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A novel type of PS (Ce6-MB@Lips) which could be excited by a NIR laser was synthesized. After effective cell internalization, the stable Ce6-MB@Lips showed high phototoxicity and ROS production capacity, and excellent ability to promote tumor cell apoptosis in vitro.

Photothermal therapy (PTT) is a safe and efficient anti-tumor treatment. A photothermal agent (PTA) with good biocompatibility and strong photothermal properties is of great importance for PTT. In this study, near-infrared (NIR) excitable clinical indocyanine green (ICG) was utilized as a PTA and further encapsulated by another PTA polydopamine (PDA) to form highly stable and efficient ICG@PDA nanoparticles (NPs). Then the ICG@PDA NPs were modified with methoxy polyethylene glycol amine (mPEG

A novel IR-783-DOX-TMTP1 theranostic nanoplatform with strong targeting ability was prepared and used for in vivo NIR-II fluorescence imaging of intratumoral vessels and chemotherapy of cervical tumor-bearing mice.

Molecular organic dyes are classic fluorescent nanoprobes finding tremendous uses in biological and life sciences. Yet, they suffer from low brightness, poor photostability, and lack of functional groups for bioconjugation. Here, we describe a class of biocompatible dye-protein optical nanoprobes, which show long-time photostability, superbrightness, and enriched functional groups. These nanoprobes utilize apoferritin (an intracellular protein for iron stores and release) to encase appropriate molecular organic dyes to produce on-demand fluorescence in aqueous solution. A pH-driven dissociation-reconstitution process of apoferritin subunits allows substantial incorporation of hydrophilic (aggregation caused quenching, ACQ) or hydrophobic (aggregation induced enhancement, AIE) dye molecules into the protein nanocavity (8 nm), producing monodispersed dye-apoferritin nanoparticles (apo-dye-NPs, ∼12 nm). As compared with single dye monomer, single apo-dye-NPs possess hundreds of times larger molar extinction coefficient and 2 orders of magnitude higher absolute luminescence quantum yield (up to 45-fold), multiplying fluorescence brightness up to 2778-fold. We show that varying the type of incorporated dyes entails a precise control over nanoprobe emission profile tunable in a broad spectral range of 370-1300 nm. Mechanical investigations indicate that the diversified microstructures of nanocavity inner surface are able to conform ACQ dyes at reasonable space interval while providing protein-guided-stacking for AIE dyes, thus enhancing fluorescence quantum yield through confining intermolecular quenching and intramolecular rotation. Moreover, apo-dye-NPs are able to emit stable fluorescence (over 13 min) without quenching in confocal imaging of HepG2 cancer cell under ultrahigh laser irradiance (1.3 × 10sup6/supW/cmsup2/sup). These superb properties make them suitable, as demonstrated in this work, for long-term super-resolved structured illumination microscopic cell imaging (spatial resolution, 117 nm) over 48 h, near-infrared (NIR) fluorescence angiography imaging of whole-body blood vessels (spatial resolution, 380 μm), and NIR photoacoustic imaging of liveriin vivo/i.

