liver cancer were selected as the study objects. The selective imaging of differenttumor types in the same mouse body was successfully realized using thecorresponding Cy5-aptamers, which positively lays the foundation for aptamers to beapplied in in vivo imaging as a novel tumor-targeted recognition molecule in thefuture.
4. Locked nucleic acid-modified aptamer probe for serum stabilityimprovement and in vivo fluorescence tumor imaging
To address aptamers’ drawbacks in complex biological systems, such asinsufficient stability, short residence time in tumor sites and narrow imaging timewindow, TD05was selected as the model to construct an imaging and detection probewith both the recognition ability against target tumor cells and good stability in bloodthrough locked nucleic acid (LNA) and3’-3’-thymidine (3’-3’-T) modifications. By investigating the effects of various modification strategies with different positions,numbers and combinations on TD05’s properties including stability, affinity andspecificity, it was found that the combined use of LNA and3’-3’-T had a synergisticeffect, and to a certain extent, the half-lives were gradually extended with the increaseof LNA incorporated. In particular, TD05.6with7-base pair-LNA substitution and3’-3’-T modification exhibited the significantly elevated detection stability affordinga half-life of5~6h to Ramos cells in serum, which was prolonged to be over ten timesof that before modification. Moreover, the in vivo tumor imaging window was alsogreatly extended from <150min of TD05to>600min of TD05.6. This strategy holdsa potential to be developed as a versatile method to provide more aptamer probes withclinical application merits for tumor imaging and detection studies.
5. Hairpin activatable aptamer probe based on cell membrane surfaceprotein-triggered conformational alteration for in vivo fluorescence tumorimaging
In order to overcome the disadvantages of dye-labeled “always on” aptamerprobes, including long diagnosis time, low imaging contrast and limited sensitivity,the specific aptamer Sgc8c against CCRF-CEM leukemia cells was selected as themodel and a hairpin activatable aptamer probe based on cell membrane surfaceprotein-triggered conformational alteration was successfully constructed. This probewas mainly hairpin-shaped in its free state and displayed a quenched fluorescence inthe absence of targets as a result of the proximity between fluorescent molecule andquencher respectively at two terminus of the probe. However, upon interacting withtarget cells, this probe effectively underwent a conformational reorganization with anactivated fluorescence, thus indicating the presence of target tumor cells. Flowcytometry assays demonstrated that this probe not only had the highly specific signalactivation ability against target tumor cells, but also significantly improved theanalytical sensitivity in comparison with “always on” probes. It could efficaciouslydetected samples containing as low as118CCRF-CEM cells. In particular, whenapplied in in vivo tumor imaging, this probe sufficiently decrease the signalinterference from unbound probes in non-target tissues, thus greatly enhancing thetumor imaging contrast and shortening the diagnosis time from several hours of“always on” strategy to15minutes. In addition, this probe also displayed excellentsequence specificity and tumor selectivity. Considering the expansion of aptamerdiscovery for varying cancer targets, this probe design might be potentially used as a versatile method to develop highly sensitive and speicfic tumor imaging probes.
6. Split activatable aptamer probe based on cell membrane surfaceprotein-triggered conformational alteration for tumor cell and in vivo imagingand detection
Also using Sgc8c as the model, the split activatable aptamer probe wassuccessfully constructed by cutting Sgc8c into two nucleic acid fragments at theproper site. In the absence of targets, there was no obvious interaction between thesetwo fragments. But after introduncing target tumor cells, these two fragments couldbe induced by the target protein on cell membrane to form the recognitionconformation similar to the intact Sgc8c, thus binding to target cells. Through flowcytometry analysis, it was found that the specific affinity of this probe against targetcells was obviously sensitive to temperatures. Utilizing its targeted recognition abilityat low temperatures, a simple, convenient, washing-free, sensitive and specificimaging and detection technique for CCRF-CEM cells was successfully developed bycombining with the fluorescence enhancement effect based on the proximity of G-richsequence to silver nanoclusters. Utilizing the feature that the binding ability of thisprobe to target cells gradually decreaced with the temperature lifted from on ice to37℃, a selective capture and temperature-controlled release method was successfullyconstructed by using96-well microplates modified with this probe as the capturevessel. It was demonstrated that this method had good biocompatibility to cells andcould be effectively applied in the cyclic capture and release of target cells and theseparation and recovery of different tumor cells in mixed samples. Finally, throughfurther modification with a PEG linker to conjugate the two fragments of this probeand change the interaction into intramolecular interaction, its inactivation problem atphysiological conditions was successfully addressed. In addition, by integrated withthe Cy3-Cy5donor-acceptor pair-based FRET effect, this probe showed thepreliminary feasibility for tumor cell detection and imaging.
