Real time monitoring peripheral nerve function with ICG and BDA-ICG by NIR-II fluorescence imaging.

Neuroanatomical tract tracers are important for studying axoplasmic transport and the complex interconnections of the nervous system. Though traditional fluorescent tracers are widely used, they have several prominent drawbacks when imaging, including low resolutions and low tissue penetrations and inability to be supervised dynamically within a long peripheral nerve during the long term. Here, we explored the potential of ICG as a neural tracer for axoplasmic transport and for the first time demonstrated that ICG could be used to detect transport function within peripheral nerve by near-infrared region II (NIR-II) imaging. On basis of this finding, a novel bi-directional neural tracer biotinylated dextran amine-indocyanine green (BDA-ICG) was prepared and characterized with better long-term stability and higher nerve-to-background ratio than ICG in vivo, and successfully imaged the injured peripheral nerve from the healthy one within 24 h. Our results show that BDA-ICG are promising neural tracers and clinically available dyes with NIR-II emission tail characteristics as ICG.

Nature Products Chlorophyll Derivatives for NIR-II Fluorescence Bioimaging and Plant-imaging.

The second near-infrared window (NIR-II, 1000 - 1700 nm) fluorescence imaging has attracted significant attention in research fields because of its unique advantages compared with conventional optical windows (400 - 900 nm). A variety of NIR-II fluorophores have been actively studied because they serve as a key component of fluorescence imaging. Among them, organic small molecule NIR-II fluorophores display outstanding imaging performance and many advantages, but types of small molecule NIR-II fluorophores with high biocompatibility are still quite limited. Novel molecular scaffolds based NIR-II dyes are highly desired. Herein, we hypothesized that chlorophyll is a new promising molecular platform for discovery NIR-II fluorophores. Thus, seven derivatives of derivatives were selected to characterize their optical properties. Interestingly, six chlorophyll derivatives displayed NIR-II fluorescence imaging capability. This characteristic allowed the successful NIR-II imaging of green leaves of various plants. Furthermore, most of these fluorophores showed capacity to monitor viscosity change because of their sensitive for viscosity. For demonstration of its biomedical applications, these probes were successfully used for NIR-II fluorescence-guided surgical resection of lymph nodes. In summary, chlorophylls are novel valuable tool molecules for NIR-II fluorescence imaging and have potential to expand their applications in biomedical field and plant science.

Clinically Translatable Solid-State Dye for NIR-II Imaging of Medical Devices.

Medical devices are commonly implanted underneath the skin, but how to real-time noninvasively monitor their migration, integrity, and biodegradation in human body is still a formidable challenge. Here, the study demonstrates that benzyl violet 4B (BV-4B), a main component in the FDA-approved surgical suture, is found to produce fluorescence signal in the first near-infrared window (NIR-I, 700-900 nm) in polar solutions, whereas BV-4B self-assembles into highly crystalline aggregates upon a formation of ultrasmall nanodots and can emit strong fluorescence in the second near-infrared window (NIR-II, 1000-1700 nm) with a dramatic bathochromic shift in the absorption spectrum of ≈200 nm. Intriguingly, BV-4B-involved suture knots underneath the skin can be facilely monitored during the whole degradation process in vivo, and the rupture of the customized BV-4B-coated silicone catheter is noninvasively diagnosed by NIR-II imaging. Furthermore, BV-4B suspended in embolization glue achieves hybrid fluorescence-guided surgery (hybrid FGS) for arteriovenous malformation. As a proof-of-concept study, the solid-state BV-4B is successfully used for NIR-II imaging of surgical sutures in operations of patients. Overall, as a clinically translatable solid-state dye, BV-4B can be applied for in vivo monitoring the fate of medical devices by NIR-II imaging.

Fibroblast Activation Protein Targeting Probe with Gly-Pro Sequence for PET of Glioblastoma.

