Читать книгу Handbook of Aggregation-Induced Emission, Volume 2 - Группа авторов - Страница 33

Fluorescence bioimaging is one of the most powerful bioimaging techniques for real‐time, noninvasive monitoring biomolecules of interest in their native environments with high spatial and temporal resolution. The development of fluorescent probes has facilitated the recent significant advances in cell biology and medical diagnostic imaging [68, 69]. Figure 2.8 shows some small molecules and macromolecules based on DSA applied in fluorescent bioimage.

Tian et al. reported a kind of highly emissive inorganic–organic nanoparticle with a core–shell structure for targeted cancer cell imaging [20]. The nanoparticles are coated with a folate‐functionalized silica shell, and DSA derivative 3‐1 with AIE properties serves as the fluorescent core, affording the folate‐functionalized fluorescent silica nanoparticles (FFSNPs) with a high fluorescence quantum yield (up to 20%). The FFSNPs have a small size whose diameter is ~60 nm (see Figure 2.9a), and they are monodispersed, stable in aqueous suspension. Whereas the solution of 3‐chloropropyltriethoxysilane (CPS)‐DSA adduct 3 and the suspensions of silica nanoparticles (SNPs) almost have no light emission under UV illumination, an intense yellow light is emitted from FFSNPs (see Figure 2.9b). In addition, the FFSNPs have low toxicity to living cells and thus are suitable to be utilized for targeted HeLa cell imaging (see Figure 2.9c, d) [20].

Wei et al. reported that luminescent silica nanoparticles could be easily fabricated by encapsulating an AIE dye 3‐2 (named An18) via a modified Stöber method [70]. In this method, octadecyltrimethoxysilane (C18‐Si) and 3‐2 were first self‐assembled and served as the core of the silica nanoparticles. Then, another silicate precursor, tetraethoxysilane, was further coated on the luminescent core, thus forming luminescent silica nanoparticles. The results demonstrated that An18‐SiO₂ nanoparticles have a uniform spherical morphology with a diameter of 70–80 nm. In addition, they exhibited high water dispersibility, remarkable fluorescent properties, and excellent biocompatibility, which makes them a promising candidate material for various biomedical applications. Moreover, they also facilely prepared water‐soluble and biocompatible fluorescent organic nanoparticles based on the AIE molecule 3‐2 by mixing 3‐2 and surfactant; further, such fluorescent organic nanoparticles were also utilized in cell imaging [71].

Figure 2.8 Small molecules and macromolecules of DSA derivatives applied in bioimage.

Prasad et al. prepared organically modified silica nanoparticles of ~30 nm, which are stably dispersible in aqueous solution [72]. The nanoparticles were fabricated by coencapsulating a photosensitizing anticancer drug 3‐(4‐hydroxyphenyl)propionic acid hydrazide (HPPH) and fluorescent aggregates of a two‐photon absorbing dye 3‐3. The results indicated that the dye 3‐3 aggregates can efficiently upconvert the energy of near‐IR light and transfer it to the coencapsulated HPPH molecules via the fluorescence resonance energy transfer (FRET) mechanism, which leads to the enhanced two‐photon generation of singlet oxygen in water. Therefore, the prepared nanoparticles in this work can be applied in the two‐photon photodynamic therapy. In addition, they prepared a kind of silicon dioxide nanoparticle with high luminescence using the AIE compound 3‐3 through the aggregation‐enhanced fluorescence approach, and two‐photon fluorescence imaging was performed on HeLa cells [73].

Figure 2.9 (a) Scanning electron microscopy (SEM) images of monodispersed folate‐functionalized fluorescent silica nanoparticles (FFSNPs) with particle sizes around 60 nm; (b) solution of 3‐chloropropyltriethoxysilane (CPS)‐DSA adduct 3 and suspensions of silica nanoparticles (SNPs) and FFSNPs in ethanol; photograph taken upon irradiation with a UV light of 365 nm; (c) folate‐mediated delivery of FFSNPs to folate receptor‐positive cancer cells. A fraction of the FFSNPs will traffic into the cancer cells by receptor‐mediated endocytosis (left side of the figure), while the remainder will remain on the cell surfaces (right side of the figure); two types of strategies can be envisioned: (d) confocal fluorescence images of FFSNPs for HeLa cells: overlay of fluorescence image and bright‐field image [20].

