叶酸受体介导的肿瘤靶向光学成像技术

叶酸受体(FR)在肿瘤细胞中都有过度表达,而在正常组织中保守表达,利用叶酸受体与叶酸及其类似物高亲合力结合的特性,将叶酸偶联荧光探针输送到肿瘤组织,从而实现肿瘤组织的特异性靶向光学成像。本文阐述了叶酸荧光探针的结构及其用于标记肿瘤细胞的作用机制,介绍了近十年来叶酸受体介导的肿瘤靶向光学成像技术,例如有机荧光染料,染料掺杂纳米颗粒,量子点,磁性纳米微粒以及多功能纳米粒子等在肿瘤靶向成像中的研究及应用进展,指出了当前研究中的主要发展方向和仍需解决的问题。     

叶酸功能化介孔碳纳米球负载阿霉素的细胞靶向传递及可控释放

以粒径90 nm的介孔碳纳米球作为靶向传药载体, 采用酸化处理改进了材料表面的亲疏水性及在溶液中的分散性, 通过壳寡糖功能化, 并利用EDC-NHS将叶酸修饰到介孔碳纳米球表面. 通过共聚焦激光扫描显微镜及流式细胞仪对实验体系的系统研究, 结果表明基于叶酸功能化的介孔碳纳米球能够有效提高负载药物对于HeLa细胞的跨膜转运效率, 叶酸阳性表达的HeLa细胞对于叶酸修饰的介孔碳纳米小球的吞噬效率明显高于叶酸阴性表达的MCF-7细胞. 对HeLa细胞毒性的定量分析表明叶酸的靶向作用在提高介孔碳纳米球内吞效率的同时, 进一步提高了阿霉素对于HeLa细胞的毒性.     

包裹RuBpy二氧化硅荧光纳米探针的制备及其用于肝癌细胞的识别

制备了两种不同官能团修饰的偶联亲和素的包裹钌联吡啶(RuBpy)二氧化硅荧光纳米探针A和探针B,并分别用于肝癌细胞的识别。通过反相微乳液法制备得到表面修饰不同官能团的纳米颗粒,然后通过亲和素与羧基化包裹RuBpy二氧化硅纳米颗粒相互连接而制备得到探针A;通过亲和素与PEG修饰的荧光二氧化硅纳米颗粒相互作用而制备得到探针B。与探针A不同的是,探针B通过一个长链PEG分子将亲和素与荧光二氧化硅纳米颗粒偶联在一起。利用免疫荧光成像法将这两种探针分别用于人肝癌细胞的识别,结果表明,含有长链PEG分子的探针B更能够有效地识别肝癌细胞表面肿瘤标志物癌胚抗原(CEA)。     

NaYF_4:Yb/Er-碳纳米管功能纳米材料的制备、表征及其在肿瘤细胞的靶向检测和治疗中的应用

将叶酸分子(FA)和2,3-二巯基丁二酸(DMSA)修饰的稀土上转换发光纳米粒子NaYF4:Yb/Er通过酰胺键偶联在多壁碳纳米管(MWCNT)的表面,得到NaYF4:Yb/Er-MWCNT-FA功能化复合纳米材料,并通过透射电子显微镜(TEM)、X射线衍射(XRD)、紫外-可见吸收光谱(UV-vis)、荧光光谱(PL)和共聚焦激光扫描显微镜等手段表征了其形貌、结构、发光性能和靶向成像性能.共聚焦激光扫描显微镜结果表明,相对于正常的HLF细胞,所制备的复合材料能够靶向检测叶酸受体高表达的宫颈癌Hela细胞.此外,将阿霉素进一步通过ππ堆垛吸附在此复合材料后,该载药体系具有明显的抗肿瘤活性,能够实现对肿瘤细胞的一步检测和治疗.     

