Development of a novel 18F-labeled small molecule probe for PET imaging of mesenchymal epithelial transition receptor expression.

The mesenchymal epithelial transition factor (c-Met) is frequently overexpressed in numerous cancers and has served as a validated anticancer target. Inter- and intra-tumor heterogeneity of c-Met, however, challenges the use of anti-MET therapies, highlighting an urgent need to develop an alternative tool for visualizing whole-body c-Met expression quantitatively and noninvasively. Here we firstly reported an 18F labeled, small-molecule quinine compound-based PET probe, 1-(4-(5-amino-7-(trifluoromethyl) quinolin-3-yl) piperazin-1-yl)-2-(fluoro-[18F]) propan-1-one, herein referred as [18F]-AZC. METHODS: [18F]-AZC was synthesized via a one-step substitution reaction and characterized by radiochemistry methods. [18F]-AZC specificity and affinity toward c-Met were assessed by cell uptake assay, with or without cold compound [19F]-AZC or commercial c-Met inhibitor blocking. MicroPET/CT imaging and biodistribution studies were conducted in subcutaneous murine xenografts of glioma. Additionally, [18F]-AZC was then further evaluated in orthotopic glioma xenografts, by microPET/CT imaging accompanied with MRI and autoradiography for co-registration of the tumor. Immunofluorescence staining was also carried out to qualitatively evaluate the c-Met expression in tumor tissue, co-localizes with H&E staining. RESULTS: This probe shows easy radiosynthesis, high stability in vitro and in vivo, high targeting affinity, and favorable lipophilicity and brain transport coefficient. [18F]-AZC demonstrates excellent tumor imaging properties in vivo and can delineate c-Met positive glioma specifically at 1 h after intravenous injection of the probe. Moreover, favorable correlation was observed between the [18F]-AZC accumulation and the amount of c-Met expression in tumor. CONCLUSION: This novel imaging probe could be applied as a valuable tool for management of anti-c-Met therapies in patients in the future.

Engineering Oxaliplatin Prodrug Nanoparticles for Second Near-Infrared Fluorescence Imaging-Guided Immunotherapy of Colorectal Cancer.

Colorectal cancer (CRC) ranks as the third common and the fourth lethal cancer type worldwide. Immune checkpoint blockade therapy demonstrates great efficacy in a subset of metastatic CRC patients, but precise activation of the antitumor immune response at the tumor site is still challenging. Here a versatile prodrug nanoparticle for second near-infrared (NIR-II) fluorescence imaging-guided combinatory immunotherapy of CRC is reported. The prodrug nanoparticles are constructed with a polymeric oxaliplatin prodrug (PBOXA) and a donor-spacer-acceptor-spacer-donor type small molecular fluorophore TQTCD. The later displays large Stokes shift (>300 nm), fluorescence emission over 1000 nm, and excellent photothermal conversion performance for NIR-II fluorescence imaging-guided photothermal therapy (PTT). The prodrug nanoparticles show seven times higher intratumoral OXA accumulation than free oxaliplatin. TQTCD-based PTT and PBOXA-induced chemotherapy trigger immunogenic cell death of the tumor cells and elicit antitumor immune response in a spatiotemporally controllable manner. Further combination of the prodrug nanoparticle-based PTT/chemotherapy with programmed death ligand 1 blockade significantly promotes intratumoral infiltration of the cytotoxic T lymphocytes and eradicates the CRC tumors. The NIR-II fluorescence imaging-guided immunotherapy may provide a promising approach for CRC treatment.

Synthesis and Biological Evaluation of Rhein-Based MRI Contrast Agents for in Vivo Visualization of Necrosis.

