NIR-II Self-Luminous Molecular Probe for In Vivo Inflammation Tracking and Cancer PDT Effect Self-Evaluating

Optical imaging in the second near-infrared (NIR-II, 900–1700 nm) window has been extensively investigated for bioimaging. However, a strong autofluorescence background from real-time excitation light significantly reduces the images’ quality of NIR-II fluorescence (FL) imaging. To resolve this issue, a NIR-II self-luminous small molecule (CLPD) based on bioluminescence (BL) resonance energy transfer (BRET) mechanism is first developed. The reactive oxygen species (ROS) can trigger NIR-II BL and reduce the NIR-II FL signals of the CLPD simultaneously, enabling ROS-correlated ratiometric BL/FL imaging. CLPD is used for high-contrast NIR-II BL imaging of osteoarthritis as well as guiding the treatment process by ratiometric BL/FL imaging. Moreover, CLPD is applied for NIR-II BL imaging of tumor triggered by the generated ROS during PDT. A correlation between the ratiometric NIR-II BL/FL signal and tumor size is constructed, providing a trustworthy tool for early assessment of PDT effect. Overall, this study presents a novel NIR-II self-luminous small molecular probe for in vivo imaging and provides a strategy for design a self-evaluation system of therapeutic effect.     

A Nitric Oxide (NO) Nanoreporter for Noninvasive Real-Time Imaging of Macrophage Immunotherapy

Macrophage-centered therapeutic approaches that rely on immune modulation of tumor associated macrophages (TAMs) from a pro-tumorigenic phenotype (M2) to an anti-tumorigenic phenotype (M1) have facilitated a paradigm shift in macrophage immunotherapy. However, limited clinical success has been achieved due to the low response rates observed in different types of cancers. The ability to measure immune response in real time is critical in order to differentiate responders from non-responders; however, there are currently no platforms to monitor real-time macrophage immunotherapy response. Hence, there is an immediate need to develop imaging techniques that can longitudinally monitor macrophage immunotherapy response. Nitric oxide (NO) produced as a result of activation of macrophages to an anti-tumorigenic state is considered as a hallmark of M1 and can be a direct indication of response. In this study, a NO nanoreporter (NO-NR) is reported that enables real-time monitoring of macrophage immunotherapy drugs in vitro and in vivo. Furthermore, it is observed that sustained inhibition of colony stimulating factor 1 receptor (CSF1R) using a CSF1R inhibitor–NO-NR system leads to enhanced efficacy and better imaging signal. In conclusion, a first-of-its-kind NO nanoreporter tool is reported that can be used as an activatable imaging agent to monitor macrophage immunotherapy response in real time.     

Color‐Convertible,Unimolecular, Micelle‐Based,Activatable Fluorescent Probe for Tumor‐Specific Detection and Imaging In Vitro and In Vivo

Recent years have witnessed significant progress in molecular probes for cancer diagnosis. However, the conventional molecular probes are designed to be “always‐on” by attachment of tumor‐targeting ligands, which limits their abilities to diagnose tumors universally due to the variations of targeting efficiency and complex environment in different cancers. Here, it is proposed that a color‐convertible, activatable probe is responding to a universal tumor microenvironment for tumor‐specific diagnosis without targeting ligands. Based on the significant hallmark of up‐regulated hydrogen peroxide (H₂O₂) in various tumors, a novel unimolecular micelle constructed by boronate coupling of a hydrophobic hyperbranched poly(fluorene‐co‐2,1,3‐benzothiadiazole) core and many hydrophilic poly(ethylene glycol) arms is built as an H₂O₂‐activatable fluorescent nanoprobe to delineate tumors from normal tissues through an aggregation‐enhanced fluorescence resonance energy transfer strategy. This color‐convertible, activatable nanoprobe is obviously blue‐fluorescent in various normal cells, but becomes highly green‐emissive in various cancer cells. After intravenous injection to tumor‐bearing mice, green fluorescent signals are only detected in tumor tissue. These observations are further confirmed by direct in vivo and ex vivo tumor imaging and immunofluorescence analysis. Such a facile and simple methodology without targeting ligands for tumor‐specific detection and imaging is worthwhile to further development.     

Gadolinium Metallofullerene‐Based Activatable Contrast Agent for Tumor Signal Amplification and Monitoring of Drug Release

Activatable imaging probes are promising to achieve increased signal‐to‐noise ratio for accurate tumor diagnosis and treatment monitoring. Magnetic resonance imaging (MRI) is a noninvasive imaging technique with excellent anatomic spatial resolution and unlimited tissue penetration depth. However, most of the activatable MRI contrast agents suffer from metal ion‐associated potential long‐term toxicity, which may limit their bioapplications and clinical translation. Herein, an activatable MRI agent with efficient MRI performance and high safety is developed for drug (doxorubicin) loading and tumor signal amplification. The agent is based on pH‐responsive polymer and gadolinium metallofullerene (GMF). This GMF‐based contrast agent shows high relaxivity and low risk of gadolinium ion release. At physiological pH, both GMF and drug molecules are encapsulated into the hydrophobic core of nanoparticles formed by the pH‐responsive polymer and shielded from the aqueous environment, resulting in relatively low longitudinal relativity and slow drug release. However, in acidic tumor microenvironment, the hydrophobic‐to‐hydrophilic conversion of the pH‐responsive polymer leads to amplified MR signal and rapid drug release simultaneously. These results suggest that the prepared activatable MRI contrast agent holds great promise for tumor detection and monitoring of drug release.     

