Covalent Attachment of Aggregation-Induced Emission Molecules to the Surface of Ultrasmall Gold Nanoparticles to Enhance Cell Penetration

Three different alkyne-terminated aggregation-induced emission molecules based on a para-substituted di-thioether were attached to the surface of ultrasmall gold nanoparticles (2 nm) by copper-catalyzed azide–alkyne cycloaddition (click chemistry). They showed a strong fluorescence and were well water-dispersible, in contrast to the dissolved AIE molecules. The AIE-loaded nanoparticles were not cytotoxic and easily penetrated the membrane of HeLa cells, paving the way for an intracellular application of AIE molecules, e.g., for imaging.     

Nucleic acids induced peptide-based AIE nanoparticles for fast cell imaging

Herein, we utilized nucleic acids induced peptide co-assembly strategy to develop novel nucleic acids induced peptide-based AIE (NIP-AIE) nanoparticles. Strong fluorescent of AIE could be observed when a little amount of nucleic acids was added into the peptide solution, and the intensity could be regulated by the concentration of nucleic acids. This AIE nanoparticle with good biocompatibility could achieve fast cell imaging. It is also proved that the fluorescence intensity of AIE decreased with time, which indicates that the reducible cross-linkers of Wpc peptide by GSH and nanoparticles gradually disintegrate in cell. Based on the different of AIE fluorescence signals which regulated by the formation and disintegration of nanoparticles, this AIE system is expected to be used for real-time monitoring of drug release from peptide-based nano carriers in vivo or in vitro, and may provide a new platform for the construction of other organic AIE nanoparticles.     

Biocompatible organic dots with aggregation-induced emission for in vitro and in vivo fluorescence imaging

Fluorescent probes play a key role in modern biomedical research. As compared to inorganic quantum dots (QDs) composed with heavy metal elements, organic dye-based fluorescent nanoparticles have higher biocompatibility and are richer in variety. However, traditional organic fluorophores tend to quench fluorescence upon aggregation, which is known as aggregation-caused quenching (ACQ) effect that hinders the fabrication of highly emissive fluorescent nanoparticles. In this work, we demonstrate the synthesis of organic fluorescent dots with aggregation-induced emission (AIE) in far-red/near-infrared (FA/NIR) region. A conventional ACQ-characteristic fluorescent dye, 3,4:9,10-tetracarboxylic perylene bisimide (PBI), is converted into an AIE fluorogen through attaching two tetraphenylethylene (TPE) moieties. The fluorescent dots with surface folic acid groups are fabricated from PBI derivative (DTPEPBI), showing specific targeting effect to folate receptor-overexpressed cancer cells. In vivo studies also suggest that the folic acid-functionalized AIE dots preferentially accumulate in the tumor site through enhanced permeability and retention (EPR) effect and folate receptor-mediated active targeting effect. The low cyto-toxicity, good FR/NIR contrast and excellent targeting ability in in vitro/in vivo imaging indicate that the AIE dots have great potentials in advanced bioimaging applications.     

Facile Fabrication of PEGylated Fluorescent Organic Nanoparticles with Aggregation‐Induced Emission Feature via Formation of Dynamic Bonds and Their Biological Imaging Applications

Driven by the high demand for sensitive and specific tools for optical imaging, fluorescent nanoprobes with various working mechanisms and advanced functionalities are flourishing at an incredible speed. This work reports the design and fabrication of aggregation‐induced emission (AIE)‐active fluorescent organic nanoparticles (FNPs) via forming dynamic phenyl borate between diol containing hydrophobic AIE dye (APD‐PhCHO) and phenylboronic acid pendant hydrophilic polymers (PEGMA‐VPBA) within 30 min. The final AIE‐active APD‐PhCHO‐PEGMA‐VPBA FNPs display high water dispersibility and strong fluorescence emission because of their amphiphilic properties and AIE feature. Biological evaluation suggests that APD‐PhCHO‐PEGMA‐VPBA FNPs possess negative effect on HeLa cells and desirable optical properties for biological imaging. More importantly, phenyl borate is a dynamic bond with pH and glucose responsiveness. Furthermore, different functions can be designed and introduced into these AIE‐active systems through adoption of different monomers for good applicability of free radical polymerization. Therefore, this work provides a novel platform for preparation of multifunctional AIE‐active nanosystems with responsiveness for various biomedical applications.