Early and accurate assessment of therapeutic response to anticancer therapy plays an important role in determining treatment planning and patient management in clinic. Magnetic rseonance imaging (MRI) of necrosis that occurs after cancer therapies provides chances for that. Here, we reported three novel MRI contrast agents, GdL1, GdL2, and GdL3, by conjugating rhein with gadolinium 2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetic acid (Gd-DOTA) through different linkers. The T1 relaxivities of three probes (7.28, 7.35, and 8.03 mM-1 s-1) were found to be higher than that of Gd-DOTA (4.28 mM-1 s-1). Necrosis avidity of GdL1 was evaluated on the rat models of reperfused liver infarction (RLI) by MRI, which showed an increase of T1-weighted contrast between necrotic and normal liver during 0.5-12 h. Besides, L1 was also labeled with 64Cu to assess its necrosis avidity on rat models of RLI and muscle necrosis (MN) by a γ-counter. The uptakes of 64CuL1 in necrotic liver and muscle were higher than those in normal liver and muscle ( P < 0.05). Then, the ability of GdL1 to assess therapeutic response was tested on rats bearing Walker 256 breast carcinoma injected with a vascular disrupting agent CA4P by MR imaging. The signal intensity of tumoral necrosis was strongly enhanced, and the contrast ratio between necrotic and viable tumor was 1.63 ± 0.11 at 3 h after administration of GdL1. Besides, exposed DNA in necrosis cells may be an important mechanism of three probes targeting to necrosis cells. In summary, GdL1 may serve as a promising MRI contrast agent for accurate assessment of treatment response.

NIR-II fluorescence imaging without intended excitation light.

Nowadays, second near-infrared window (NIR-II) dyes are almost excited by laser diodes, but none of the white light (400-700 nm) excited NIR-II imaging has been studied because of the lack of suitable optical probes. Herein, a novel blue-shifted NIR-II dye, TPA-TQT, has been selected for use in multi-wavelength white light emitting diode (LED) excited NIR-II imaging. This white LED barely caused photo-quenching of the dyes, especially indocyanine green (ICG), whereas the ICG's brightness decreased by 90% under continuous 808 nm laser irradiation. Compared to single-wavelength LED, multi-wavelength LED showed a lower background and similar signal-to-background ratios. This system provided high image resolution to identify blood vessels (103 µm), lymphatic capillaries (129.8 µm), and to monitor hindlimb ischemia-reperfusion and lymphatic inflammation. Furthermore, white LED excited NIR-II fluorescence imaging-guided surgery (FIGS) was successfully performed in 4T1 tumor-bearing mice. Impressively, the lighting LED-based NIR-II FIGS was found to clearly delineate small lesions of metastatic tumors of about ∼350 µm diameter and further was able to guide surgical removal. Overall, multi-wavelength LED-based NIR-II imaging is a promising imaging strategy for tumor delineation and other biomedical applications.

Hypsochromic Shift Donor-Acceptor NIR-II Dye for High-Efficiency Tumor Imaging.

Nowadays, second near-infrared window (NIR-II) dyes' development focuses on pursuing a longer absorption/emission wavelength and higher quantum yield, which usually means an extended π conjugation system, resulting in an enormous molecular weight and poor druggability. Most researchers thought that the reduced π conjugation system would bring on a blueshift spectrum that causes dim imaging qualities. Little efforts have been made to study smaller NIR-II dyes with a reduced π conjugation system. Herein, we synthesized a reduced π conjugation system donor-acceptor (D-A) probe TQ-1006 (Em = 1006 nm). Compared with its counterpart donor-acceptor-donor (D-A-D) structure TQT-1048 (Em = 1048 nm), TQ-1006 exhibited comparable excellent blood vessels, lymphatic drainage imaging performance, and a higher tumor-to-normal tissue (T/N) ratio. An RGD conjugated probe TQ-RGD showed an extra high contrast tumor imaging (T/N ≥ 10), further proving D-A dyes' excellent NIR-II biomedical imaging applications. Overall, the D-A framework provides a promising approach to designing next-generation NIR-II fluorophores.

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.

Capillaries segmentation of NIR-II images and its application in ischemic stroke.

Fluorescence imaging in the second near-infrared window (NIR-II) offers µm resolution blood vessel information noninvasively, which is crucial for the diagnosis and surgery treatment of some blood vessel-related diseases. However, only a few blood vessel segmentation algorithms have been done for the NIR-II images so far. Here, we proposed a vessel segmentation algorithm that used multi-scale enhancement and fractional differential to enhance capillaries, and then segmented vessels based on the blood vessels' tubular characteristics. Experimental results showed that this method could effectively suppress the point and lump tissue noise influence during vascular segmentation. The accuracy of vessel identification by other algorithms dropped below 30%, while our algorithm still achieved an accuracy of around 50% in deep vessel segmentation experiments with the 6.5 mm Intralipid. So it had the advantage of accurately detecting deep and dim blood capillaries. Meanwhile, the vascular density quantization algorithm had been successfully applied to the mice's ischemic stroke evaluations for the first time. In addition, this algorithm can provide the quantified vessel features under physiological or pathological conditions, which could be used to accurately evaluate the stroke drugs' therapeutic effect in the future.