7. pH-activatable aptamer probe stimulated by the acidic tumormicro-circumstance for tumor cell and in vivo fluorescence imaging
Aiming at the weak acidic feature of tumor tissues and tumor cell lysosomes (pH4-6), simultaneously combining the targeted recognition ability of aptamers, thespecific aptamer S6against A549lung cancer cells was selected as the model. ApH-activatable aptamer probe with both the detection specificity and “signal on”architecture was successfully constructed through conjugation of Cy5-aptamer and quencher BHQ3using an acid-labile linker ATU. At the neutral condition, this probeshowed a fluorescence quenching efficiency as high as about98%, and after incubatedin buffer with pH4.5for24hours, the signal nearly recovered to100%. Bycomparison with the hairpin activatable aptamer probe, it was found that this probeexhibited much better fluorescence stability no matter in serum or inside mice.Confocal microscope imaging results indicated that this probe not only held thesite-specific acid-activatable functionality in lysosomes of live cells, but alsoafforded a variety of advantages over “always on” probes, such as highly specific,high-contrast, simple, washing-free and so on. A further in vivo fluorescence tumorimaging study also demonstrated that this probe positively had the activatableimaging functionality inside tumor tissues of mice and the activation wassequence-dependent and tumor-targeted. This design might be developed as a versitilestrategy to construct probes for the characterization and imaging study of acidicdesease sites like tumors.
8. Self-assembled aptamer-carbon nanotube activatable probe for in vivofluorescence tumor imaging
By integrating advantages of functionalized nanomaterials and aptamers as thesignal switch and the targeted recognition molecule respectively, Sgc8c was selectedas the model to construct an activatable probe for tumor imaging based on the strongadsorption and quenching effect of single-wall carbon nanotubes (SWNTs) tofluorophore-labeled DNA. In the absence of targets, Cy5-Sgc8c firmly assembled onthe surface of SWNTs and displayed a quenched fluorescence. However, with theaddition of target tumor cells, the binding of aptamer with the target protein on cellmembrane could force Cy5apart from SWNTs with an elevated fluorescence.Through flow cytometry assay and in vivo fluorescence imaging, it was found that nomatter in buffer or inside tumor-grafted mice, CCRF-CEM cells could effectivelyactivate the fluorescence signal of this probe and this activation was highlysequence-dependent and tumor-targeted. In particular, in comparison with “alwayson” probes, it was demonstrated that SWNTs could greatly reduce the nonspecificsignal background interference from unbound probes in non-target systems, thussignificantly improving the in vitro analysis sensitivity and in vivo imaging contrastfor target tumor cells. This probe design is simple and convenient, which held greatpotentials to be developed as a versatile tumor imaging method with low background,high contrast and high specificity.
9. Activatable aptamer probes-functionalized Au@Au/Ag nanoparticles for invivo fluorescence tumor imaging and the guided photothermal thaerapy
On the basis of above in vivo tumor imaging studies based on acitivatable aptamerprobes (AAPs), a preliminary study on the construction and application of theranosticprobes based on aptamer-nanomaterial functionalized assemblies was performed byfurther utilizing the antitumor properties of functionalized nanomaterials. Firstly,using Au nanorods as templates, Au@Au/Ag NPs were successfully synthesized bycoating Au nanorods with Ag layers and then etching with Au. The prepared NPsexhibited a broad and intense absorption from400to1100nm and a heatingefficiency4.5times higher than Au nanorods under a980nm laser irradiation.Thereafter, the acitivatable aptamer probe (AAP) sequence was designed by adoptingS6as the model and a theranostic probe based on AAP-modified Au@Au/Ag NPs wasthen successfully constructed after the self-assembly of DNA and NPs via “Au-S”bonding, in which Au@Au/Ag NPs served as both the “optical nano heater” and thefluorescence quencher. Through a series of in vitro and in vivo investigations, it wasdemonstrated that this probe could achieve not only the selective photothermaltherapy but also the acitvatable imaging and detection of target tumor cells. Byfurther intravenously injecting this probe into a mouse grafted with different tumors,the specific activatable imaging and the guided near-infrared photothermal therapy ofA549tumor was successfully realized. Considering the expansion of aptamerdiscovery for varying cancer targets and the broad and strong absorption spectra ofAu@Au/Ag NPs, this probe design might be explored to be a technique platform forthe synchronous imaging and therapy of multi-targets.