Purpose Indocyanine green (ICG) is a favorable fluorescence nanoprobe for its strong NIR-I fluorescence emission and good photothermal capabilities. However, the stability and tumor targeting ability of ICG is poor, which limits its further applications. To further improve the photothermal and therapeutic efficiency of ICG, bovine serum albumin (BSA) was utilized to encapsulate the ICG and the chemotherapeutic drug doxorubicin (DOX) was loaded to form the BSA@ICG-DOX theranostic nanoplatform. Methods In this study, ICG-loaded BSA nanoparticles (NPs) and the BSA@ICG-DOX NPs were fabricated using reprecipitation methods. Next, the tumour inhibition ability and biocompatibility of the NPs were evaluated. A subcutaneous xenografted nude mice model was established and imaging guided synergetic therapy was performed with the assistance of BSA@ICG-DOX NPs under 808 nm laser irradiation. Results The BSA@ICG NPs exhibited strong NIR-I fluorescence emission, excellent photothermal properties, biocompatibility, and tumor targeting ability. To further improve the therapeutic efficiency, the chemotherapeutic drug doxorubicin (DOX) was loaded into the BSA@ICG NPs to form the BSA@ICG-DOX theranostic nanoplatform. The BSA@ICG-DOX NPs were spherical with an average size of ~194.7 nm. The NPs had high encapsulation efficiency (DOX: 19.96% and ICG: 60.57%), and drug loading content (DOX: 0.95% and ICG: 3.03%). Next, excellent NIR-I fluorescence and low toxicity of the BSA@ICG-DOX NPs were verified. Targeted NIR-I fluorescence images were obtained after intravenous injection of the NPs into the subcutaneous cervical tumors of the mice. Conclusion To improve the anti-tumor efficiency of the ICG@BSA NPs, the chemotherapeutic drug DOX was loaded into the BSA@ICG NPs. The NIR excitation/emission and targeted BSA@ICG-DOX NPs enables high-performance diagnosis and chemo/photothermal therapy of subcutaneous cervical tumors, providing a promising approach for further biomedical applications., Graphical Abstract

Cervical diseases such as lymph node disease and tubal obstruction have threatened women's health. However, the traditional diagnostic methods still have shortcomings. NIR-II fluorescence imaging with advantages of low scattering, negligible autofluorescence, and high spatial resolution could be an ideal option. To obtain high quality NIR-II fluorescence imaging, selecting appropriate nanoprobes becomes the important issue. As a small molecular photothermal agent, extensive applications of ICG are rather limited because of its drawbacks. Herein, natural silk fibroin (SF) was synthesized and encapsulated ICG molecules to form SF@ICG nanoparticles (NPs). After detailed analysis, SF@ICG NPs showed excellent stability and long circulation time, as well as strong NIR-II fluorescence emission, well photo-stability, biocompatibility and well photothermal property under 808 nm laser irradiation. Furthermore, SF@ICG NPs were utilized for NIR-II fluorescence imaging of lymph node/lymphangiography and angiography of fallopian tubes. The process of fallopian tubes could be detected with high resolution and high sensitivity.

It is of great significance to study the brain structure and function in deep-tissue for neuroscience research and bio-medical applications because of the urgent demand for precise theranostics. Three-photon fluorescence microscopic (3PFM) bioimaging excited by the light in near-infrared IIb (NIR-IIb, 1,500–1,700 nm) spectral region is one of the most promising imaging techniques with the advantages of high spatial resolution, large imaging depth, and reduced scattering. Herein, a type of NIR-IIb light excitable deep-red emissive semiconducting polymer dots (P-dots) with bright 3PF and large three-photon absorption cross-section (σ3) at 1,550 nm was prepared. Then the P-dots were functionalized with polystyrene polymer polystyrene graft ethylene oxide functionalized with carboxyl groups (PS-PEG-COOH) and modified with NH2-poly(ethylene glycol) (PEG) to synthesis photochemically stable and biocompatible P-dots nanoparticles (NPs). Further the P-dots NPs were utilized for in vivo 3PFM bioimaging of cerebral vasculature with and without the brain skull under 1,550 nm femtosecond (fs) laser excitation. In vivo 3PFM bioimaging of the mice cerebral vasculature at various vertical depths was obtained. Moreover, a vivid three-dimensional structure of the mice vascular architecture beneath the skull was reconstructed. At the depth of 350 µm beneath the brain skull, 3.8 µm blood vessels could still be clearly recognized. NIR-IIb excitable P-dots assisted 3PFM bioimaging has great potential in accurate deep tissue bioimaging.