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.

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.

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.

Development of Mitochondria-Targeted Small-Molecule Dyes for Myocardial PET and Fluorescence Bimodal Imaging.

Mitochondria-targeting positron emission tomography (PET) and fluorescent dual-modal probes are rarely reported. As one of the most promising lipophilic cations, F16 and its derivatives (F16s) have never been used for myocardial imaging. In this work, 14 F16s are synthesized and evaluated for cardiac imaging. In vitro cell fluorescence imaging revealed that the lead probe 5MEF is precisely localized in the mitochondria of cardiomyocytes. In addition, it shows excellent ex vivo fluorescence imaging quality with the heart-to-muscle and heart-to-liver ratios up to ∼2. Furthermore, the radiofluorinated probe 18F-5MEF is successfully prepared and shows a high initial heart uptake of 8.66 ± 0.34 % ID/g at 5 min post injection. It displays a high heart imaging performance, a long retention time in the heart, and a low background in the most normal tissues as revealed by PET. To our knowledge, this is the first time novel F16 analogues are designed and developed for myocardial dual-modal imaging.

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.

Evaluation of Radioiodinated 1,4-Naphthoquinones as Necrosis Avid Agents for Rapid Myocardium Necrosis Imaging.

PURPOSE: Identifying necrotic myocardium in ischemic regions is of great importance for risk stratification and clinical decision-making. However, rapid noninvasive imaging of necrotic myocardium is still challenging. This study sought to evaluate the potential of 1,4-naphthoquinones to rapidly visualize necrotic myocardium and the possible mechanisms of necrosis avidity. PROCEDURES: Six 1,4-naphthoquinones were radiolabeled with iodine-131 and the necrosis avidity was estimated in mouse models with muscular necrosis by gamma counting and autoradiography. The necrotic myocardium imaging property and biodistribution of [131I]naphthazarin (6) were determined in rat models with re-perfused myocardial infarction. A possible mechanism of necrosis avidity was explored by in vitro DNA-binding and in vivo blocking experiments. RESULTS: The radiochemical purities of the six radiotracers were greater than 95 %. The uptakes in necrotic muscles of all six radiotracers were higher than those in viable muscles, and [131I]naphthazarin (6) showed the highest necrotic-to-viable ratio and necrosis-to-blood ratio at all tested time points. The necrotic myocardium could be clearly visualized by single-photon emission computed tomography/x-ray computed tomography (SPECT/CT) using [131I]naphthazarin (6) as early as 3 h post-injection. Post-mortem biodistribution showed the uptake of [131I]naphthazarin (6) in necrotic myocardium was 11.67-fold higher than that in viable myocardium. Absorption spectra and emission spectra suggested naphthazarin (6) could bind to DNA through intercalation. The uptake of [131I]naphthazarin (6) in necrotic muscle could be significantly blocked by excessive ethidium bromide (a typical DNA intercalator) and cold naphthazarin (6) with 63.49 and 71.96 % decline at 3 h post-injection in vivo, respectively. CONCLUSIONS: 1,4-Naphthoquinones retained necrosis avidity and [131I]naphthazarin (6) rapidly visualized necrotic myocardium. The necrosis avidity mechanism of [131I]naphthazarin (6) may be attributed to its binding with exposed DNA in necrotic tissues.

Novel 18F-Labeled 1-Hydroxyanthraquinone Derivatives for Necrotic Myocardium Imaging.

Rapid detection and precise evaluation of myocardial viability is necessary to aid in clinical decision making whether to recommend revascularization for patients with myocardial infarction (MI). Three novel 18F-labeled 1-hydroxyanthraquinone derivatives were synthesized, characterized, and evaluated as potential necrosis avid imaging agents for assessment of myocardial viability. Among these tracers, [18F]FA3OP emerged as the most promising compound with best stability and highest targetability. Clear PET images of [18F]FA3OP were obtained in rat model of myocardial infarction and reperfusion at 1 h after injection. In addition, the possible mechanisms of [18F]FA3OP for necrotic myocardium were discussed. The results showed [19F]FA3OP may bind DNA to achieve targetability to necrotic myocardium by intercalation. In summary, [18F]FA3OP was a more promising "hot spot imaging" tracer for rapid visualization of necrotic myocardium.