一、基于二氧化硅荧光纳米颗粒新型标记物的肿瘤细胞溶酶体定位与示踪成像研究
为克服传统染料标记物所存在的信号强度低、易光漂白、标记寿命短等缺陷，满足细胞水平生命活动的实时、原位、动态和长时间成像与表征要求，利用二氧化硅荧光纳米颗粒（DSiNPs）的优越性能，在系统开展不同表面电荷DSiNPs在肿瘤细胞内的被动靶向定位与成像研究基础上，发展了一种新型肿瘤细胞溶酶体表征与示踪方法。首先采用反向微乳液法以四甲基罗丹明（TAMRA）为核，成功制备了粒径相当而表面电荷完全相反的两种DSiNPs。通过颗粒的荧光信号同步指示作用，结合细胞器常规标记物进行荧光共定位成像研究结果表明，表面带正电荷的TAMRA-NH_2-DSiNPs由于缓冲容量较大，在进入溶酶体后呈现出部分逃逸和被内质网捕获的特点；而表面带负电荷的TAMRA-DSiNPs则可长时间滞留于溶酶体中。随后以Hela宫颈癌细胞为模型，经条件优化后发现，TAMRA-DSiNPs能特异性定位于溶酶体内，且标记性能不受核材料限制，有望用于亚细胞结构的多色荧光成像研究。尤其与常规标记物（包括大分子Alexa488-dextran和小分子LysoTracker Green）相比，其不仅具有光稳定性强、循环寿命长且生物相容性好等一系列优点，而且还显示出优越的对细胞固定化和透化处理过程的耐受性能。最后，TAMRA-DSiNPs即被成功用于经氯喹处理的Hela细胞内溶酶体示踪以及多种不同细胞系的溶酶体表征研究，为其进一步在肿瘤发生发展过程中溶酶体相关生命活动的成像与表征中的应用提供了有力支持。
二、基于Annexin V功能化二氧化硅荧光纳米颗粒新型标记方法的早期凋亡肿瘤细胞成像研究
利用DSiNPs所具备的荧光信号强、光稳定性好、细胞毒性低等优点，结合“配体-受体”相互作用，基于Annexin V功能化DSiNPs与凋亡早期细胞膜上外翻磷脂酰丝氨酸（PS）的特异性结合，发展了一种灵敏、特异、稳定而简便的新型早期凋亡肿瘤细胞成像与表征方法。首先采用反向微乳液法以异硫氰酸罗丹明B（RBITC）为核，成功制备了包裹RBITC的二氧化硅纳米颗粒（RBITC-DSiNPs）。经TEM表征显示，该颗粒粒径为50±5nm、分散性好。通过进一步在其表面共价交联Annexin V，成功构建了一种主动靶向型早期凋亡肿瘤细胞识别与标记探针。结合激光共聚焦荧光成像考察发现，该探针不仅可有效区分紫杉醇诱导的早期凋亡MCF-7乳腺癌细胞和未经处理的MCF-7细胞，还能实现经药物诱导不同时间的早期凋亡细胞表面PS外翻程度的原位表征与示踪。尤其与Cy3染料标记方法相比，该探针显示出更为优越的光稳定性，在长达20分钟的激光连续照射后，仍能保持清晰而明亮的荧光标记信号，有望在肿瘤细胞凋亡相关研究以及抗癌药物的筛选等方面发挥重要作用。
三、基于cell-SELEX技术筛选的Aptamer探针的肿瘤活体荧光成像研究
为克服传统生物抗体的局限并开发新型肿瘤活体成像分子探针，采用基于cell-SELEX技术筛选的肿瘤细胞特异性Aptamer，通过直接修饰近红外荧光染料Cy5构建了一系列肿瘤靶向识别与标记探针，并系统开展了包括血液瘤和实体瘤在内的多种肿瘤的活体荧光成像研究。首先以Ramos淋巴瘤及其Aptamer TD05为模型，经流式细胞分析证实，Cy5-TD05在小鼠血清中能较好地保持对体外培养和原代培养Ramos细胞的结合性能。活体荧光成像结果也表明，其不仅具有对小鼠体内Ramos肿瘤的靶向成像能力，而且还显示出优越的序列依赖性和肿瘤特异性。在此基础上，为进一步拓展Aptamer在肺癌、肝癌等其他类型肿瘤荧光成像中的应用，利用其相应Cy5标记Aptamer探针成功实现了同一只小鼠体内不同类型肿瘤的选择性成像效果，为Aptamer作为一类新型肿瘤靶向识别分子今后在活体成像中的应用奠定了基础。
四、基于锁核酸修饰Aptamer探针的血液稳定性改善与肿瘤活体荧光成像研究
为解决Aptamer在用于复杂生物体系时仍然存在的稳定性不足、肿瘤部位滞留时间短、成像时间窗口窄等问题，以TD05为模型，采用锁核酸（LNA）和反转T碱基（3’-3’-T）修饰方法，成功构建了一种同时具有靶肿瘤细胞识别能力和血液稳定性的肿瘤成像探针。通过考察一系列不同取代组合、位置和数量的修饰策略对TD05抗酶切性能、亲和力和特异性的影响发现，LNA和3’-3’-T的共同修饰对其血清稳定性具有明显的协同改善效果，且一定程度上LNA取代数越多则对Aptamer半衰期的延长效果越明显。尤其经7对LNA取代和3’-3’-T修饰的TD05.6探针在37度血清的生理条件下显示出长达5-6小时的识别能力半衰期，提高至修饰前的10倍以上，并成功将活体肿瘤成像时间窗口从修饰前的105分钟延长到10小时以上。这一探针修饰策略将有望发展成为一种通用的方法为肿瘤活体成像研究提供更多具有临床实用价值的Aptamer探针。
五、基于细胞膜表面蛋白触发构型变化的发夹型激活式Aptamer探针的肿瘤活体荧光成像研究
为进一步解决上述染料直接标记“always on”Aptamer探针存在的诊断时间长、成像对比度不高、灵敏度有限等不足，以CCRF-CEM白血病细胞的特异性Aptamer Sgc8c为模型，巧妙地设计了一种基于细胞膜表面蛋白触发构型变化的发夹型激活式Aptamer探针。该探针在游离状态下主要为发夹构型，当没有目标物存在时，探针本体两端的荧光分子和淬灭分子相互靠近而导致荧光熄灭；而在与靶细胞作用后，该探针可有效发生构型重组而导致荧光激活，从而指示靶肿瘤细胞的存在。经流式细胞分析证实，该探针具有对靶肿瘤细胞的高特异性信号激活性能，尤其与“always on”探针相比，可显著提高分析灵敏度，有效检测低至118个CCRF-CEM细胞数的样品。而在应用于肿瘤活体成像时，该探针则不仅可明显降低来自非靶组织中未结合探针的信号干扰从而使肿瘤成像对比度得到明显增强，并将诊断时间从“always on”模式的数小时缩短至15分钟；而且还具有优越的序列特异性和肿瘤靶向性。鉴于Aptamer筛选技术对靶标范围的不断拓展，这一探针设计将有望作为一种通用的方法用于发展高灵敏、高特异性的肿瘤活体成像探针。
六、基于细胞膜表面蛋白触发构型变化的裂开型激活式Aptamer探针的肿瘤细胞和活体成像与检测研究
以Sgc8c为模型，通过在适当位点将其分裂为两条核酸片段，成功构建了一种基于细胞膜表面蛋白触发构型变化的裂开型激活式Aptamer探针。该探针在没有靶标存在时，两条游离核酸片段之间不会发生明显的相互作用；而当体系中引入靶肿瘤细胞后，细胞膜表面的靶蛋白会诱导其形成与完整Sgc8c类似的识别构型从而与靶细胞结合。经流式细胞分析发现，该探针对靶细胞的特异性亲和力具有明显的温度敏感性。利用低温下该探针的靶向识别性能，结合富G序列对银纳米簇的邻近荧光增强效应，发展了一种简单、方便、免洗、灵敏而特异的CCRF-CEM细胞成像与检测技术。利用温度从冰上逐渐上升至37度时该探针与靶细胞的结合能力逐渐下降的特点，选取探针修饰的96孔板作为捕获容器，建立了一种肿瘤细胞选择性捕获与温控释放方法；并证实该方法不仅具有良好的细胞亲和性，而且可有效用于靶细胞的循环捕获与释放以及混合体系中不同肿瘤细胞的分离和回收。最后，通过进一步修饰PEG linker将该探针的两条游离核酸片段相连，使分子间作用转换为分子内作用，成功克服了其在生理条件下容易失活的难题，并结合Cy3-Cy5供受体对的FRET效应初步展示了一定的肿瘤细胞检测与成像可行性。