High treatment accuracy is the key to efficient cancer treatment. Photodynamic therapy (PDT) and photothermal therapy (PTT) are two kinds of popular, precise treatment methods. The combination of photodynamic and photothermal therapy (PDT/PTT) can greatly enhance the precise therapeutic efficacy. In this work, protoporphyrin IX (PpIX) was selected as the PDT agent (photosensitizer), and new indocyanine green (IR-820) was selected as the PTT agent. Further, the two kinds of theranostic agents were encapsulated by biological-membrane-compatible liposomes to form PpIX-IR-820@Lipo nanoparticles (NPs), a new kind of PDT/PTT agent. The PpIX-IR-820@Lipo NPs exhibited good water solubility, a spherical shape, and high fluorescence peak emission in the near-infrared spectral region (700-900 nm, NIR). The cellular toxicity of PpIX-IR-820@Lipo NPs for human cervical cancer cells (HeLa) and human cervical epithelial cells (H8) was detected by the CCK-8 method, and low cytotoxicity was observed for the PpIX-IR-820@Lipo NPs. Then, the excellent cellular uptake of PpIX-IR-820@Lipo NPs was confirmed by laser scanning confocal microscopy. Moreover, the PDT/PTT property of PpIX-IR-820@Lipo NPs was illustrated via 2',7'-dichlorofluorescin diacetate (DCFH-DA) and annexin V-fluorescein isothiocyanate (annexin V-FITC), as indicator probes. The PDT/PTT synergistic efficiency of PpIX-IR-820@Lipo NPs on HeLa cells was verified, exhibiting a high efficiency of 70.5%. Thus, the novel theranostic PpIX-IR-820@Lipo NPs can be used as a promising PDT/PTT synergistic theranostic nanoplatform in future cervical cancer treatment.

Fluorescence imaging has been widely used in the fields of biomedicine and clinical diagnosis. Compared with traditional fluorescence imaging in the visible spectral region (400-760 nm), near-infrared (NIR, 700-1 700 nm) fluorescence imaging is more helpful to improve the signal-to-noise ratio and the sensitivity of imaging. Highly-sensitive fluorescent probes are required for high-quality fluorescence imaging, and the rapid development of nanotechnology has led to the emergence of organic dyes with excellent fluorescent properties. Among them, organic fluorescent probes with the advantages of high safety, good biocompatibility, and high optical stability, are more favorable than inorganic fluorescent probes. Therefore, NIR fluorescence imaging assisted with organic fluorescent probes can provide more structural and dynamic information of biological samples to the researchers, which becomes a hot spot in the interdisciplinary research field of optics, chemistry and biomedicine. This review summarizes the application of NIR organic fluorescent probes in cervical cancer imaging. Several typical organic fluorescent probes (such as indocyanine green, heptamethine cyanine dye, rhodamine and polymer fluorescent nanoparticles) assisted NIR fluorescence imaging and their applications in cervical cancer diagnosis were introduced, and the future development and application of these techniques were discussed.

Photothermal therapy (PTT) has attracted wide attention due to its noninvasiveness and its thermal ablation ability. As photothermal agents are crucial factor in PTT, those with the characteristics of biocompatibility, non-toxicity and high photothermal stability have attracted great interest. In this work, new indocyanine green (IR-820) was utilized as a photothermal agent and near-infrared (NIR) fluorescence imaging nanoprobe. To improve the biocompatibility, poly(styrene-co-maleic anhydride) (PSMA) was utilized to encapsulate the IR-820 molecules to form novel IR-820@PSMA nanoparticles (NPs). Then, the optical and thermal properties of IR-820@PSMA NPs were studied in detail. The IR-820@PSMA NPs showed excellent photothermal stability and biocompatibility. The cellular uptaking ability of the IR-820@PSMA NPs was further confirmed in HeLa cells by the NIR fluorescent confocal microscopic imaging technique. The IR-820@PSMA NPs assisted PTT of living HeLa cells was conducted under 793 nm laser excitation, and a high PTT efficiency of 73.3% was obtained.