As an important cancer-associated fibroblast-specific biomarker, fibroblast activation protein (FAP) has become an attractive target for tumor diagnosis and treatment. However, most FAP-based radiotracers showed inadequate uptake and short retention in tumors. In this study, we designed and synthesized a novel FAP ligand (DOTA-GPFAPI-04) through assembling three functional moieties: a quinoline-based FAP inhibitor for specifically targeting FAP, a FAP substrate Gly-Pro as a linker for increasing the FAP protein interaction, and a 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) chelator for radiolabeling with different radionuclides. The FAP targeting ability of DOTA-GPFAPI-04 was investigated by molecular docking studies. DOTA-GPFAPI-04 was then radiolabeled with 68Ga to give [68Ga]Ga-DOTA-GPFAPI-04 for positron emission tomography (PET) imaging of glioblastoma. [68Ga]Ga-DOTA-GPFAPI-04 exhibited a purity of >98% and high stability analyzed by radio-HPLC in saline and mouse serum. Cell uptake studies demonstrated the targeting specificity of the probe. Further in vivo pharmacokinetic studies in normal mice demonstrated the quick clearance of the probe. Moreover, compared with the widely studied [68Ga]Ga-FAPI-04, [68Ga]Ga-DOTA-GPFAPI-04 showed much higher U87MG tumor uptake values (4.467 ± 0.379 for [68Ga]Ga-DOTA-GPFAPI-04 and 1.267 ± 0.208% ID/g for [68Ga]Ga-FAPI-04 at 0.5 h post-injection, respectively). The area under the curve based on time-activity curve (TAC) analysis for tumor radioactivity in small animal models was 422.5 for [68Ga]Ga-DOTA-GPFAPI-04 and 98.14 for [68Ga]Ga-FAPI-04, respectively, demonstrating that the former had longer tumor retention time. The tumor-to-muscle (T/M) ratio for [68Ga]Ga-DOTA-GPFAPI-04 reached 9.15 in a U87MG xenograft animal model. PET imaging and blocking assays showed that [68Ga]Ga-DOTA-GPFAPI-04 had specific tumor uptake. In summary, this study demonstrates the successful synthesis and evaluation of a novel FAPI targeting probe, [68Ga]Ga-DOTA-GPFAPI-04, with a Gly-Pro sequence. It shows favorable in vivo glioblastoma imaging properties and relatively long tumor retention, highlighting DOTA-GPFAPI-04 as a promising molecular scaffold for developing FAP targeting tumor theranostic agents.

Development of a Novel 18F-Labeled Probe for PET Imaging of Estrogen Receptor ß.

Estrogen receptor beta (ERß) is an important ER subtype that plays crucial roles in many physiological and pathological disorders. Herein, we developed the probe [18F]PVBO for in vivo ERß targeted PET imaging and obtained promising results. The nonradioactive PVBO showed a 12.5-fold stronger binding affinity to ERß than to ERα in vitro. In vitro assays revealed the specific uptake of [18F]PVBO by DU145 cells. The uptake of [18F]PVBO by DU145 xenografts increased during the 120 min dynamic scanning, with a maximum uptake of 2.80 ± 0.30% ID/g. Based on time activity curves (TACs), the injection of [18F]PVBO with unlabeled PVBO or ERB-041 resulted in a significant signal reduction with the tumor/muscle (T/M) ratio <1 at 30, 60, 75, and 120 min post-injection (p < 0.05). [18F]PVBO demonstrates the feasibility of noninvasively imaging ERß-positive tumors by small-animal PET and provides a new strategy for visualizing ERß in vivo.

Bioinspired large Stokes shift small molecular dyes for biomedical fluorescence imaging.

Long Stokes shift dyes that minimize cross-talk between the excitation source and fluorescent emission to improve the signal-to-background ratio are highly desired for fluorescence imaging. However, simple small molecular dyes with large Stokes shift (more than 120 nanometers) and near-infrared (NIR) emissions have been rarely reported so far. Here, inspired by the chromophore chemical structure of fluorescent proteins, we designed and synthesized a series of styrene oxazolone dyes (SODs) with simple synthetic methods, which show NIR emissions (>650 nanometers) with long Stokes shift (ranged from 136 to 198 nanometers) and small molecular weight (<450 daltons). The most promising SOD9 shows rapid renal excretion and blood-brain barrier passing properties. After functioning with the mitochondrial-targeted triphenylphosphonium (TPP) group, the resulting SOD9-TPP can be engineered for head-neck tumor imaging, fluorescence image-guided surgery, brain neuroimaging, and on-site pathologic analysis. In summary, our findings add an essential small molecular dye category to the classical dyes.