七、基于肿瘤酸性微环境刺激响应的pH激活式Aptamer探针的肿瘤细胞和活体荧光成像研究
针对肿瘤组织的弱酸性特点以及肿瘤细胞溶酶体内的酸性环境（pH4-6），同时结合Aptamer的靶向识别性能，以A549肺癌细胞的特异性Aptamer S6为模型，利用可在低pH条件下水解的ATU（3,9-双(3-氨丙基)-2,4,8,10-四氧杂螺[5.5]十一烷）小分子将Cy5标记Aptamer与荧光淬灭分子BHQ3相连，成功构建了一种同时具备检测特异性和“signal on”信号模式的pH激活式Aptamer探针。该探针在中性环境中的荧光淬灭效率高达约98%，而当置于pH4.5的缓冲液中24小时即可发生几乎100%信号恢复。通过与发夹型激活式Aptamer探针比较发现，该探针无论在小鼠血清中还是裸鼠体内，均显示出优越的荧光稳定性。激光共聚焦显微成像结果则表明，该探针不仅具备在活细胞溶酶体内的定点酸性激活性能，而且与“always on”探针相比，具有成像特异性高、对比度高、简单、免洗等一系列优点。进一步的活体肿瘤荧光成像结果也证实，该探针完全具备在裸鼠体内肿瘤组织中发生信号激活的成像性能，且该激活行为具有高度的序列特异性和肿瘤靶向性，有望发展成为一种通用探针设计平台用于肿瘤等酸性疾病区域的表征和成像研究。
八、基于Aptamer-碳纳米管自组装激活式探针的肿瘤活体荧光成像研究
结合功能化纳米材料和Aptamer分别作为信号转换器件和靶向识别分子的优势，以Sgc8c为模型，利用单壁碳纳米管（SWNTs）对荧光标记DNA的强吸附性能和高效淬灭作用，成功构建了一种基于Aptamer-碳纳米管自组装功能体的激活式肿瘤成像探针。当体系中不存在靶标时，Cy5-Sgc8c牢固附着于SWNTs表面，Cy5荧光被淬灭；而在加入靶肿瘤细胞后，由于Aptamer与细胞表面的靶蛋白结合使Cy5离开SWNTs表面，荧光得以恢复。通过流式细胞术、活体荧光成像技术等考察发现，无论是在缓冲液体系中还是裸鼠移植瘤模型内，CCRF-CEM细胞均可有效激活该探针的荧光信号，且激活性能具有高度的序列特异性和肿瘤靶向性。尤其通过与“always on”探针对比证实，SWNTs可有效降低非靶体系中未结合探针的非特异性信号背景干扰，从而明显改善靶肿瘤细胞的体外分析信倍比和活体成像对比度。这一探针设计简单方便，有望发展成为一种低背景、高对比、高特异且具有普遍适用性的肿瘤成像方法。
九、基于激活式Aptamer探针功能化Au@Au/Ag纳米颗粒的肿瘤活体荧光成像及其引导下的近红外光热治疗研究
在上述基于激活式Aptamer探针（AAP）的肿瘤活体成像研究基础上，结合功能化纳米材料的肿瘤杀伤性能，初步探讨了基于Aptamer-纳米材料功能组装体的“诊疗一体化”探针构建与应用研究。首先以金纳米棒（Au NRs）为模板，采用先包银后刻蚀金的方法成功合成了Au@Au/Ag球形纳米颗粒（Au@Au/AgNPs）。该颗粒在400-1100nm范围内显示出强烈的光吸收性能，且在980nm光激发下具有较Au NRs高约4.5倍的产热效率。随后，利用该颗粒同时作为光热转换元件和荧光淬灭元件，并以S6为模型设计竞争型AAP序列，通过“Au-S”作用下颗粒与DNA的自组装行为，成功构建了基于AAP功能化Au@Au/Ag NPs的“诊疗一体化”多功能探针。经过一系列体内外考察证实，该探针不仅具有对靶肿瘤细胞的选择性光热治疗效果，而且可有效实现对体内外靶肿瘤细胞的激活式成像与检测。进一步以尾静脉注射方式将该探针导入植有不同肿瘤的裸鼠体内，成功实现了对A549阳性肿瘤的特异性激活式成像及其引导下的近红外光热治疗。鉴于Aptamer的靶标种类丰富而Au@Au/Ag NPs的吸收光谱宽且强，该探针设计将有望发展成为一种具有多目标同时成像与治疗潜能的技术平台。
Due to its rising incidence and high mortality, the malignant tumor is a seriousthreat to human health and life. To explore novel diagnosis and prognostic monitoringmethods for early-stage tumors, especially to reveal the laws and mechanisms intumor occurrence and development through non-invasive, in-situ and real-timeimaging and characterization at the cell level and in vivo, holds great significance forthe improvement of its cure and survival rate. In recent years, the rapid progress ofnanotechnology and molecular engineering technology plus their continuousintercrossing with life science provides an opportunity for developing novel tumorimaging and characterization methods. On the one hand, a variety of functionalizednanomaterials with unique signal generation and enhancement properties afford agreat potential to overcome the background interference and realize the amplificationof targets. On the other hand, the tumor specific aptamer, a newly emerging andgradually enriching probe, provides an ideal kind of targeted recognition molecule tomeet the selectivity demand in clinical applications, resulting from a lot of advantagesover traditional biological antibodies. In this thesis, aiming at the problems andchallenges confronted