     

Fabrication of Double Emission Enhancement Fluorescent Nanoparticles with Combined PET and AIEE Effects

The major challenge in the fabrication of fluorescent silica nanoparticles (FSNs) based on dye-doped silica nanoparticles (DDSNs) is aggregation-caused fluorescence quenching. Here, we constructed an FSN based on a double emission enhancement (DEE) platform. A thio-reactive fluorescence turn-on molecule, N-butyl-4-(4-maleimidostyryl)-1,8-naphthalimide (CS), was bound to a silane coupling agent, (3-mercaptopropyl)-trimethoxysilane (MPTMS), and the product N-butyl-4-(3-(trimethoxysilyl-propylthio)styryl)-1,8-naphthalimide (CSP) was further used to fabricate a core–shell nanoparticle through the Stöber method. We concluded that the turn-on emission by CSP originated from the photoinduced electron transfer (PET) between the maleimide moiety and the CSP core scaffold, and the second emission enhancement was attributed to the aggregation-induced emission enhancement (AIEE) in CSP when encapsulated inside a core–shell nanoparticle. Thus, FSNs could be obtained through DEE based on a combination of PET and AIEE effects. Systematic investigations verified that the resulting FSNs showed the traditional solvent-independent and photostable optical properties. The results implied that the novel FSNs are suitable as biomarkers in living cells and function as fluorescent visualizing agents for intracellular imaging and drug carriers.     

Synthesis of an AIE-active fluorogen and its application in cell imaging

A fluorogen named 1-decyl-1-methyl-2,5-bis{4-[(N,N-diethylamino)methyl]phenyl}-3,4-diphenylsilole (3) was synthesized. It emitted weakly as isolated molecule but strongly as supramolecular aggregate, showing a characteristic behavior of aggregation-induced emission (AIE). The molecules of 3 formed highly emissive nanoparticles in aqueous media, which quickly and selectively marked cytoplasm of HeLa cells and posed no toxicity to the living cells. The fluorogen is thus a promising candidate material for cell imaging as a sensitive, selective and cytocompatible biosensor. Supported by the Research Grants Council of Hong Kong (Grant Nos. 603008, 601608 and 602707), the National Natural Science Foundation of China (Grant No. 20634020) and the CAO GuangBiao Foundation of Zhejiang University.     

Biocompatible and noncytotoxic nucleoside-based AIEgens sensor for lighting-up nucleic acids

Aggregation-induced emission luminogens (AIEgens) have been used in biomacromolecules detection. Herein, TPE-dC and TPE-dU acted as the nucleoside-based AIEgens sensors in the first case, which can be used to detect ctDNA and rRNA in vitro and light up the nucleus in vivo depending on the intermolecular binding affinity. This AIE process enables the quantitative analysis or visualization of nucleic acids in solution or gels state, respectively. Furthermore, confocal laser scanning microscopy (CLSM) images of L929 cells stained with TPE-dC or TPE-dU clearly shows that nucleoside-based AIEgens bio-probes can pass the cell membranes to reach the cell nucleus, without cytotoxicity at the imaging condition (incubation time > 12 h, and 10 μmol/L of concentration). Since the nucleus is rich in DNA/RNA, fluorescence turn-on mode has a great potential in nucleus imaging and clinical diagnosis.     

载药荧光纳米粒子的制备及在乳腺癌MCF-7细胞系的应用及效果评价

利用两亲性聚乙二醇-聚乳酸共聚物（PEG-PDLLA）包覆荧光染料（DPBA）和紫杉醇（PTX）,通过自组装方法制得载药荧光纳米粒子DPBA/PTX@PEG-PDLLA.纳米粒子尺寸均一,具有良好的生物相容性.对纳米粒子的发光性质、载药量和体外药物释放等进行了表征,并考察了纳米粒子对乳腺癌细胞MCF-7的抑制效果,观察了MCF-7细胞对纳米粒子的摄取情况.结果表明,DPBA/PTX@PEG-PDLLA纳米粒子具有较强的红光发射,不仅可以用于MCF-7肿瘤细胞质荧光成像,而且对肿瘤细胞的增殖具有一定的抑制能力.     

Protein detection and quantitation by tetraphenylethene-based fluorescent probes with aggregation-induced emission characteristics

Three functionalized derivatives of tetraphenylethylene (TPE), namely, 1,2-bis(4-methoxyphenyl)-1,2-diphenylethene (1), 1,2-bis(4-hydroxyphenyl)-1,2-diphenylethene (2), and 1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene sodium salt (3), were synthesized and their fluorescence properties were investigated. All the TPE molecules are nonluminescent in the solution state but are induced to emit efficiently by aggregate formation. This novel process of aggregation-induced emission (AIE) is rationalized to be caused by the restriction of intramolecular rotations of the dye molecules in the aggregate state. The possibility of utilizing the AIE effect for protein detection and quantification is explored using bovine serum albumin (BSA) as a model protein, with salt 3 being found to perform as a stable, sensitive, and selective bioprobe.     