J-aggregates albumin-based NIR-II fluorescent dye nanoparticles for cancer phototheranostics.

Phototheranostics, relying on energy conversions of fluorophores upon excitation, integrating diagnostic fluorescence imaging and photo-driven therapy, represents a promising strategy for cancer precision medicine. Compared with the first near-infrared biological window (NIR-I), fluorophores imaged in the second window (NIR-II, 1000-1700 â€‹nm) exhibit a higher temporal and spatial resolution and tissue penetration depth. Polymethine cyanine-based dye IR1061 is a typical NIR-II small-molecule organic fluorophore, but its low water solubility and short circulation time limiting its biological applications. Therefore, human serum albumin (HSA) nanoparticles with great biocompatibility and biosafety were employed to fabricate hydrophobic IR1061, which exhibited red-shifted absorption band as typical for J-aggregates. Moreover, IR1061@HSA nanoparticles can be successfully used for NIR-II imaging to noninvasively visualize the tumor vascular networks, as well as real-time intraoperative image-guided tumor resection. Interestingly, benefiting from the high photothermal conversion efficiency brought by J-aggregates, IR1061@HSA nanoparticles were also explored for photothermal therapy (PTT) and cause efficient thermal ablation of tumors. Overall, IR1061@HSA, as a novel J-aggregates albumin-based NIR II dye nanoparticle with high biocompatibility, provides an integrated versatile platform for cancer phototheranostics with promising clinical translation prospects.

A Small-Molecule Based Organic Nanoparticle for Photothermal Therapy and Near-Infrared-IIb Imaging.

Near-infrared window IIb (NIR-IIb, 1500-1700 nm) fluorescence imaging demonstrates attractive properties including low scattering, low absorption, and deep tissue penetration, and photothermal therapy (PTT) is also a promising modality for cancer treatment. However, until now, there is no report on theranostic systems based on small organic molecules combining fluorescence imaging in the NIR-IIb and PTT, highlighting the challenge and strong need for development of such agents. Herein, we report a novel small molecule NIR-IIb dye IT-TQF with a D-A-D structure, which exhibited high fluorescence intensity in the NIR-IIb window. To further translate IT-TQF into an effective theranostic agent, IT-TQF was encapsulated into DSPE-PEG2000 to construct IT-TQF NPs. The physical and photochemical properties of the nanoprobe were investigated in vitro, and the in vivo NIR-IIb imaging and PTT performance were evaluated in normal, subcutaneous, orthotopic, and metastatic tumor mice models. IT-TQF NP-based NIR-IIb imaging demonstrated high spatial resolution and high tissue penetration depth, and small normal blood vessels (55.3 µm) were successfully imaged in the NIR-IIb window. Subcutaneous, orthotopic, and metastatic tumors were all clearly delineated. A high tumor signal-to-background ratio (SBR) of 9.42 was achieved for orthotopic osteosarcoma models, and the erosions of bone tissue caused by tumor cells were precisely visualized. Moreover, NIR-II image-guided surgery was successfully performed to completely remove the orthotopic tumor. Importantly, IT-TQF NPs displayed high PTT efficacy (photothermal conversion efficiency: 47%) for effective treatment of tumor mice. In conclusion, IT-TQF NPs are a novel and promising phototheranostic agent in the NIR-IIb window, and the nanoprobe has high potential for a broad range of biomedical applications.

Organic single molecule based nano-platform for NIR-II imaging and chemo-photothermal synergistic treatment of tumor.

Integrating multiple functionalities of near-infrared second window fluorescence imaging (NIR-Ⅱ FLI), chemotherapy, and photothermal treatment (PTT) into a single molecule is desirable but still a highly challenging task. Herein, inspired by the results that hyperthermia can enhance the cytotoxicity of some alkylating agents, we designed and synthesized the novel compound NM. By introducing nitrogen mustard's active moiety bis(2-chlorethyl)amino into Donor-Acceptor-Donor (D-A-D) electronic structure, the unimolecular system not only behaviored as a chemotherapeutic agent but also exhibited good PTT and NIR-Ⅱ FLI abilities. The hydrophobic agent NM was encapsulated by DSPE-PEG2000 to generate the nano-platform NM-NPs. The current study on in vitro and in vivo experiments indicated that NM-NPs make vessels visualize clearly in the NIR-II zone and achieve complete tumor elimination through chemo-photothermal synergistic treatment. Overall, this study provides a new innovative strategy for developing superior, versatile phototheranostics for cancer theranostics.