Source: Reproduced from Ref. [20] with permission from the Royal Society of Chemistry.

Tian et al. successfully designed and synthesized a novel fluorescent molecule 3‐4 based on a diselenide bond and DSA AIEgens. The compound 3‐4 can self‐assemble into uniform nanoparticles via the generally used nanoprecipitation method, and the nanoparticles emit a bright orange fluorescence. The redox‐responsive fluorescent nanoparticles based on the diselenide bond and DSA AIEgens have potential applications in cell imaging and reduction‐sensitive drug delivery for selective cancer therapy [74].

Lu et al. developed a fluorescence‐amplified AIE probe by incorporating a hydrophobic AIE fluorophore 2‐1 into biocompatible F127‐FA nanoparticles. A significant FRET effect took place through encapsulating donor 2‐1 and acceptor bis(4‐(N‐(2‐naphthyl)phenylamino)‐phenyl)fumaronitrile (NPAPF) simultaneously into the nanoparticles, which resulted in the notable increase of acceptor emission. Furthermore, 2‐1 and NPAPF coloaded F127‐FA nanoparticles showed a large Stokes shift, spherical morphology, and low cytotoxicity [75].

Stable AIEgen‐based nanoparticles of different shapes were prepared by assembling the copolymer and DSA and applied in noninvasive long‐term imaging. The nanoparticles have excellent physical and photostability under physiological conditions. The in vitro experimental results verified that these AIE‐active organic nanoparticles are biocompatible and internalized through various pathways of cellular uptake. The long‐term imaging ability was verified by the in vitro and in vivo experiments. It was confirmed that the rod‐like nanoparticles were significantly more internalized than the spherical particles, and they exhibited a better imaging effect [76].

Tian et al. prepared fluorescent nanoparticles by constructing a coassembly of human papillomavirus (HPV) capsid protein L1 and a complex consisting of DNA and an AIE molecule 3‐5. The virus‐like particles (VLPs) of HPV encapsulate the complex via electrostatic interaction. The coassembled nanoparticles, 3‐5‐DNA@VLPs, demonstrated a homogeneous size of ∼53 nm and enhanced fluorescence. Notably, the strong cell‐entry ability of VLPs endowed the AIE–DNA complex to enter the cell easily, which led to efficient brighter imaging in HeLa and normal 293T cell lines. Therefore, this work supplied a powerful approach to deliver the AIEgen into cells, which not only provided a simple, fast, biocompatible, and highly efficient fluorescence tool for in vivo cell imaging but also largely extended the bioapplications of AIEgens [77].

AIEgen‐based poly(l‐lactic‐co‐glycolic acid) (PLGA) magnetic nanoparticles to localize cytokine vascular endothelial growth factor (VEGF) were developed. The nanoparticles, as a novel theranostic system, could be utilized for simultaneous photothermal therapy and magnetic resonance imaging, which was applicable to early diagnostics and treatment of cancer cells. The system was constructed by loading the oleic acid‐Fe₃O₄ and an AIEgen, triphenylamine‐divinylanthracene‐dicyano, inside the PLGA shell, which was then modified with anti‐VEGF‐A antibodies. By adjusting the proportion of AIEgens and Fe₃O₄, this system showed a strong red emission desirable for early cancer cell diagnosis, as well as magnetic properties suitable for application in magnetic resonance imaging [78].