Photothermal therapy (PTT) is a popular tumor therapy method, which is based on efficient photothermal nanoagents (PTNs). Clinical Indocyanine Green (ICG), as a Food and Drug Administration (FDA) approved agent, is an often-used PTN, meanwhile it is also a good near-infrared (NIR) fluorescence contrast agent. However, the further applications of ICG in biomedical fields are limited due to its poor stability. In this study, ICG was encapsulated by the amphiphilic polymer poly(styrene-co-maleic anhydride) (PSMA) to form ICG@PSMA nanoparticles. Furthermore, optical and thermal characteristics of ICG@PSMA nanoparticles were studied in detail. Strong NIR fluorescence and excellent photothermal properties of ICG@PSMA nanoparticles under 808 nm laser irradiation were measured. Besides, favorable biocompatibility of ICG@PSMA nanoparticles was demonstrated on a human cervical cancer cell line (HeLa) via cell viability studies. Hence, ICG@PSMA nanoparticles were further applied to enhanced PTT of living HeLa cells under 808 nm excitation, and a high PTT efficiency of ∼70% was obtained. The novel ICG nanoparticles as a promising PTT nanoplatform could offer an opportunity for further tumour treatments.

Three-photon luminescence (3PL) imaging with near-infrared (NIR) excitation is quite promising for its deep penetration, high resolution, and good signal-to-noise ratio (SNR). In this report, a type of red emissive fluorophore TPEPT with aggregation-induced emission (AIE) properties was synthesized, and it was found to possess a large three-photon absorption (3PA) cross-section of 6.33 × 10−78 cm6 s2 under 1550 nm femtosecond laser excitation. TPEPT was then encapsulated with mPEG5000-DSPE to form AIE nanoparticles, and the chemical stability, optical properties and toxicity were studied afterwards. TPEPT nanoparticles were then applied for 3PL in vivo vascular imaging of mouse brain under 1550 nm fs laser excitation, and a fine three-dimensional (3D) reconstruction with a depth of 500 μm was achieved.

Second near-infrared (NIR-II, 1000-1700 nm) fluorescence bioimaging has attracted tremendous scientific interest and already been used in many biomedical studies. However, reports on organic NIR-II fluorescent probes for in vivo photoinduced imaging and simultaneous therapy, as well as the long-term tracing of specific biological objects, are still very rare. Herein we designed a single-molecular and NIR-II-emissive theranostic system by encapsulating a kind of aggregation-induced emission luminogen (AIEgen, named BPN-BBTD) with amphiphilic polymer. The ultra-stable BPN-BBTD nanoparticles were employed for the NIR-II fluorescence imaging and photothermal therapy of bladder tumors in vivo. The 785 nm excitation triggered photothermal therapy could completely eradicate the subcutaneous tumor and inhibit the growth of orthotopic tumors. Furthermore, BPN-BBTD nanoparticles were capable of monitoring subcutaneous and orthotopic tumors for a long time (32 days). Single-molecular and NIR-II-emitted aggregation-induced emission nanoparticles hold potential for the diagnosis, precise treatment, and metastasis monitoring of tumors in the future.

Fluorescent carbon dots (FCDs) have attracted tremendous attention in biological applications. The short-wave infrared (SWIR, 900–1700 nm) spectral range is considered as a novel optical tissue window due to low photon scattering. In this work, we investigated the fluorescence characteristics of FCDs based on the SWIR spectral range. SWIR emissions were observed from FCDs for the first time, when long-wavelength excitation (e.g. longer than 731 nm) was adopted. Wavelength-tunable two-photon fluorescence could also be obtained from the FCDs, under 1000–1560 nm SWIR femtosecond (fs) excitation. Interestingly, when an fs excitation wavelength as long as 1560 nm was adopted, two typical nonlinear optical signals, namely two-photon fluorescence and third harmonic generation (THG), could be observed. Based on the one-photon SWIR fluorescence, FCDs were successfully utilized for in vivo sentinel node mapping and tumour imaging. Under SWIR fs excitation, FCD-assisted two-photon fluorescence microscopy realized deep-tissue imaging of zebrafish embryos and the brain neuron networks of mice. SWIR excited and emitting FCDs have potential as fluorescent probes for deep-tissue and high-contrast functional bioimaging and related applications in the future.