Acceptor engineering for NIR-II dyes with high photochemical and biomedical performance.

It is highly important and challenging to develop donor-acceptor-donor structured small-molecule second near-infrared window (NIR-II) dyes with excellent properties such as water-solubility and chem/photostability. Here, we discovery an electron acceptor, 6,7-di(thiophen-2-yl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline (TQT) with highest stability in alkaline conditions, compared with conventional NIR-II building block benzobisthiadiazole (BBT) and 6,7-diphenyl-[1,2,5] thiadiazolo[3,4-g]quinoxaline (PTQ). The sulfonated hydrophilic dye, FT-TQT, is further synthesized with 2.13-fold increased quantum yield than its counterpart FT-BBT with BBT as acceptor. FT-TQT complexed with FBS is also prepared and displays a 16-fold increase in fluorescence intensity compared to FT-TQT alone. It demonstrates real-time cerebral and tumor vessel imaging capability with µm-scale resolution. Dynamic monitoring of tumor vascular disruption after drug treatment is achieved by NIR-II fluorescent imaging. Overall, TQT is an efficient electron acceptor for designing innovative NIR-II dyes. The acceptor engineering strategy provides a promising approach to design next generation of NIR-II fluorophores which open new biomedical applications.

Liver injury long-term monitoring and fluorescent image-guided tumor surgery using self-assembly amphiphilic donor-acceptor NIR-II dyes.

Real-time monitoring of liver dysfunction represents a significant unmet demand in clinical and preclinical research. The second near-infrared window (NIR-II, 1000-1700 nm) fluorescent imaging is an attractive method for biomedical imaging and may be a promising approach for liver dysfunction monitoring. Herein, we designed and synthesized a small-molecule NIR-II dye TQT 1009 with an asymmetric donor-acceptor (D-A) core. By introducing four kinds of polyethylene glycol (PEG) with different length and molecular weights (nK, n = 0.5, 2, 5, 10) to TQT1009, the dye was self-assembled into different nanoparticles named as TQP nK with regulated size and controllable circulation lifetime in vivo. In general, TQP nK showed a super high contrast ratio for blood vessels, bones, intestines, lymph, and tumor imaging. The best-selected probe, TQP 10K, exhibited ultralong in vivo circulation time (>96 h) which was suitable for long-term quantitative monitoring of liver and vessel function at a single dose, implying the excellent prospects compared with ICG, which was quickly eliminated in blood within a few minutes. Meanwhile, TQP 10K also achieved NIR-II surgical navigation of tumor in an extended time window (>7 d). Overall, our results demonstrate the self-assembly PEGylated amphiphilic TQP nK provide a new probe design strategy for liver function monitoring and image-guided tumor surgery in a prolonged time window.

A phosphorescent probe for in vivo imaging in the second near-infrared window.

In the second near-infrared spectral window (NIR-II; with wavelengths of 1,000-1,700 nm), in vivo fluorescence imaging can take advantage of reduced tissue autofluorescence and lower light absorption and scattering by tissue. Here, we report the development and in vivo application of a NIR-II phosphorescent probe that has lifetimes of hundreds of microseconds and a Stokes shift of 430 nm. The probe is made of glutathione-capped copper-indium-selenium nanotubes, and in acidic environments (pH 5.5-6.5) switches from displaying fluorescence to phosphorescence. In xenograft models of osteosarcoma and breast cancer, intravenous or intratumoral injections of the probe enabled phosphorescence imaging at signal-to-background ratios, spatial resolutions and sensitivities higher than NIR-II fluorescence imaging with polymer-stabilized copper-indium-sulfide nanorods. Phosphorescence imaging may offer superior imaging performance for a range of biomedical uses.

Synergistic strategy of rare-earth doped nanoparticles for NIR-II biomedical imaging.