Besides the AIE small molecules, there are also some AIE macromolecules applied in the fluorescent bioimaging. A series of random copolymers (polymer 3‐6 in Figure 2.8) based on poly[N‐(2‐hydroxypropyl)methacrylamide] (PHPMA) and hydrophobic AIE fluorophores DSA were prepared. It indicated that enhancing the molar fractions of the hydrophobic AIE fluorophores can lead to increasing the quantum efficiencies of the copolymers. These polymers were almost nonemissive in the solvent of dimethyl sulfoxide (DMSO) but emitted strong fluorescence in the mixed solvent of DMSO/water. It was confirmed that these polymers formed small micelles with an average diameter of about 10 nm in the aqueous solutions. Although the DSA fluorophore is water‐insoluble, when it chemically conjugates with the biocompatible polymer PHPMA, the prepared final copolymers, which are noncytotoxic to living cells, can be applied in biological condition for bioimaging [23]. In addition, by using PHPMA and DSA fluorophores, they prepared a new series of random copolymers (polymer 3‐7 in Figure 2.8). Similarly, the fluorescence quantum yields of the AIE copolymers in aqueous solutions increase with increasing the molar fractions of the hydrophobic AIE fluorophores and/or the trifluoromethyl moieties. It was the first report that the AIE fluorophores were integrated with fluorine‐containing polymers to manipulate the quantum yields of the AIE fluorophores [79].

Tian et al. prepared new polymer dots (AIE Pdots) through a self‐assembly process by using an AIE‐conjugated block copolymer 3‐8. The copolymer contains an AIE fluorophore DSA, hydrophobic poly(ɛ‐caprolactone) segments, hydrophilic poly(ethylene glycol) segments, and folate groups. The AIE Pdots have an average diameter of 15 nm and exhibited a good monodispersity in H₂O. They possess a high solid‐state fluorescence quantum efficiency of Φ = 27.0%. Moreover, the AIE Pdots presented good stability and low toxicity to living cells. The biological imaging results showed that the folic acid‐functionalized AIE Pdots can be applied in targeted HeLa intracellular imaging [80].

Tian et al. successfully designed and synthesized an AIE macromolecule 3‐9 by combining the red‐emissive fluorophores with propeller‐shaped AIE fluorophores based on DSA. Then, the ultrabright red‐emissive AIE dot 3‐9@PS‐PVP (3‐9 in poly(styrene)‐poly(4‐vinylpyridine nanoparticles)) was fabricated by using 3‐9 as the core and biocompatible PS‐PVP as the encapsulation matrix. The prepared AIE dots presented a spherical morphology and uniform small size. In addition, they have good stability in water and little cytotoxicity to living cell. Furthermore, the dots can stain both the cytoplasm and the nuclei and emit a strong red fluorescence signal. Figure 2.10a–c shows the confocal laser scanning microscopy images of HeLa cells after incubation with the AIE dots for 16 hours at 37 °C. Figure 2.10d illustrates a possible mechanism of the uptake of the AIE dots by the nuclei [81]. The pyridine salt on the outer layer of the nanoparticles carries positive charges, which was obtained through protonating the PVP chains by the hydrochloric acid. This pyridine salt is like the quaternary ammonium salt, which has a high affinity with negatively charged DNA (binding constant of ∼10⁵ M⁻¹) [82]. Therefore, the surface of the dots could absorb DNA or RNA. Benefiting from their small size simultaneously, the dots could go along with DNA or RNA into the nuclei when the genetic substance delivery process occurs [83–85].

Figure 2.10 Confocal laser scanning microscopy images of HeLa cells after incubation with 3‐9@PS‐PVP (3‐9 in poly(styrene)‐poly(4‐vinylpyridine nanoparticles) (with a fluorogen loading of 10%) for 16 hours at 37 °C. (a) Fluorescence image; (b) bright‐field image; and (c) overlay of (a) and (b). Concentration of AIE dots: 0.15 mg/ml. (d) Illustration of the possible mechanism of the uptake of the AIE dots by the nuclei [81].

Source: Reproduced from Ref. [81] with permission from the Royal Society of Chemistry.

Wei et al. utilized phospholipid monomer firstly to fabricate cross‐linked fluorescent polymeric nanoparticles by using the AIE dye 3‐10 based on DSA fluorophore [86]. The prepared nanoparticles exhibited strong fluorescence and stable dispersibility in physiological solution below the critical micellar concentration. Moreover, the nanoparticles also have excellent biocompatibility, which makes them have potential applications in cell imaging.