Imaging the brain with high integrity is of great importance to neuroscience and related applications. X-ray computed tomography (CT) and magnetic resonance imaging (MRI) are two clinically used modalities for deep-penetration brain imaging. However, their spatial resolution is quite limited. Two-photon fluorescence microscopic (2PFM) imaging with its femtosecond (fs) excitation wavelength in the traditional near-infrared (NIR) region (700-1000 nm) is able to realize deep-tissue and high-resolution brain imaging. However, it requires craniotomy and cranial window or skull-thinning techniques due to photon scattering of the excitation light. Herein, based on a type of aggregation-induced emission luminogen (AIEgen) DCDPP-2TPA with a large three-photon absorption (3PA) cross section at 1550 nm and deep-red emission, we realized through-skull three-photon fluorescence microscopic (3PFM) imaging of mouse cerebral vasculature without craniotomy and skull-thinning. Reduced photon scattering of a 1550 nm fs excitation laser allowed it to effectively penetrate the skull and tightly focus onto DCDPP-2TPA nanoparticles (NPs) in the cerebral vasculature, generating bright three-photon fluorescence (3PF) signals. In vivo 3PF images of the cerebral vasculature at various vertical depths were obtained, and a vivid 3D reconstruction of the vascular architecture beneath the skull was built. As deep as 300 μm beneath the skull, small blood vessels of 2.4 μm could still be recognized.

With high contrast and deep penetration, two-photon fluorescence (2PF) imaging has become one of the most promising in vivo fluorescence imaging techniques. To obtain good imaging contrast, fluorescent nanoprobes with good 2PF properties are highly needed. In this work, bright 2PF polymer dots (P dots) were applied for in vivo mouse brain imaging. Deep-red emissive P dots with PFBT as the donor and PFDBT5 as the acceptor were synthesized and used as a contrast agent. P dots were further encapsulated by poly(styrene-co-maleic anhydride) (PSMA) and grafted with poly(ethylene glycol) (PEG). The P dots-PEG exhibit large two-photon absorption (2PA) cross-sections (δ≥8500 g), good water dispersibility, and high biocompatibility. P dots-PEG was further utilized first time for in vivo vascular imaging of mouse ear and brain, under 690–900 nm femtosecond (fs) laser excitation. Due to the large 2PA cross-section and deep-red emission, a large imaging depth (~720 µm) was achieved.

Nanoparticle-assisted near-infrared (NIR) bioimaging and two-photon fluorescence microscopy (TPFM) are two important technologies in biophotonic research. In this work, we synthesize the dye named 2-(4-bromophenyl)-3-(4-(4-(diphenylamino)styryl)phenyl)fumaronitrile (TPABDFN), which had a large two-photon absorption cross-section and bright NIR emission. The dyes were then encapsulated with poly(styrene- co -maleic anhydride) (PSMA), forming fluorescent nanoparticles. The TPABDFN-PSMA nanoparticles possessed high chemical and optical stability, good biocompatibility, as well as large two-photon absorption cross-section (5.56 × 10 5 GM). Furthermore, we combined NIR bioimaging and TPFM together, and utilized TPABDFN-PSMA nanoparticles as fluorescent contrast agents for two-photon excited NIR microscopic imaging, with a 1040 nm-femtosecond laser. In vivo angiography of mice ear and brain was performed. Due to the deep penetration capability of both 1040 nm-excitation and NIR emission light, a very large in vivo microscopic imaging depth (∼1.2 mm) was achieved. NIR emissive and biocompatible TPABDFN-PSMA nanoparticles have great potential in disease diagnosis and clinical therapies, where deep-tissue imaging is required.

A selenium-containing FR/NIR AIE luminogen with efficient solid-state emission is reported. Its AIE dots exhibit high brightness, large Stokes shift, good biocompatibility and satisfactory photostability, making them the first selenium-containing FR/NIR nanoprobes with AIE characteristics for in vivo bioimaging applications with high contrast and a high penetration depth.