in the exploration of novel tumor cell and in vivo imaging andcharacteriazation techniques including signal amplification, targeted recognition,signal activation and multi-functionality, a series of probes have been designed andconstructed by utilizing advantages of functionalized nanomaterials and aptamers. Onthis basis, a sensitive, specific, in-situ and real-time study on fluorescence tumorimaging and characteriazation has been systematically performed from the subcellularand cellular level to in vivo level. The detailed description is listed as follows:
1. Fluorescent silica nanoparticles-based novel marker for lysosome location,tracking and imaging in tumor cells
In order to overcome limitations of traditional fluorescent dyes, such as low signalintensity, easy photobleaching and short labeling life, and to satisfy demands forreal-time, in-situ, dynamic and long-time imaging and characterization of lifeactivities at the cell level, advantages of fluorescent silica nanoparticles (DSiNPs)have been utilized. On the basis of a systematic study on the passive-targeted locationand imaging of DSiNPs with different surface charges in tumor cells, a novellysosome characterization and tracking method was developed. Firstly, two kinds of DSiNPs with comparative diameters and opposite surface charges were successfullyprepared through a reverse microemulsion method using TAMRA as the core material.Utilizing the synchronous indication effect of NPs’ fluorescence combined withtraditional markers of organelles, a colocalization fluorescence imaging investigationwas conducted. It was revealed that due to a higher buffer capacity,TAMRA-NH2-DSiNPs with positive surface charge could enter into and then partiallyescape from lysosomes followed by the capture of endoplasmic reticulum. In contrast,TAMRA-DSiNPs with negative surface charge could locate in lysosomes for a longtime. After a further condition optimization using Hela cervix cancer cells as themodel, results showed that TAMRA-DSiNPs could specifically locate in lysosomes ofHela cells and the labeling pattern would not be affected by core materials, whichdisplays a potential application in multi-color staining of subcellular structures. Inparticular, compared with traditional markers, including big molecule Alexa488-dextran and small molecule LysoTracker Green, TAMRA-DSiNPs exhibitedseveral advantages like high photostability, long circulating-life and goodbiocompatibility. Moreover, TAMRA-DSiNPs-based lysosome marker also presentedexcellent tolerance to the cell fixation and permeabilization treatments. Subsequently,TAMRA-DSiNPs have been successfully applied to track lysosomes inchloroquine-treated cells and label lysosomes in different cell lines, which stronglysupported its further application in imaging and characterization of lysosomes-relatedlife activities in the tumor occurrence and development process.
2. Annexin V-functionalized fluorescent silica nanoparticles-based novellabeling method for imaging of early-stage apoptotic tumor cells