A novel fluorescent silica tracer for biological silicification studies.

BACKGROUND: Biological silica production has drawn intense attention and several molecules involved in biosilicification have been identified. Cellular mechanisms, however, remain unknown mainly due to the lack of probes required for obtaining information on live specimens. RESULTS: The fluorescence spectra of the compound 2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole (PDMPO) are affected by the presence of >3.2 mM silicic acid. Increase in intensity and shift in the fluorescence coincide with the polymerization of Si. The unique PDMPO-silica fluorescence is explored here to visualize Si deposition in living diatoms. The fluorophore is selectively incorporated and co-deposited with Si into the newly synthesized frustules (the outer silica shells) showing an intense green fluorescence. CONCLUSIONS: We suggest that a fluorescence shift is due to an interaction between PDMPO and polymeric silicic acid. PDMPO is an excellent probe for imaging newly deposited silica in living cells and has also a potential for a wide range of applications in various Si-related disciplines, including biology of living organisms as diatoms, sponges, and higher plants, clinical research (e.g. lung fibrosis and cancer, bone development, artificial bone implantation), and chemistry and physics of materials research.     

One-step preparation of branched PEG functionalized AIE-active luminescent polymeric nanoprobes

The synthesis of amphiphilic aggregation-induced emission(AIE) dyes based organic nanoparticles has recently attracted increasing attention in the biomedical fields. These AIE dyes based nanoparticles could effectively overcome the aggregation caused quenching effect of conventional organic dyes, making them promising candidates for fabrication of ultrabright organic luminescent nanomaterials. In this work, AIE-active luminescent polymeric nanoparticles(4-NH_2-PEG-TPE-E LPNs) were facilely fabricated through Michael addition reaction between tetraphenylethene acrylate(TPE-E) and 4-arm-poly(ethylene glycol)-amine(4-NH_2-PEG) in rather mild ambient. The 4-NH_2-PEG can not only endow these AIE-active LPNs good water dispersibility, but also provide functional groups for further conjugation reaction. The size, morphology and luminescent properties of 4-NH_2-PEG-TPE-E LPNs were characterized by a series of techniques in detail. Results suggested that these AIE-active LPNs showed spherical morphology with diameter about 100–200 nm. The obtained 4-NH_2-PEG-TPE-E LPNs display high water dispersibility and strong fluorescence intensity because of their self assembly and AIE properties of TPE-E.Biological evaluation results demonstrated that 4-NH_2-PEG-TPE-E LPNs showed negative toxicity toward cancer cells and good fluorescent imaging performance. All of these features make 4-NH_2-PEG-TPE-E LPNs promising candidates for biological imaging and therapeutic applications.     

High-Performance Quinoline-Malononitrile Core as a Building Block for the Diversity-Oriented Synthesis of AIEgens

In vivo fluorescent monitoring of physiological processes with high-fidelity is essential in disease diagnosis and biological research, but faces extreme challenges due to aggregation-caused quenching (ACQ) and short-wavelength fluorescence. The development of high-performance and long-wavelength aggregation-induced emission (AIE) fluorophores is in high demand for precise optical bioimaging. The chromophore quinoline-malononitrile (QM) has recently emerged as a new class of AIE building block that possesses several notable features, such as red to near-infrared (NIR) emission, high brightness, marked photostability, and good biocompatibility. In this minireview, we summarize some recent advances of our established AIE building block of QM, focusing on the AIE mechanism, regulation of emission wavelength and morphology, the facile scale-up and fast preparation for AIE nanoparticles, as well as potential biomedical imaging applications.     

Glycosylated star-shaped conjugated oligomers for targeted two-photon fluorescence imaging

A glucopyranose functionalized star-shaped oligomer, N-tris{4,4',4'-[(1E)-2-(2-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-9,9-bis(6-2-amido-2-deoxy-1-thio-β-D-glucopyranose-hexyl)-9H-fluoren-7-yl)vinyl]phenyl}phenylamine (TVFVBN-S-NH(2)), is synthesized for two-photon fluorescence imaging. In water, TVFVBN-S-NH(2) self-assembles into nanoparticles with an average diameter of ～49?nm and shows a fluorescence quantum yield of 0.21. Two-photon fluorescence measurements reveal that TVFVBN-S-NH(2) has a two-photon absorption cross-section of ～1100 GM at 780?nm in water. The active amine group on the glucopyranose moiety allows further functionalization of TVFVBN-S-NH(2) with folic acid to yield TVFVBN-S-NH(2) FA with similar optical and physical properties as those for TVFVBN-S-NH(2). Cellular imaging studies reveal that TVFVBN-S-NH(2) FA has increased uptake by MCF-7 cells relative to that for TVFVBN-S-NH(2), due to specific interactions between folic acid and folate receptors on the MCF-7 cell membrane. This study demonstrates the effectiveness of glycosylation as a molecular engineering strategy to yield water-soluble materials with a large two-photon absorption (TPA) cross-section for targeted cancer-cell imaging.     

