Defining a Polymethine Dye for Fluorescence Anisotropy Applications in the Near‐Infrared Spectral Range

Fluorescence anisotropy in the near‐infrared (NIR) spectral range is challenging because of the lack of appropriate NIR fluorescent labels. We have evaluated polymethine fluorescent dyes to identify a leading candidate for NIR anisotropy applications. The NIR dye LS601 demonstrated low fluorescence anisotropy values (r) as a result of its relatively long fluorescent lifetime 1.3 ns. The r value of LS601 unbound and coupled to biological macromolecules was found to have a sufficient dynamic range from 0.24 to 0.37, demonstrating the feasibility of fluorescence anisotropy in the NIR. The viability of fluorescence anisotropy using a NIR label was demonstrated by characterization of dye–protein conjugates. These results open the door to a number of applications in drug discovery, fluorescence anisotropy imaging and contrast agent development.     

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.     

An enzyme-responsive metal-enhanced near-infrared fluorescence sensor based on functionalized gold nanoparticles

Near-infrared (NIR) fluorescence imaging is promising due to the high penetration depths and minimal levels of autofluorescence in living systems. However, it suffers from low fluorescent quantum yield, and metal-enhanced fluorescence (MEF) is considered to be a promising technique to overcome this. Stimuli-responsive NIR fluorescence enhancement shows remarkable potential for applications in medical imaging and diagnosis. Herein, we successfully fabricated an enzyme-responsive near-infrared sensor based on MEF by functionalizing gold nanoparticles with NIR fluorophores and enzyme-responsive self-aggregation moieties. The NIR fluorescence of fluorophores on the gold nanoparticles was significantly enhanced due to increases both in the light scattering intensity and in the radiative decay rate (k _r) of the NIR fluorophores, along with relatively small variation in the nonradiative decay rate. This novel strategy for NIR fluorescent sensors should be particularly promising for NIR fluorescence imaging of enzyme activities and early diagnosis based on rationally designed nanomaterials.     

Semiconducting polymer dots as near‐infrared fluorescent probes for bioimaging and sensing

In recent years, semiconducting polymer dots (Pdots) have emerged as a new type of ultrabright fluorescent probes, which have been proved to be very useful for biomedical imaging. Pdots possess several exceptional advantages including high fluorescence brightness, fast radiative rate, excellent photostability, and negligible cytotoxicity. Among these new types of Pdots, the near‐infrared (NIR) fluorescent Pdots appear to be the most urgent and important owing to their promising deep‐tissue imaging in the clinic. This mini‐review highlights the recent progress in the design of NIR‐emitting Pdots and their biomedical applications both in vitro and in vivo.     

Near IR Fluorescent Polystyrene/Albumin Core/Shell Nanoparticles for Specific Targeting of Colonic Neoplasms

Previous studies have shown that albumin has a high affinity towards tumours, and, as a result, many drug/albumin conjugates, as well as albumin nanoparticles, have been studied as antineoplastic drug carriers. Numerous studies have also shown the high affinity of cyanine dyes for albumin. This work combines the former and the latter for the preparation of NIR fluorescent and photostable nanoparticles as diagnostic biomaterials. Tumour‐specific labelling by NIR fluorescent polystyrene/albumin core/shell nanoparticles is demonstrated, without the presence of additional targeting moieties, and they possess great potential for clinical applications.

     

Synthesis and Characterization of Far‐Red/NIR‐Fluorescent BODIPY Dyes,Solid‐State Fluorescence,and Application as Fluorescent Tags Attached to Carbon Nano‐onions

A series of π‐extended distyryl‐substituted boron dipyrromethene (BODIPY) derivatives with intense far‐red/near‐infrared (NIR) fluorescence was synthesized and characterized, with a view to enhance the dye’s performance for fluorescence labeling. An enhanced brightness was achieved by the introduction of two methyl substituents in the meso positions on the phenyl group of the BODIPY molecule; these substituents resulted in increased structural rigidity. Solid‐state fluorescence was observed for one of the distyryl‐substituted BODIPY derivatives. The introduction of a terminal bromo substituent allows for the subsequent immobilization of the BODIPY fluorophore on the surface of carbon nano‐onions (CNOs), which leads to potential imaging agents for biological and biomedical applications. The far‐red/NIR‐fluorescent CNO nanoparticles were characterized by absorption, fluorescence, and Raman spectroscopies, as well as by thermogravimetric analysis, dynamic light scattering, high‐resolution transmission electron microscopy, and confocal microscopy.     