Taking advantages of DSiNPs like strong fluorescence intensity, goodphotostability and low cell cytoxicity, and combining the “ligand-receptor”interaction, a novel, sensitive, specific, stable and facile method for early-stageapoptotic tumor cell imaging and characterization has been developed based on thespecific binding between the Annexin V-modified DSiNPs and the externalizedphosphatidylserine (PS) on the membrane of early-stage apoptotic tumor cells. Firstly,rhodamine B isothiocyanate (RBITC)-doped silica nanoparticles (RBITC-DSiNPs)were successfully prepared through a reverse microemulsion method using RBITC asthe core material. TEM characterization showed that RBITC-DSiNPs were uniformand well-dispersed with a diameter of50±5nm. By further modifying Annexin V ontoNP’s surface, an active-targeted recognition and labeling probe for early-stage apoptotic cells was successfully constructed. Through confocal fluorescence imaging,it was found that this probe not only effectively identified the paclitaxel-inducedearly-stage apoptotic MCF-7breast cancer cells from untreated MCF-7cells, but alsoachieved the in-situ characterization and tracking of PS’s externalization progress atthe apoptotic cell surface along with the extended induction time. In particular,compared with the pure Cy3-based labeling method, this probe exhibited much betterphotostability. Its fluorescent labeling signal could still be clear and bright after acontinuous laser irradiation as long as20minutes. This probe might play an importantrole in tumor cell apoptosis-related researches and the screening of anti-tumor drugs.
3. Aptamers generated by cell-SELEX for in vivo fluorescence tumor imaging
To address the limitations of traditional biological antibodies and explore novelmolecular probes for in vivo tumor imaging, a series of tumor-targeted recognitionand labeling probes were constructed by adopting tumor specific aptamers evoledfrom cell-SELEX and directly modifying the near-infrared fluorescent dye Cy5. Then,a systematic in vivo fluorescence imaging study was performed for several tumorsincluding blood cancer and solid cancer. Ramos lymphoma and its aptamer TD05waschosen as the model firstly. Flow cytometry assays demonstrated that Cy5-TD05inmouse serum could well retain its recognition ability to both in vitro and ex vivocultured Ramos cells. In vivo fluorescence imaging results also showed thatCy5-TD05could achieve the targeted imaging of Ramos tumor inside mice withexcellent sequence dependence and tumor selectivity. On this basis, to further expandaptamers’ application in fluorescence imaging of other tumor types, both lung cancerand liver cancer were selected as the study objects. The selective imaging of differenttumor types in the same mouse body was successfully realized using thecorresponding Cy5-aptamers, which positively lays the foundation for aptamers to beapplied in in vivo imaging as a novel tumor-targeted recognition molecule in thefuture.