HA-AuNPs/FDF用于透明质酸酶的高灵敏检测、肿瘤靶向细胞荧光成像和光疗

本工作构建了一种新型复合纳米诊疗剂HA-AuNPs/FDF,用于透明质酸酶(HAase)的高灵敏荧光检测、肿瘤靶向荧光成像和光动力/光热协同治疗.吡咯并吡咯二酮基共轭小分子(FDF)与肿瘤靶向生物分子透明质酸(HA)功能化的金纳米粒子(HA-AuNPs)通过静电作用自组装形成HA-Au NPs/FDF. FDF在近红外光激发下产生较强的荧光, HA-AuNPs会通过荧光共振能量转移效应(FRET)猝灭FDF的荧光.然而,肿瘤细胞中过表达的透明质酸酶(HAase)能使HA逐渐降解, FDF被释放,从而荧光逐渐恢复. HA-AuNPs/FDF的荧光恢复程度与HAase的浓度有很好的线性关系,可用于快速定量检测HAase.而且, HA-AuNPs/FDF作为透明质酸酶激活的荧光探针成功地用于人宫颈癌肿瘤HeLa细胞的靶向荧光成像,细胞实验结果证实它能通过光动力/光热协同治疗有效抑制HeLa细胞的增殖.该体系为实现精准高效的肿瘤诊疗拓展了思路.     

基于聚苯胺纳米纤维复合膜界面构建电化学细胞传感器及其对癌细胞的识别检测

合成了聚苯胺纳米纤维,直径在50～70 nm之间;基于静电作用构建聚苯胺纳米纤维-纳米金复合膜界面,并在此界面上层层组装修饰叶酸分子,构建叶酸功能化传感界面,基于叶酸分子与癌细胞表面过量表达的叶酸受体之间的特异性识别作用,将此传感界面应用于对癌细胞的识别和捕获。结果表明:叶酸功能化传感界面能够特异性识别和捕获叶酸受体过量表达的癌细胞。采用电化学阻抗技术,以HeLa细胞为模型,应用于对癌细胞的识别和检测,细胞在1.0×104～6.4×106cells/mL浓度范围内与阻抗变化值ΔRct呈良好的线性关系;检出限为2000 cells/mL。本方法简单、快速灵敏、重现性和稳定性良好;制备的传感器可以再生使用。     

靶向性纳米金探针对宫颈癌细胞的激光扫描共聚焦散射成像

制备了粒径均一的纳米金颗粒, 再对其表面进行叶酸修饰, 制得具有靶向性的纳米金探针. 利用激光扫描共聚焦显微镜(LSCM), 对靶向性纳米金的细胞特异性散射成像进行研究. 实验结果表明, 人宫颈癌细胞(Hela)对纳米金-叶酸的摄取作用强于对纳米金的摄取, 但随着时间的延长, 两者的差别逐渐减小. 表明在适当的时间内纳米金-叶酸探针对宫颈癌细胞具有良好的靶向性.     

聚甲基丙烯酰胺包覆的ZnO发光量子点及其在细胞成像中的应用

通过溶胶-凝胶法合成了在水溶液中稳定发光、荧光颜色可调的ZnO@polymer核壳型纳米粒子, 并研究了这种量子点对人类宫颈癌HeLa细胞的毒性以及细胞吞噬后激光共聚焦成像的效果. 这种荧光探针的外壳是一种通过配位键与ZnO内核结合的共聚物, 该共聚物外层是亲水的聚甲基丙烯酰胺, 内层是疏水的聚甲基丙烯酸酯. 细胞毒性实验证明, 该材料有良好的生物兼容性, 适用于生物荧光标记. 共聚焦荧光成像结果显示, 这些纳米粒子可以穿透HeLa细胞膜并在细胞质中稳定发光.     

基于碳点多功能纳米载药体系的制备及pH响应释药性能研究

采用一步微波法成功制备了表面带氨基的荧光纳米碳点CDots, 并通过酰胺化反应将靶向基团叶酸接枝到碳点表面, 成功获得中间产物CDots-FA. 在此基础上, 通过已合成四臂端酰肼基化合物2与抗肿瘤药物阿霉素(DOX)连接, 实现在碳点表面的阿霉素药物分子的化学键合, 最终获得多功能纳米载药体系DOX-CDots-FA. 利用原子力显微镜(AFM)、高分辨透射电镜(HR-TEM)和荧光光谱仪对荧光纳米碳点CDots的性能进行表征, 并通过核磁共振、紫外-可见吸收光谱对DOX-CDots-FA结构、接枝率进行了表征. 同时对纳米载药体系DOX-CDots-FA体外药物释放行为、细胞毒性及细胞摄取成像进行了系统的研究. 结果表明, DOX-CDots-FA具有良好的pH响应性. 叶酸靶向基团能加速DOX-CDots-FA被HeLa (FR+)细胞摄取, 并表现出更强的细胞毒性. 同时细胞摄入成像实验表明, 在叶酸靶向作用下, DOX-CDots-FA通过内吞作用进入HeLa细胞, 随后阿霉素被释放出来并进入细胞核区域, 抑制细胞的生长, 从而实现靶向治疗, 降低毒副作用.     