High‐Performance Quinoline‐Malononitrile Core as a Building Block for the Diversity‐Oriented Synthesis of AIEgens

In vivo fluorescent monitoring of physiological processes with high‐fidelity is essential in disease diagnosis and biological research, but faces extreme challenges due to aggregation‐caused quenching (ACQ) and short‐wavelength fluorescence. The development of high‐performance and long‐wavelength aggregation‐induced emission (AIE) fluorophores is in high demand for precise optical bioimaging. The chromophore quinoline‐malononitrile (QM) has recently emerged as a new class of AIE building block that possesses several notable features, such as red to near‐infrared (NIR) emission, high brightness, marked photostability, and good biocompatibility. In this minireview, we summarize some recent advances of our established AIE building block of QM, focusing on the AIE mechanism, regulation of emission wavelength and morphology, the facile scale‐up and fast preparation for AIE nanoparticles, as well as potential biomedical imaging applications.     

Structural and process controls of AIEgens for NIR-II theranostics

Aggregation-induced emission (AIE) is a cutting-edge fluorescence technology, giving highly-efficient solid-state photoluminescence. Particularly, AIE luminogens (AIEgens) with emission in the range of second near-infrared window (NIR-II, 1000–1700 nm) have displayed salient advantages for biomedical imaging and therapy. However, the molecular design strategy and underlying mechanism for regulating the balance between fluorescence (radiative pathway) and photothermal effect (non-radiative pathway) in these narrow bandgap materials remain obscure. In this review, we outline the latest achievements in the molecular guidelines and photophysical process control for developing highly efficient NIR-II emitters or photothermal agents with aggregation-induced emission (AIE) attributes. We provide insights to optimize fluorescence efficiency by regulating multi-hierarchical structures from single molecules (flexibilization) to molecular aggregates (rigidification). We also discuss the crucial role of intramolecular motions in molecular aggregates for balancing the functions of fluorescence imaging and photothermal therapy. The superiority of the NIR-II region is demonstrated by fluorescence/photoacoustic imaging of blood vessels and the brain as well as photothermal ablation of the tumor. Finally, a summary of the challenges and perspectives of NIR-II AIEgens for in vivo theranostics is given.

Structural and process controls of NIR-II AIEgens realize manipulating of radiative (R) and nonradiative (NR) decay for precise theranostics.     

Facilely prepared aggregation-induced emission (AIE) nanocrystals with deep-red emission for super-resolution imaging

Organic nanocrystals (NCs) with high brightness are highly desirable for biological imaging. However, the preparation of NCs by a facile and fast method is still challenging. Herein, an aggregation-induced emission (AIE) luminogen of 4,4′-(5,6-difluorobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(N,N-bis(4-methoxyphenyl)aniline) (DTPA-BT-F) in the deep-red region is designed with intensive crystalline features to obtain NCs by kinetically controlled nanoprecipitation. The prepared AIE NCs with high brightness and good photo-stability are then applied in super-resolution imaging via stimulated emission depletion (STED) nanoscopy. As observed, the nanostructures in lysosomes of both fixed and live cells are well visualized with superior lateral resolutions under STED nanoscopy (full width at half maximum values, 107 and 108 nm) in contrast to that in confocal imaging (548 and 740 nm). More importantly, dynamic monitoring and long-term tracking of lysosomal movements in live HeLa cells, such as lysosomal contact, can also be carried out by using DTPA-BT-F NCs at a superior resolution. To the best of our knowledge, this is the first case of AIE NCs prepared by nanoprecipitation for STED nanoscopy, thus providing a new strategy to develop high performance imaging agents for super-resolution imaging.

AIE nanocrystals with high brightness in the deep-red region were facilely prepared by kinetically controlled nanoprecipitation. These nanocrystals were then applied in super-resolution cellular imaging via STED nanoscopy.     