4. Locked nucleic acid-modified aptamer probe for serum stabilityimprovement and in vivo fluorescence tumor imaging
To address aptamers’ drawbacks in complex biological systems, such asinsufficient stability, short residence time in tumor sites and narrow imaging timewindow, TD05was selected as the model to construct an imaging and detection probewith both the recognition ability against target tumor cells and good stability in bloodthrough locked nucleic acid (LNA) and3’-3’-thymidine (3’-3’-T) modifications. By investigating the effects of various modification strategies with different positions,numbers and combinations on TD05’s properties including stability, affinity andspecificity, it was found that the combined use of LNA and3’-3’-T had a synergisticeffect, and to a certain extent, the half-lives were gradually extended with the increaseof LNA incorporated. In particular, TD05.6with7-base pair-LNA substitution and3’-3’-T modification exhibited the significantly elevated detection stability affordinga half-life of5~6h to Ramos cells in serum, which was prolonged to be over ten timesof that before modification. Moreover, the in vivo tumor imaging window was alsogreatly extended from <150min of TD05to>600min of TD05.6. This strategy holdsa potential to be developed as a versatile method to provide more aptamer probes withclinical application merits for tumor imaging and detection studies.
5. Hairpin activatable aptamer probe based on cell membrane surfaceprotein-triggered conformational alteration for in vivo fluorescence tumorimaging
In order to overcome the disadvantages of dye-labeled “always on” aptamerprobes, including long diagnosis time, low imaging contrast and limited sensitivity,the specific aptamer Sgc8c against CCRF-CEM leukemia cells was selected as themodel and a hairpin activatable aptamer probe based on cell membrane surfaceprotein-triggered conformational alteration was successfully constructed. This probewas mainly hairpin-shaped in its free state and displayed a quenched fluorescence inthe absence of targets as a result of the proximity between fluorescent molecule andquencher respectively at two terminus of the probe. However, upon interacting withtarget cells, this probe effectively underwent a conformational reorganization with anactivated fluorescence, thus indicating the presence of target tumor cells. Flowcytometry assays demonstrated that this probe not only had the highly specific signalactivation ability against target tumor cells, but also significantly improved theanalytical sensitivity in comparison with “always on” probes. It could efficaciouslydetected samples containing as low as118CCRF-CEM cells. In particular, whenapplied in in vivo tumor imaging, this probe sufficiently decrease the signalinterference from unbound probes in non-target tissues, thus greatly enhancing thetumor imaging contrast and shortening the diagnosis time from several hours of“always on” strategy to15minutes. In addition, this probe also displayed excellentsequence specificity and tumor selectivity. Considering the expansion of aptamerdiscovery for varying cancer targets, this probe design might be potentially used as a versatile method to develop highly sensitive and speicfic tumor imaging probes.
6. Split activatable aptamer probe based on cell membrane surfaceprotein-triggered conformational alteration for tumor cell and in vivo imagingand detection
Also using Sgc8c as the model, the split activatable aptamer probe wassuccessfully constructed by cutting Sgc8c into two nucleic acid fragments at theproper site. In the absence of targets, there was no obvious interaction between thesetwo fragments. But after introduncing target tumor cells, these two fragments couldbe induced by the target protein on cell membrane to form the recognitionconformation similar to the intact Sgc8c, thus binding to target cells. Through flowcytometry analysis, it was found that the specific affinity of this probe against targetcells was obviously sensitive to temperatures. Utilizing its targeted recognition abilityat low temperatures, a simple, convenient, washing-free, sensitive and specificimaging and detection technique for CCRF-CEM cells was successfully developed bycombining with the fluorescence enhancement effect based on the proximity of G-richsequence to silver nanoclusters. Utilizing the feature that the binding ability of thisprobe to target cells gradually decreaced with the temperature lifted from on ice to37℃, a selective capture and temperature-controlled release method was successfullyconstructed by using96-well microplates modified with this probe as the capturevessel. It was demonstrated that this method had good biocompatibility to cells andcould be effectively applied in the cyclic capture and release of target cells and theseparation and recovery of different tumor cells in mixed samples. Finally, throughfurther modification with a PEG linker to conjugate the two fragments of this probeand change the interaction into intramolecular interaction, its inactivation problem atphysiological conditions was successfully addressed. In addition, by integrated withthe Cy3-Cy5donor-acceptor pair-based FRET effect, this probe showed thepreliminary feasibility for tumor cell detection and imaging.