手术导航用荧光探针

荧光成像技术因具有操作简便、分辨率高、安全性好且可实时成像等优势,在术中导航中具有广阔的应用前景.虽然目前还没有靶向荧光探针在临床上得到批准,但已经有相当一部分荧光探针进入了临床试验阶段.最早进入临床试验的是一些偶联肿瘤靶向配体的荧光染料,例如近红外菁染料(IRDye800CW)标记的肿瘤特异抗体,叶酸标记的异硫氰酸荧...     

Doubly Caged Linker for AND‐Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification

In situ quantification of the conjugation efficiency of azide‐terminated synthetic polymers/imaging probes and thiol‐functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self‐sorting bridging unit. Orthogonal dual “click” coupling of C1 with azide‐ and thiol‐functionalized precursors led to highly fluorescent bioconjugates, whereas single‐click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody–probe conjugates from an anti‐carcinoembryonic antigen and a quinone‐caged profluorescent naphthalimide derivative, the dual “click” coupling process with C1 was monitored on the basis of the emission turn‐on of C1 , whereas prominent changes in FRET ratios occurred for antibody–imaging‐probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.     

Preparation of Fluorescent Organometallic Porphyrin Complex Nanogels of Controlled Molecular Structure via Reverse‐Emulsion Click Chemistry

Here, we are the first to report a novel approach to preparing well‐defined poly(ethylene glycol) (PEG) fluorescent nanogels, with well‐defined molecular structures and desired functionalities via reverse (mini)emulsion copper(I)‐catalyzed azide‐alkyne cycloaddition (REM‐CuAAC). Nanogels with hydroxyl groups and Ga‐porphyrin complex (Ga‐porphyrin‐OH nanogels), as well as with Ga‐porphyrin complex and folate functional groups (Ga‐porphyrin‐FA), are successfully prepared. Nanogels of 30 and 120 nm in diameter are obtained and they exhibit an emission maxima within the wavelength range 700–800 nm. The nanogels could find uses in near infrared (NIR) imaging attributable to their fluorescence and their functionality for cell affinity.     

Gd3+与RGD共修饰量子点用于胰腺癌细胞的荧光及MR双模态成像

结合磁共振成像(MRI)和荧光成像技术,以钆离子(Gd3+)、量子点及精氨酸(R)-甘氨酸(G)-天冬氨酸(D)(RGD)多肽等为功能单元,采用纳米载体组装技术构建了MRI弛豫率/荧光效率高和靶向性强的Gd3+与RGD共修饰的量子点双模态纳米探针(QDs@Gd3+-RGD),并将其用于胰腺癌细胞的双模态成像.实验结果表明,QDs@Gd3+-RGD双模态纳米探针具有较高的弛豫率,且能对胰腺癌patu8988细胞进行荧光和T1-weighted MR成像.     

Organic Nanoprobes for Fluorescence and 19F Magnetic Resonance Dual‐Modality Imaging