Synthesis and characterization of monodisperse, mesoporous, and magnetic sub-micron particles doped with a near-infrared fluorescent dye

Recently, multifunctional silica nanoparticles have been investigated extensively for their potential use in biomedical applications. We have prepared sub-micron monodisperse and stable multifunctional mesoporous silica particles with a high level of magnetization and fluorescence in the near infrared region using an one-pot synthesis technique. Commercial magnetite nanocrystals and a conjugated-NIR-dye were incorporated inside the particles during the silica condensation reaction. The particles were then coated with polyethyleneglycol to stop aggregation. X-ray diffraction, N₂ adsorption analysis, TEM, fluorescence and absorbance measurements were used to structurally characterize the particles. These mesoporous silica spheres have a large surface area (1978 m²/g) with 3.40 nm pore diameter and a high fluorescence in the near infrared region at λ=700 nm. To explore the potential of these particles for drug delivery applications, the pore accessibility to hydrophobic drugs was simulated by successfully trapping a hydrophobic ruthenium dye complex inside the particle with an estimated concentration of 3 wt%. Fluorescence imaging confirmed the presence of both NIR dye and the post-grafted ruthenium dye complex inside the particles. These particles moved at approximately 150 μm/s under the influence of a magnetic field, hence demonstrating the multifunctionality and potential for biomedical applications in targeting and imaging.     

Multifunctional Uniform Core–Shell Fe3O4@mSiO2 Mesoporous Nanoparticles for Bimodal Imaging and Photothermal Therapy

Multimodal imaging and simultaneous therapy is highly desirable because it can provide complementary information from each imaging modality for accurate diagnosis and, at the same time, afford an imaging‐guided focused tumor therapy. In this study, indocyanine green (ICG), a near‐infrared (NIR) imaging agent and perfect NIR light absorber for laser‐mediated photothermal therapy, was successfully incorporated into superparamagnetic Fe₃O₄@mSiO₂ core–shell nanoparticles to combine the merit of NIR/magnetic resonance (MR) bimodal imaging properties with NIR photothermal therapy. The resultant nanoparticles were homogenously coated with poly(allylamine hydrochloride) (PAH) to make the surface of the composite nanoparticles positively charged, which would enhance cellular uptake driven by electrostatic interactions between the positive surface of the nanoparticles and the negative surface of the cancer cell. A high biocompatibility of the achieved nanoparticles was demonstrated by using a cell cytotoxicity assay. Moreover, confocal laser scanning microscopy (CLSM) observations indicated excellent NIR fluorescent imaging properties of the ICG‐loaded nanoparticles. The relatively high r₂ value (171.6 mM ^?1 s^?1) of the nanoparticles implies its excellent capability as a contrast agent for MRI. More importantly, the ICG‐loaded nanoparticles showed perfect NIR photothermal therapy properties, thus indicating their potential for simultaneous cancer diagnosis as highly effective NIR/MR bimodal imaging probes and for NIR photothermal therapy of cancerous cells.     

Inorganic–Organic Hybrid Nanoparticles with Biocompatible Calcium Phosphate Thin Shells for Fluorescence Enhancement

Polymeric micelles consisting of asymmetric triblock copolymers were successfully used for fabrication of robust hybrid nanoparticles with highly biocompatible calcium phosphate shells. The hydrophobic polystyrene core encapsulates hydrophobic fluorescent dyes such as Nile red. The anionic polyacrylic acid provides the site for the mineralization reaction of calcium phosphate. The polyethylene glycol corona stabilizes the hybrid nanoparticles. Fluorescent dyes can be used as imaging agents for determining the location of the nanoparticles and to give an observable indication of drug delivery, while the calcium phosphate shell can enhance the fluorescence of the encapsulated dye.     

Near-infrared dyes,nanomaterials and proteins

Taking the advantage of reduced scattering and low autofluorescence background, the NIR fluorescence probes, such as fluorescence proteins, organic molecules and nanoparticles, not only hold the promise of in vivo imaging of biological processes in physiology and pathology with high signal-to-noise ratio, but also for clinical diagnosis. In this review, we provide an overview of the recent progress on NIR probes, focusing on fundamental mechanisms of NIR dyes and nanoparticles, and protein engineering strategies for NIR proteins.     

Keto‐benzo[h]‐Coumarin‐Based Near‐Infrared Dyes with Large Stokes Shifts for Bioimaging Applications

Fluorescence imaging is a promising tool for the visualization of biomolecules in living systems and there is great demand for new fluorescent dyes that absorb and emit in the near‐infrared (NIR) region. Herein, we constructed three new fluorescent dyes ( NBC dyes) based on keto‐benzo[h]coumarin ( k‐BC ) and benzopyrilium salts. These dyes showed large Stokes shifts (>100 nm) and NIR emission (>800 nm). The relationship between the structures and optical properties of these dyes was further investigated by using density functional theory calculations at the B3LYP/6‐3G level of theory. Fluorescence images indicated that the fabricated dyes exhibited good photostability and low cytotoxicity and, thus, have potential applications as imaging agents in living cells and animals.     

