Various biological imaging modalities have become essential tools in life science research, preclinical research, and clinical practice. The emergence of enormous in vivo imaging technologies such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-beam emission computed tomography (SPECT) plays a significant role in disease diagnosis, progression monitoring, and prognosis, bringing the possibility of molecular imaging into medical observation.
Although they have unlimited penetration depth, the above techniques suffer from disadvantages such as limited spatial resolution, long operation times, and low sensitivity. Additionally, the equipment is often very expensive and induces radiation. On the other hand, as a radiation-free technique, fluorescence imaging has been widely used for in vivo imaging due to its high spatiotemporal resolution and labeling specificity. However, the performance of FL imaging is deteriorated by the strong absorption, scattering, and autofluorescence of biological tissues in the visible (400?700 nm) or NIR-I (700?900 nm) regions and shows unsatisfactory penetration depth, spatial resolution and signal-to-noise ratio (SNR), limiting its further application in in vivo imaging.
FL in the second near-infrared region (NIR-II, 1000?1700 nm), on the contrary, shows appealing advantages due to its deeper penetration depth (>10 mm), improved spatial resolution (about 3 μm), and higher signal-to-noise ratio (about 20), unveiling great clinical translation. Since single-walled carbon nanotubes (SWNTs) were first applied to NIR-II fluorescence imaging in small animals, the development of NIR-II fluorescent probes with high molar absorbance coefficients, high fluorescence quantum yields, good stability, and good biocompatibility has been a research hotspot. In the past decade, NIR-II fluorescent probes were primarily classified into two categories: organic and inorganic probes. The NIR-II inorganic fluorescent probes (e.g., single-walled carbon nanotubes, quantum dots, and rare earth doped conversion materials) have strong heavy metal toxicity, are typically poor in biocompatibility, and have difficulty completing physiological metabolism in vivo, limiting their potential applications in clinical practice. On the other side, organic NIR-II fluorescent probes are free of heavy metal ions and have clear structures as well as better biocompatibility, making them more suitable and promising for clinical translation. Some representative examples are D-A-D small molecules, cyanine dyes, and conjugated polymers. Hence, to guide the future development of this field more rationally, it is important and necessary to summarize the molecular structure design concepts and biomedical imaging applications of organic NIR-II fluorescent probes.
In this review, we systematically summarize the molecular structure design concepts and biomedical imaging applications of organic NIR-II fluorescent probes reported in the current literatures. The research progress of organic NIR-II fluorescent probes is classified into anatase dyes, D-A-D organic small molecules, and conjugated polymers.
First, the molecular design strategies of cyanine dyes with NIR-II emission wavelengths are summarized in terms of red-shifting absorption/emission wavelength, improving fluorescence quantum yield, enhancing biocompatibility, and chemical stability, respectively. Up to now, the reasonable and result-oriented design strategies to achieve cyanine dyes with NIR-II emission wavelengths primarily include: 1) extending the effective conjugation system, 2) modifying the donor and acceptor units, and 3) constructing fluorophore J-polymer. The strategies to enhance the fluorescence brightness primarily include: 1) introducing spatial site resistance, 2) forming complexes with proteins, and 3) enhancing the rigidity of molecular structures. The effective strategies to improve biocompatibility primarily include: 1) encapsulating hydrophobic fluorescent molecules by nanoprecipitation using amphiphilic materials and 2) introducing hydrophilic groups on hydrophobic fluorescent molecules utilizing molecular engineering.
Second, the development process of D-A-D small molecules in terms of donor/acceptor unit modulation and fluorescence quantum efficiency enhancement is also presented. In 2016, Dai's team reported for the first time that the water-soluble small molecule CH1055-PEG could be used for NIR-II fluorescence imaging. Since then, a series of small molecules with NIR-II emission have been designed by modulating the electron-giving/absorbing ability of donor/acceptor units. Moreover, strategies have been proposed to enhance fluorescence quantum efficiency, such as by introducing shielding units, suppressing TICT states, constructing hydrophobic nonpolar environments, and building fluorescent small molecules with AIE properties.
Subsequently, we summarize the molecular design strategies of organic conjugated polymers with high brightness and further discuss their applications in bioimaging, primarily including tumor imaging, dynamic angiography, and photothermal therapy.
Finally, the issues and challenges that need to be addressed to identify the clinical translation of NIR-II fluorescence imaging techniques are discussed.
NIR-II fluorescence imaging has been widely used in basic scientific research and preclinical practice. Organic NIR-II fluorescent probes are highly amenable to clinical translation due to their excellent biocompatibility, good synthetic reproducibility, and extremely high chemical modifiability. To date, a series of NIR-II fluorescent probes with excellent performance have been developed and applied for in vivo imaging with a high signal-to-noise ratio, deep-tissue penetrating ability, and high spatial and temporal resolution. However, most organic NIR-II fluorescent probes reported in the literatures are not yet well established and have limitations in clinical applications. To expand the biological applications of NIR-II fluorescent probes and to achieve true clinical translation, the following challenges must be overcome: 1) the development of liver/kidney metabolizable probes to address long-term probe safety; 2) the development of endogenous NIR-II fluorescent proteins for long-term biomonitoring; and 3) the development and optimization of NIR-II fluorescent imaging systems.