固载四苯基乙烯及其衍生物聚集体的聚乙烯醇薄膜对有机挥发气氛的检测

以聚乙烯醇(PVA)为粘胶剂,将四苯基乙烯(TPE)及其衍生物聚集体固载在滤纸上,以有效发挥AIE的特性.为了考察PVA浓度对四苯基乙烯及其衍生物聚集态下发光性能的影响规律,本文研究了PVA浓度分别与TPE、1,1,2,2-四-(4-(5-溴戊氧基)苯基)乙烯(TPE-OR)、(4-二苯基)苯基二苯并富烯(BpPDBF)三种化合物荧光强度之间的关系.实验结果表明,即使水含量不同,三种化合物均对PVA浓度依赖性存在着荧光强度极大值.说明提高PVA浓度,增加溶液粘度,将会使TPE及其衍生物分子内旋转进一步受限,但同时也会妨碍TPE及其衍生物形成更大的聚集体,即荧光强度的变化是这两种作用的综合体现.另外,固载于滤纸上的TPE、TPE-OR、BpPDBF薄膜可以用于检测特定有机溶剂及硝基化合物的挥发气氛,并具有比较灵敏的响应.     

Study of aggregation induced emission of cyano-substituted oligo (p-phenylenevinylene) by femtosecond time resolved fluorescence

The aggregation induced emission (AIE) mechanism of the cyano-substituted oligo (p-phenylenevinylene)1,4-bis [1-cyano-2-(4-(diphenylamino) phenyl) vinyl] benzene (TPCNDSB) is investigated by time resolved fluorescence technique. By reconstructing the time resolved emission spectra (TRES), it is found that in solvent of low polarity, the emission is mainly from the local emission (LE) state with high quantum yield, but in high polarity solvent, the emission is mainly from the intramolecular charge transfer (ICT) state, which is a relatively dark state, with low quantum yield. In crystal form, the restriction of transfer from LE state to ICT state results in efficient AIE.     

“Turn-On” Activatable AIE Dots for Tumor Hypoxia Imaging

A hypoxia-responsive fluorescence probe of amphiphilic PEGylated azobenzene caged tetraphenylethene (TPE) for tumor cell imaging is reported; it possesses excellent solubility in aqueous medium due to the easy formation of micelles by self-assembly. The fluorescence resonance energy transfer (FRET) process ensures that the fluorescence of the azobenene caged AIE fluorogen is quenched efficiently. When cultured with tumor cells, the azo-bond is reduced under hypoxia conditions and the fluorescence of AIE fluorogen recovers dramatically. Besides using UV light, NIR light can also be used as the excited light resource to generate the fluorescence due to the two-photon fluorescence imaging process.     

Subcellular Localization of Sulfonated Tetraphenyl Porphines in Colon Carcinoma Cells by Spectrally Resolved Imaging

Subcellular localization of the dye, 5,10,15,20-tetra(4-sul-fonatophenyl)porphine (TPPS₄) and the more hydrophobic dye, 5,10,15,20-tetra(1-sulfonatophenyl)porphine (TPPS₁), in murine colon carcinoma cells was studied by spectrally resolved imaging (SRI) combined with image processing techniques. Spectrally resolved imaging enabled the acquisition of multipixel fluorescence spectra (>10⁴) from a single cell. Demarcation of specific localization sites and segregation of the irrelevant fluorescence were based on the pixel spectra and by operating the functions of spectral similarity mapping (SSM), principal component analysis (PCA) and spectral classification. The SRI revealed the fine details of the photochemical process that clarify some aspects of subcellular damage. The SRI depicted the differences between TPPS₄ and TPPS, with respect to their initial localization and their fate at the end of the photochemical effect. The dye TPPS₄ was localized initially in lysosomal vesicles, and upon irradiation fluorescence was seen in the nucleus as well as in vesicles. Some of the vesicles were closely related to the nucleus, as resolved by SSM, PCA and spectral classification. Additional light exposure stimulated relocalization of TPPS₄ into the nucleus as well as into the nucleolus, which was clearly depicted by SSM and PCA. Spectral classification showed a third, weak residual cytoplasmic array around the nucleus. The dye TPPS, concentrated in a Golgi-like complex and was resolved in the nuclear envelope and in small vesicles: it was not redistributed into other compartments upon photosensitization. Serum supplementation to the incubation media of colon carcinoma cells treated with TPPS₄ or TPPS, did not change the localization patterns. Pixel spectra of the two dyes in the cells showed spectral shifts and expanded shoulders due to microenvironmental effects. Thus, the chemical nature of the sulfonated phenyl porphines, and not their interaction with serum proteins, was the main determinant of their binding to the lysosomes, nucleus, nucleolus, nuclear envelope or Golgi.