新型铕配合物二氧化硅荧光纳米粒子DPPDA-Eu3+/SiO2的制备与表征

本文首先合成配位体4,7-二苯基-1,10-菲罗啉-2,9-二羧酸(DPPDA,C₂₆H₁₆N₂O₄)及铕配合物DPPDA-Eu³⁺((C₂₆H₁₆N₂O₄)₂Eu·15H₂O),然后采用反相微乳液法,通过正硅酸乙酯和3-氨丙基三甲氧基硅烷的共水解、聚合作用成功制备出表面带氨基的二氧化硅包裹铕配合物DPPDA-Eu³⁺的核壳型荧光纳米颗粒DPPDA-Eu³⁺/SiO₂。利用透射电子显微镜、荧光光谱、紫外-可见光谱等手段进行表征,并进行了光稳定性、荧光泄露与氨基测定等实验,结果表明所制备的纳米粒子呈规则球状,大小均匀,粒径为80±8 nm,具有良好的单分散性和光稳定性,不易发生荧光分子从二氧化硅壳层中泄露,纳米粒子表面带有氨基,可不需要进行表面修饰而直接与生物分子反应。该纳米粒子可望作为一种新型的稀土荧光探针应用于时间分辨荧光免疫分析、生物芯片及生物传感器等。     

Photoswitchable Fluorescent Nanoparticles: Preparation,Properties and Applications

This minireview highlights recent advances of research dedicated to photoswitchable fluorescent nanoparticles and their applications. Recently, several strategies have been developed to synthesize nanoparticles with optically switchable emission properties: either fluorescence on/off or dual‐alternating‐color fluorescence photoswitching. The underlying mechanisms of fluorescence photoswitching enable many different types of photoswitchable fluorescent nanoparticles to change fluorescence colors, thus validating the basis of the initial photoswitching design. Among all possible applications, the usage of photoswitchable fluorescent nanoparticles to empower super‐resolution fluorescence imaging and to label biological targets was subsequently reviewed. Finally, we summarize the important areas regarding future research and development on photoswitchable fluorescent nanoparticles.     

A Three‐Component Assembly Promoted by Boronic Acids Delivers a Modular Fluorophore Platform (BASHY Dyes)

The modular assembly of boronic acids with Schiff‐base ligands enabled the construction of innovative fluorescent dyes [boronic acid salicylidenehydrazone (BASHY)] with suitable structural and photophysical properties for live cell bioimaging applications. This reaction enabled the straightforward synthesis (yields up to 99 %) of structurally diverse and photostable dyes that exhibit a polarity‐sensitive green‐to‐yellow emission with high quantum yields of up to 0.6 in nonpolar environments. These dyes displayed a high brightness (up to 54 000 m ^?1 cm^?1). The promising structural and fluorescence properties of BASHY dyes fostered the preparation of non‐cytotoxic, stable, and highly fluorescent poly(lactide‐co‐glycolide) nanoparticles that were effectively internalized by dendritic cells. The dyes were also shown to selectively stain lipid droplets in HeLa cells, without inducing any appreciable cytotoxicity or competing plasma membrane labeling; this confirmed their potential as fluorescent stains.     

Dual-Emissive Platinum(II) Metallacage with a Sensitive Oxygen Response for Imaging of Hypoxia and Imaging-Guided Chemotherapy

Imaging of hypoxia in vivo helps with accurate cancer diagnosis and evaluation of therapeutic outcomes. A Pt^II metallacage with oxygen-responsive red phosphorescence and steady fluorescence for in vivo hypoxia imaging and chemotherapy is reported. The therapeutic agent and diagnostic probe were integrated into the metallacage through heteroligation-directed self-assembly. Nanoformulation by encapsulating the metallacage into nanoparticles greatly enhanced its stability the in physiological environment, rendering biomedical applications feasible. Apart from enhanced red phosphorescence upon hypoxia, the ratio between red and blue emissions, which only varies with intracellular oxygen level, provides a more precise standard for hypoxia imaging and detection. Moreover, in vivo explorations demonstrate the promising potential applications of the metallacage-loaded nanoparticles as theranostic agents for tumor hypoxia imaging and chemotherapy.     

Near-infrared fluorescence spectroscopy of single-walled carbon nanotubes and its applications

Semiconducting single-walled carbon nanotubes (SWCNTs) emit fluorescence at near-infrared (NIR) wavelengths that are characteristic of the specific diameter and the chiral angle. While providing a convenient method for structural identification of semiconducting SWCNTs, NIR fluorescence of SWCNTs also offers a powerful approach for sensor development and in vivo or real-time imaging of biological systems.This article provides an introductory overview of the approaches to obtaining individually dispersed semiconducting SWCNTs with reasonably good purity, which is a critical step in acquiring NIR fluorescence spectra. It also summarizes the progress since 2002 in sensor design and applications in bioimaging in vitro and in vivo using NIR fluorescence of semiconducting SWCNTs.     