Featuring simultaneous multicolor imaging for multiple targets, a synergistic strategy has become promising for fluorescence imaging applications. Visible and first near infrared (NIR-I, 700-900 nm) fluorophores have been explored for multicolor imaging to achieve good multi-target capacity, but they are largely hampered by the narrow imaging bands available (400-900 nm, bandwidth 500 nm), the broad emission spectra of many fluorophores, shallow tissue penetration and scattering loss. With attractive characteristic emission peaks in the second NIR window (NIR-II, 1000-1700 nm), a narrow emission spectrum, and deeper tissue penetration capability, rare-earth doped nanoparticles (RENPs) have been considered by us to be outstanding candidates for multicolor bioimaging. Herein, two RENPs, NaYF4:Yb20Er2@NaYF4 and NaYF4:Nd5@NaYF4, were prepared and modified with polyethylene glycol (PEG) to explore simultaneous imaging in the NIR-IIb (1530 nm, under 980 nm laser excitation) and the NIR-II (1060 nm, under 808 nm laser excitation) windows. The PEGylated-RENPs (RENPs@PEG) were able to simultaneously visualize the circulatory system, trace the lymphatic system, and evaluate the skeletal system. Our study demonstrates that RENPs have high synergistic imaging capability in multifunctional biomedical applications using their NIR-II fluorescence. Importantly, the two RENPs@PEG are complementary to each other for higher temporal resolution in NaYF4:Nd5@NaYF4@PEG and higher spatial resolution in NaYF4:Yb20Er2@NaYF4@PEG, which may provide more comprehensive and accurate imaging diagnosis. In conclusion, RENPs are highly promising nanomaterials for multicolor imaging in the NIR-II window.

Azide-Dye Unexpected Bone Targeting for Near-Infrared Window II Osteoporosis Imaging.

Azide is an important chemical functional group and has been widely used in chemical biology. However, the impact of azide on the in vivo behaviors of compounds has been rarely studied. Herein, azide was introduced into a fluorescent dye for the near-infrared window two (NIR-II) bone imaging. Specifically, we designed and synthesized the small-molecule NIR-II dyes, N3-FEP-4T capped with azide and FEP-4T without azide capping. In vitro assays revealed that N3-FEP-4T showed 5- and 5.6- times higher hydroxyapatite accumulation and macrophage uptake than those of FEP-4T, respectively. Moreover, N3-FEP-4T displayed higher bone uptakes and much better bone NIR-II imaging quality, demonstrating the specific bone-targeting ability of the azide-containing probe. N3-FEP-4T was then further successfully used for osteoporosis NIR-II imaging. Overall, our study provides insights into the impact of azide on the in vivo behavior of azide-containing compounds and opens a new window for biological application of azide.

Smart Self-Assembly Amphiphilic Cyclopeptide-Dye for Near-Infrared Window-II Imaging.

Development of novel nanomaterials for disease theranostics represents an important direction in chemistry and precision medicine. Fluorescent molecular probes in the second near-infrared window (NIR-II, 1000-1700 nm) show high promise because of their exceptional high detection sensitivity, resolution, and deep imaging depth. Here, a sharp pH-sensitive self-assembling cyclopeptide-dye, SIMM1000, as a smart nanoprobe for NIR-II imaging of diseases in living animals, is reported. This small molecule assembled nanoprobe exhibits smart properties by responding to a sharp decrease of pH in the tumor microenvironment (pH 7.0 to 6.8), aggregating from small nanoprobe (80 nm at pH 7.0) into large nanoparticles (>500 nm at pH 6.8) with ≈20-30 times enhanced fluorescence compared with the non-self-assembled CH-4T. It yields micrometer-scale resolution in blood vessel imaging and high contrast and resolution in bone and tumor imaging in mice. Because of its self-aggregation in acidic tumor microenvironments in situ, SIMM1000 exhibits high tumor accumulation and extremely long tumor retention (>19 days), while being excretable from normal tissues and safe. This smart self-assembling small molecule strategy can shift the paradigm of designing new nanomaterials for molecular imaging and drug development.

Biodegradable Silica-Based Nanotheranostics for Precise MRI/NIR-II Fluorescence Imaging and Self-Reinforcing Antitumor Therapy.