Two-photon excited fluorescence microscopy and nanoparticle-assisted photodynamic therapy (PDT) are two important areas in biomedical research, and their combination can be more beneficial. A type of red emissive photosensitizer (PS) with aggregation-induced emission (AIE) features, which is called tetraphenylethylene (TPE-red), was synthesized and further encapsulated with poly(styrene-co-maleic anhydride) (PSMA) to form nanoparticles. Two-photon fluorescence, as well as two-photon excited reactive oxygen species (ROS) generation by TPE-red–PSMA nanoparticles, was characterized. A large two-photon absorption cross-section was observed at 1040 nm with a femtosecond (fs) laser. PDT via two-photon excitation was well realized on tumor cells, using TPE-red–PSMA nanoparticles as PSs under 1040 nm fs laser excitation. Based on our study, we believe that two-photon excited PDT with AIE-active PSs has great potential applications in deep tissue imaging-guided therapy.

The emerging second near-infrared (NIR-II) fluorescence imaging has shown great promise for in vivo imaging of small animals such as mice, yet the onward movement to large-size animals such as rats is still undeveloped. In this work, for the first time, we report comprehensive NIR-II deciphering of brain blood vasculature and main organs of living rats based on aggregation-induced emission (AIE) dots. The AIE dots possess simultaneously high brightness, excellent stability with various treatments, and good biocompatibility. Thanks to the long emission range and high brightness, in vivo NIR-II fluorescence microscopic imaging with the AIE dots beyond 1200 nm allows for visualizing rat brain vasculature with high spatial resolution (∼4 μm) and deep penetration (700 μm), representing the highest penetration depth and best resolution of rat brain imaging yet achieved in the field. The NIR-II AIE dots also enable the detection of tiny vascular blockage in real time at the early stage of cerebral thrombosis. Furthermore, in vivo non-invasive monitoring of rat’s gastrointestinal tract and bladder in a high-contrast manner helps to better understand the digestive system and excretory processes. This work will inspire new insights into the development of advanced NIR-II technique for large animal imaging, rendering great promise for real applications.

Visualization of the brain in its native environment is important for understanding common brain diseases. Herein, bright luminogens with remarkable aggregation-induced emission (AIE) characteristics and high quantum yields of up to 42.6% in the solid state are synthesized through facile reaction routes. The synthesized molecule, namely BTF, shows ultrabright far-red/near-infrared emission and can be fabricated into AIE dots by a simple nanoprecipitation procedure. Due to their high brightness, large Stokes shift, good biocompatibility, satisfactory photostability, and large three-photon absorption cross section, the AIE dots can be utilized as efficient fluorescent nanoprobes for in vivo brain vascular imaging through the intact skull by a three-photon fluorescence microscopy imaging technique. This is the first example of using AIE dots for the visualization of the cerebral stroke process through the intact skull of a mouse with high penetration depth and good image contrast. Such good results are anticipated to open up a new venue in the development of efficient emitters with strong nonlinear optical effects for noninvasive bioimaging of living brain.

Fluorescence bioimaging in the second near-infrared spectral region (NIR-II, 1000–1700 nm) can provide advantages of high spatial resolution and large penetration depth, due to low light scattering. However, NIR-II fluorophores simultaneously possessing high brightness, good stability, and biocompatibility are very rare. Hydrophobic NIR-II emissive PbS@CdS quantum dots (QDs) are surface-functionalized, via a silica and amphiphilic polymer (Pluronic F-127) dual-layer coating method. The as-synthesized PbS@CdS@SiO2@F-127 nanoparticles (NPs) are aqueously dispersible and possess a quantum yield of ≈5.79%, which is much larger than those of most existing NIR-II fluorophores. Thanks to the dual-layer protection, PbS@CdS@SiO2@F-127 NPs show excellent chemical stability in a wide range of pH values. The biocompatibility of PbS@CdS@SiO2@F-127 NPs is studied, and the results show that the toxicity of the NPs in vivo could be minimal. PbS@CdS@SiO2@F-127 NPs are then utilized for in vivo and real-time NIR-II fluorescence microscopic imaging of mouse brain. The architecture of blood vessels is visualized and the imaging depth reaches 950 µm. Furthermore, in vivo NIR-II fluorescence imaging of gastrointestinal tract is achieved, by perfusing PbS@CdS@SiO2@F-127 NPs into mice at a rather low dosage. This work illustrates the potential of ultrastable, biocompatible, and bright NIR-II QDs in biomedical and clinical applications, which require deep tissue imaging.