A unique class of near-infrared functional fluorescent dyes with carboxylic-acid-modulated fluorescence ON/OFF switching: rational design, synthesis, optical properties, theoretical calculations, and applications for fluorescence imaging in living animals

Fluorescence imaging is one of the most powerful techniques for monitoring biomolecules in living systems. Fluorescent sensors with absorption and emission in the near-infrared (NIR) region are favorable for biological imaging applications in living animals, as NIR light leads to minimum photodamage, deep tissue penetration, and minimum background autofluorescence interference. Herein, we have introduced a new strategy to design NIR functional dyes with the carboxylic-acid-controlled fluorescence on-off switching mechanism by the spirocyclization. Based on the design strategy, we have developed a series of Changsha (CS1-6) NIR fluorophores, a unique new class of NIR functional fluorescent dyes, bearing excellent photophysical properties including large absorption extinction coefficients, high fluorescence quantum yields, high brightness, good photostability, and sufficient chemical stability. Significantly, the new CS1-6 NIR dyes are superior to the traditional rhodamine dyes with both absorption and emission in the NIR region while retaining the rhodamine-like fluorescence ON-OFF switching mechanism. In addition, we have performed quantum chemical calculations with the B3LYP exchange functional employing 6-31G* basis sets to shed light on the structure-optical properties of the new CS1-6 NIR dyes. Furthermore, using CS2 as a platform, we further constructed the novel NIR fluorescent TURN-ON sensor 7, which is capable of imaging endogenously produced HClO in the living animals, demonstrating the value of our new CS NIR functional fluorescent dyes. We expect that the design strategy may be extended for development of a wide variety of NIR functional dyes with a suitable fluorescence-controlled mechanism for many useful applications in biological studies.     

A near-infrared fluorescent sensor for detection of cyanide in aqueous solution and its application for bioimaging

A new NIR fluorescent sensor based on an amine-substituted heptamethine cyanine dye displayed a highly selective fluorescence enhancement with cyanide in aqueous solutions, and was applied for the imaging of anthropogenic and biogenic cyanide.     

Application of Deuteporfin in the Metastatic Lymph Node Mapping of Pancreatic Cancer: An in vivo Study

For most cancer patients, the presence of metastatic lymph nodes usually indicates regional recurrence and poor prognosis. Therefore, lymph node mapping is a requisite for disease staging, prognosis prediction and decision making in the treatment of cancer. Deuteporfin, a second‐generation photosensitizer, has a maximum excitation wavelength that can reach the near infrared (NIR) region (650–700 nm). We aimed to take advantage of these aspects of deuteporfin and use it as a fluorescent probe for metastatic lymph node mapping in vivo using NIR fluorescent imaging. In our study, we further investigated whether a photosensitizer could be used as a tracer for metastatic lymph node mapping of pancreatic cancer based on previous reports. Compared to normal tissues, tumor tissues including primary tumors and metastatic lymph nodes had a higher uptake ability of deuteporfin (P < 0.05). Our research confirmed this targeting property of deuteporfin using in vivo fluorescent imaging. Consistent with observations from in vivo imaging experiments, frozen sections of metastatic lymph nodes intuitively displayed significantly higher and wider distributions of deuteporfin than normal sections.     

Fluorescent Phthalocyanine-Encapsulated Bovine Serum Albumin Nanoparticles: Their Deployment as Therapeutic Agents in the NIR Region

In recent times, researchers have aimed for new strategies to combat cancer by the implementation of nanotechnologies in biomedical applications. This work focuses on developing protein-based nanoparticles loaded with a newly synthesized NIR emitting and absorbing phthalocyanine dye, with photodynamic and photothermal properties. More precisely, we synthesized highly reproducible bovine serum albumin-based nanoparticles (75% particle yield) through a two-step protocol and successfully encapsulated the NIR active photosensitizer agent, achieving a good loading efficiency of 91%. Making use of molecular docking simulations, we confirm that the NIR photosensitizer is well protected within the nanoparticles, docked in site I of the albumin molecule. Encouraging results were obtained for our nanoparticles towards biomedical use, thanks to their negatively charged surface (−13.6 ± 0.5 mV) and hydrodynamic diameter (25.06 ± 0.62 nm), favorable for benefitting from the enhanced permeability and retention effect; moreover, the MTT viability assay upholds the good biocompatibility of our NIR active nanoparticles. Finally, upon irradiation with an NIR 785 nm laser, the dual phototherapeutic effect of our NIR fluorescent nanoparticles was highlighted by their excellent light-to-heat conversion performance (photothermal conversion efficiency 20%) and good photothermal and size stability, supporting their further implementation as fluorescent therapeutic agents in biomedical applications.