7. pH-activatable aptamer probe stimulated by the acidic tumormicro-circumstance for tumor cell and in vivo fluorescence imaging
Aiming at the weak acidic feature of tumor tissues and tumor cell lysosomes (pH4-6), simultaneously combining the targeted recognition ability of aptamers, thespecific aptamer S6against A549lung cancer cells was selected as the model. ApH-activatable aptamer probe with both the detection specificity and “signal on”architecture was successfully constructed through conjugation of Cy5-aptamer and quencher BHQ3using an acid-labile linker ATU. At the neutral condition, this probeshowed a fluorescence quenching efficiency as high as about98%, and after incubatedin buffer with pH4.5for24hours, the signal nearly recovered to100%. Bycomparison with the hairpin activatable aptamer probe, it was found that this probeexhibited much better fluorescence stability no matter in serum or inside mice.Confocal microscope imaging results indicated that this probe not only held thesite-specific acid-activatable functionality in lysosomes of live cells, but alsoafforded a variety of advantages over “always on” probes, such as highly specific,high-contrast, simple, washing-free and so on. A further in vivo fluorescence tumorimaging study also demonstrated that this probe positively had the activatableimaging functionality inside tumor tissues of mice and the activation wassequence-dependent and tumor-targeted. This design might be developed as a versitilestrategy to construct probes for the characterization and imaging study of acidicdesease sites like tumors.
8. Self-assembled aptamer-carbon nanotube activatable probe for in vivofluorescence tumor imaging
By integrating advantages of functionalized nanomaterials and aptamers as thesignal switch and the targeted recognition molecule respectively, Sgc8c was selectedas the model to construct an activatable probe for tumor imaging based on the strongadsorption and quenching effect of single-wall carbon nanotubes (SWNTs) tofluorophore-labeled DNA. In the absence of targets, Cy5-Sgc8c firmly assembled onthe surface of SWNTs and displayed a quenched fluorescence. However, with theaddition of target tumor cells, the binding of aptamer with the target protein on cellmembrane could force Cy5apart from SWNTs with an elevated fluorescence.Through flow cytometry assay and in vivo fluorescence imaging, it was found that nomatter in buffer or inside tumor-grafted mice, CCRF-CEM cells could effectivelyactivate the fluorescence signal of this probe and this activation was highlysequence-dependent and tumor-targeted. In particular, in comparison with “alwayson” probes, it was demonstrated that SWNTs could greatly reduce the nonspecificsignal background interference from unbound probes in non-target systems, thussignificantly improving the in vitro analysis sensitivity and in vivo imaging contrastfor target tumor cells. This probe design is simple and convenient, which held greatpotentials to be developed as a versatile tumor imaging method with low background,high contrast and high specificity.
9. Activatable aptamer probes-functionalized Au@Au/Ag nanoparticles for invivo fluorescence tumor imaging and the guided photothermal thaerapy
On the basis of above in vivo tumor imaging studies based on acitivatable aptamerprobes (AAPs), a preliminary study on the construction and application of theranosticprobes based on aptamer-nanomaterial functionalized assemblies was performed byfurther utilizing the antitumor properties of functionalized nanomaterials. Firstly,using Au nanorods as templates, Au@Au/Ag NPs were successfully synthesized bycoating Au nanorods with Ag layers and then etching with Au. The prepared NPsexhibited a broad and intense absorption from400to1100nm and a heatingefficiency4.5times higher than Au nanorods under a980nm laser irradiation.Thereafter, the acitivatable aptamer probe (AAP) sequence was designed by adoptingS6as the model and a theranostic probe based on AAP-modified Au@Au/Ag NPs wasthen successfully constructed after the self-assembly of DNA and NPs via “Au-S”bonding, in which Au@Au/Ag NPs served as both the “optical nano heater” and thefluorescence quencher. Through a series of in vitro and in vivo investigations, it wasdemonstrated that this probe could achieve not only the selective photothermaltherapy but also the acitvatable imaging and detection of target tumor cells. Byfurther intravenously injecting this probe into a mouse grafted with different tumors,the specific activatable imaging and the guided near-infrared photothermal therapy ofA549tumor was successfully realized. Considering the expansion of aptamerdiscovery for varying cancer targets and the broad and strong absorption spectra ofAu@Au/Ag NPs, this probe design might be explored to be a technique platform forthe synchronous imaging and therapy of multi-targets.
引文
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