Multimodal imaging techniques have been demonstrated to be greatly advantageous in achieving accurate diagnosis and gained increasing attention in recent decades. Herein, we present a new strategy to integrate the complementary modalities of ¹⁹F magnetic resonance imaging (¹⁹F MRI) and fluorescence imaging (FI) into a polymer nanoprobe composed of hydrophobic fluorescent organic core and hydrophilic fluorinated polymer shell. The alkyne‐terminated fluorinated copolymer (Pn) of 2,2,2‐trifluoroethyl acrylate (TFEA) and poly(ethylene glycol) methyl ether acrylate (PEGA) was first prepared via atom transfer radical polymerization (ATRP). The PEGA plays an important role in both improving ¹⁹F signal and modulating the hydrophilicity of Pn. The alkynyl tail in Pn is readily conjugated with azide modified tetra‐phenylethylene (TPE) through click chemistry to form azo polymer (TPE‐azo‐Pn). The core‐shell nanoprobes (TPE‐P3N) with an average particle size of 57.2 ± 8.8 nm are obtained via self‐assembly with ultrasonication in aqueous solution. These nanoprobes demonstrate high water stability, good biocompatibility, strong fluorescence and good ¹⁹F MRI performance, which present great potentials for simultaneous fluorescence imaging and ¹⁹F–MR imaging.     

Red carbon dots as label-free two-photon fluorescent nanoprobes for imaging of formaldehyde in living cells and zebrafishes

Direct,in situ selective detection of intracellular formaldehyde(FA)is of great significance for understanding its function in FA-related diseases.Herein,red carbon dots(RCD)are reported as label-free two-photon fluorescent nanoprobes for detecting and imaging of FA.Upon addition of FA,the-NH2 groups of RCD could quickly and specially react with aldehydes to form Schiff base and then the strong fluorescence of RCD with blue-shift emission is recovery due to the destruction of the hydrogen bond interaction between RCD and water.In addition,the nanoprobes exhibit outsta nding photo stability,rapid response(<1 min),high sensitivity(~9.9μmol/L)and excellent selectivity toward FA over other aldehyde group compounds.Notably,owing to the good cell-membrane permeability and biocompatibility,as well as the large two-photon absorption cross-section,the as-prepared RCD can be used as label-free nanoprobes for selectively detecting and imaging FA in living cells and zebrafishes through one-photon and two-photon excitation.Moreover,RCD could stain the tissue of zebrafishes at depths interval of up to 240μm under two-photon excitation.This research implied that RCD are promising tools for directly and in situ imaging FA in vivo,thus providing critical insights into FA-related pathophysiological processes.     

Biocompatible fluorescent polyamine‐based cyclophosphazene hybrid nanospheres for targeted cell imaging

Fluorescent nanoprobes are highly desirable toolkit for bioimaging applications. This study reports the first example for the synthesis of a nontoxic prototypical fluorescent organic compound 2‐benzo[d]thiazol‐2‐yl)‐3‐(2‐chloro‐4‐(dimethylamino)phenyl)acrylonitrile (BCA) and its entrapment into the poly[cyclotriphosphazene‐co‐polyethyleneimine] cross‐linked (PCPEI) nanospheres named as BCA@PCPEI for targeted cell imaging application. The as‐prepared BCA@PCPEI nanospheres were thoroughly characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), fourier transform infrared (FTIR), thermogravimetric analysis (TGA), and phosphorus‐31 nuclear magnetic resonance (³¹P‐NMR) analyses. The surface functional analysis of the nanospheres was performed by X‐ray photoelectron spectroscopy (XPS), which proves that the content ratios of elements belong to the precursors concentrations. The as‐prepared nanospheres displayed emission at 606 nm with bright orange fluorescence at any concentration. Moreover, the nanospheres were also less cytotoxic and maintained remarkable cell viability up to 100 μg/mL. Owing to the fluorescence with higher emission, this material has shown excellent cell imaging performance with better targeting ability to HeLa cells.     

Bioorthogonal Turn‐On Probe Based on Aggregation‐Induced Emission Characteristics for Cancer Cell Imaging and Ablation

Bioorthogonal turn‐on probes have been widely utilized in visualizing various biological processes. Most of the currently available bioorthogonal turn‐on probes are blue or green emissive fluorophores with azide or tetrazine as functional groups. Herein, we present an alternative strategy of designing bioorthogonal turn‐on probes based on red‐emissive fluorogens with aggregation‐induced emission characteristics (AIEgens). The probe is water soluble and non‐fluorescent due to the dissipation of energy through free molecular motion of the AIEgen, but the fluorescence is immediately turned on upon click reaction with azide‐functionalized glycans on cancer cell surface. The fluorescence turn‐on is ascribed to the restriction of molecular motion of AIEgen, which populates the radiative decay channel. Moreover, the AIEgen can generate reactive oxygen species (ROS) upon visible light (λ=400–700 nm) irradiation, demonstrating its dual role as an imaging and phototherapeutic agent.     