Multi-modality cancer diagnosis techniques based on the second near-infrared window fluorescence (NIR-II FL, 1000-1700 nm) imaging have become the focus of research attention. For such multimodality probes, how to take advantage of the tumor microenvironments (TME) characteristics to better image diseases and combine efficient therapeutics to achieve theranostics is still a big challenge. Herein, a novel TME-activated nanosystem (FMSN-MnO2 -BCQ) employing degradable silica-based nanoplatform is designed, adjusting the ratio of intratumoral hydrogen peroxide (H2 O2 )/glutathione (GSH) for magnetic resonance imaging (MRI)/NIR-II FL imaging and self-reinforcing chemodynamic therapy (CDT). Innovative bovine serum albumin (BSA)-modified fusiform-like mesoporous silica nanoparticles (FMSN) is fabricated as a carrier for NIR-II small molecule (CQ4T) and MRI reporter MnO2 . Remarkably, the BSA modification helped to achieve the dual-functions of high biocompatibility and enhance NIR-II fluorescence. The FMSN-MnO2 -BCQ with FMSN framework featuring a stepwise degradability in tumor interior released MnO2 and BCQ nanoparticles. Through the specific degradation of MnO2 by the TME, the produced Mn2+ ions are effectively exerted Fenton-like activity to generate hydroxyl radical (•OH) from endogenous H2 O2 to eradicate tumor cells. More importantly, the GSH depletion due to the synergistic effect of tetrasulfide bond and MnO2 in turn induced the oxidative cytotoxicity for self-reinforcing CDT.

A thiopyrylium salt for PET/NIR-II tumor imaging and image-guided surgery.

All tumor imaging modalities have resolution limits below which deeply situated small metastatic foci may not be identified. Moreover, incomplete lesion excision will affect the outcomes of the patients. Scintigraphy is adept in locating lesions, and second near-infrared window (NIR-II) imaging may allow precise real-time tumor delineation. To achieve complete excision of all lesions, multimodality imaging is a promising method for tumor identification and management. Here, a NIR-II thiopyrylium salt, XB1034, was first synthesized and bound to cetuximab and trans-cyclooctene (TCO) to produce XB1034-cetuximab-TCO. This probe provides excellent sensitivity and high temporal resolution NIR-II imaging in mice bearing tumors developed from human breast cancer cells MDA-MB-231. To enable PET imaging, 68 Ga-NETA-tetrazine is subsequently injected into the mice to undergo a bio-orthogonal reaction with the preinjected XB1034-cetuximab-TCO. PET images achieved in the tumor models using the pretargeting strategy are of much higher quality than those obtained using the direct radiolabeling method. Moreover, real-time NIR-II imaging allows accurate tumor excision and sentinel lymph node mapping. In conclusion, XB1034 is a promising molecular imaging probe for tumor diagnosis and treatment.

Mitochondria-targeted delocalized lipophilic cation complexed with human serum albumin for tumor cell imaging and treatment.

Small molecule 5BMF is a novel mitochondria-targeted delocalized lipophilic cation (DLC) with good anti-tumor activity and fluorescence emission suitable for bioimaging. In this study, human serum albumin (HSA) complexed with 5BMF (5BMF@HSA) has been developed to further improve its solubility (from 1.61 to 5.41 mg/mL), and the fluorescent intensity of 5BMF@HSA was improved over 2 times. Nearly 10-fold 5BMF was released from 5BMF@HSA complex in acidic condition when compared with neutral/basic environment. Intracellular distribution of 5BMF was altered by HSA as its signals were observed in lysosomes where free 5BMF barely localized. Both 5BMF@HSA and 5BMF showed selective toxicity toward tumor cells in µM and nM range and effectively inhibited tumor growth in mice model. In summary, 5BMF@HSA shows improved solubility in aqueous buffer and enhanced fluorescence emission, and maintains tumor inhibition capability. It is a promising protein complex for tumor cell imaging and tumor treatment.

Tuning the near infrared II emitting wavelength of small molecule dyes by single atom alteration.

A series of small molecule dyes demonstrate the feasibility of manipulating Near Infrared II emission by simply altering the donors' heteroatoms, which involved both electronegativity and intramolecular steric effects. Furthermore, these dyes show high resolution and stability for in vivo imaging after being complexed with human serum albumin.