量子点-卟啉纳米复合体系在肿瘤细胞成像中的应用

本文合成了高荧光量子产率、单分散性好的水溶性CdTe量子点(quantum dots,QDs),并与α,β,γ,δ-四(1-甲基吡啶嗡-4-基)卟吩对甲苯磺酸盐(TMPyP)组装成QDs-TMPyP纳米复合物,研究了该复合物检测DNA的机理以及肿瘤细胞成像。结果显示,QDs-TMPyP纳米复合物通过光致电子转移机制检测DNA,当CdTe QDs和CdTe QDs-TMPyP浓度低于1.0μmol/L时,HeLa肿瘤细胞存活率达92%以上,表现出低的细胞毒性。0.2μmol/L CdTe QDs-TMPyP作用于肿瘤细胞时,细胞生长状态良好,对细胞内能谱分析发现细胞内含有Cd和Te原子。CdTe QDs-TMPyP复合物比CdTe QDs更易被HeLa细胞摄取,利用量子点荧光成功实现了细胞核内成像,为宫颈癌细胞药物输送和细胞成像的深入研究打下基础。     

A Golgi Apparatus-Targeting,Naphthalimide-Based Fluorescent Molecular Probe for the Selective Sensing of Formaldehyde

Formaldehyde (FA) is a colorless, flammable, foul-smelling chemical used in building materials and in the production of numerous household chemical goods. Herein, a fluorescent chemosensor for FA is designed and prepared using a selective organ-targeting probe containing naphthalimide as a fluorophore and hydrazine as a FA-binding site. The amine group of the hydrazine reacts with FA to form a double bond and this condensation reaction is accompanied by a shift in the absorption band of the probe from 438 nm to 443 nm upon the addition of FA. Further, the addition of FA is shown to enhance the emission band at 532 nm relative to the very weak fluorescent emission of the probe itself. Moreover, a high specificity is demonstrated towards FA over other competing analytes such as the calcium ion (Ca²⁺), magnesium ion (Mg²⁺), acetaldehyde, benzaldehyde, salicylaldehyde, glucose, glutathione, sodium sulfide (Na₂S), sodium hydrosulfide (NaHS), hydrogen peroxide (H₂O₂), and the tert-butylhydroperoxide radical. A typical two-photon dye incorporated into the probe provides intense fluorescence upon excitation at 800 nm, thus demonstrating potential application as a two-photon fluorescent probe for FA sensing. Furthermore, the probe is shown to exhibit a fast response time for the sensing of FA at room temperature and to facilitate intense fluorescence imaging of breast cancer cells upon exposure to FA, thus demonstrating its potential application for the monitoring of FA in living cells. Moreover, the presence of the phenylsulfonamide group allows the probe to visualize dynamic changes in the targeted Golgi apparatus. Hence, the as-designed probe is expected to open up new possibilities for unique interactions with organ-specific biological molecules with potential application in early cancer cell diagnosis.     

Star polymers by photoinduced copper‐catalyzed azide–alkyne cycloaddition click chemistry

Well‐defined star polymers consisting of tri‐, tetra‐, or octa‐arms have been prepared via coupling‐onto strategy using photoinduced copper(I)‐catalyzed 1,3‐dipolar cycloaddition click reaction. An azide end‐functionalized polystyrene and poly(methyl methacrylate), and an alkyne end‐functionalized poly(ε‐caprolactone) as the integrating arms of the star polymers are prepared by the combination of controlled polymerization and nucleophilic substitution reactions; whereas, multifunctional cores containing either azide or alkyne functionalities were synthesized in quantitatively via etherification and ring‐opening reactions. By using photoinduced copper‐catalyzed azide–alkyne cycloaddition (CuAAC) click reaction, reactive linear polymers are simply attached onto multifunctional cores to form corresponding star polymers via coupling‐onto methodology. The chromatographic, spectroscopic, and thermal analyses have clearly demonstrated that successful star formations can be obtained via photoinduced CuAAC click reaction. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 1687–1695