Fluoro-Photoacoustic Polymeric Renal Reporter for Real-Time Dual Imaging of Acute Kidney Injury

Photoacoustic (PA) imaging agents detect disease tissues and biomarkers with increased penetration depth and enhanced spatial resolution relative to traditional optical imaging, and thus hold great promise for clinical applications. However, existing PA imaging agents often encounter the issues of slow body excretion and low-signal specificity, which compromise their capability for in vivo detection. Herein, a fluoro-photoacoustic polymeric renal reporter (FPRR) is synthesized for real-time imaging of drug-induced acute kidney injury (AKI). FPRR simultaneously turns on both near-infrared fluorescence (NIRF) and PA signals in response to an AKI biomarker (γ-glutamyl transferase) with high sensitivity and specificity. In association with its high renal clearance efficiency (78% at 24 h post-injection), FPRR can detect cisplatin-induced AKI at 24 h post-drug treatment through both real-time imaging and optical urinalysis, which is 48 h earlier than serum biomarker elevation and histological changes. More importantly, the deep-tissue penetration capability of PA imaging results in a signal-to-background ratio that is 2.3-fold higher than NIRF imaging. Thus, the study not only demonstrates the first activatable PA probe for real-time sensitive imaging of kidney function at molecular level, but also highlights the polymeric probe structure with high renal clearance.     

Semiconducting Polymer Nanoreporters for Near-Infrared Chemiluminescence Imaging of Immunoactivation

Real-time in vivo imaging of immunoactivation is critical for longitudinal evaluation of cancer immunotherapy, which, however, is rarely demonstrated. This study reports semiconducting polymer nanoreporters (SPNRs) with superoxide anion (O₂^•−)-activatable chemiluminescence signals for in vivo imaging of immunoactivation during cancer immunotherapy. SPNRs are designed to comprise an SP and a caged chemiluminescence phenoxy-dioxetane substrate, which respectively serve as the chemiluminescence acceptor and donor to enable intraparticle chemiluminescence resonance energy transfer. SPNRs are intrinsically fluorescent but only become chemiluminescent upon activation by O₂^•−. Representing the first O₂^•−-activatable near-infrared chemiluminescent reporter, SPNR3 sensitively differentiates higher O₂^•− levels in immune cells from other cells including cancer and normal cells. Following systemic administration, SPNR3 passively accumulates into tumors in living mice and activates the chemiluminescence signals responding to the concentration of O₂^•− in the tumor microenvironment. Moreover, the enhancement of in vivo chemiluminescence signal after cancer immunotherapy is correlated with increased population of T cells in the tumor, proving its feasibility in tracking of T cell activation. Thus, SPNRs represent the first kind of chemiluminescent reporters competent for in vivo imaging of immunoactivation.     

High‐Contrast Fluorescence Detection of Metastatic Breast Cancer Including Bone and Liver Micrometastases via Size‐Controlled pH‐Activatable Water‐Soluble Probes

Breast cancer metastasis is the major cause of cancer death in women worldwide. Early detection would save many lives, but current fluorescence imaging probes are limited in their detection ability, particularly of bone and liver micrometastases. Herein, probes that are capable of imaging tiny (<1 mm) micrometastases in the liver, lung, pancreas, kidneys, and bone, that have disseminated from the primary site, are reported. The influence of the poly(ethylene glycol) (PEG) chain length on the performance of water‐soluble, pH‐responsive, near‐infrared 4,4′‐di?uoro‐4‐bora‐3a ,4a ‐diaza‐s ‐indacene (BODIPY) probes is systematically investigated to demonstrate that PEG tuning can provide control over micrometastasis tracking with high tumor‐to‐background contrast (up to 12/1). Optimized probes can effectively visualize tumor boundaries and successfully detect micrometastases with diameters <1 mm. The bone‐metastasis‐targeting ability of these probes is further enhanced by covalent functionalization with bisphosphonate. This improved detection of both bone and liver micrometastases (<2 mm) with excellent tumor‐to‐normal contrast (5.2/1). A versatile method is thus introduced to directly synthesize modular water‐soluble probes with broad potential utility. Through a single intravenous injection, these materials can image micrometastases in multiple organs with spatiotemporal resolution. They thus hold promise for metastasis diagnosis, image‐guided surgery, and theranostic PEGylated drug therapies.     

A Bioresponsive Near‐Infrared Fluorescent Probe for Facile and Persistent Live‐Cell Tracking

Molecular imaging significantly transforms the field of biomedical science and facilitates the visualization, characterization, and quantification of biologic processes. However, it is still challenging to monitor cell localization in vivo, which is essential to the study of tumor metastasis and in the development of cell‐based therapies. While most conventional small‐molecule fluorescent probes cannot afford durable cell labeling, transfection of cells with fluorescent proteins is limited by their fixed fluorescence, poor tissue penetration, and interference of autofluorescence background. Here, a bioresponsive near‐infrared fluorescent probe is reported as facile and reliable tool for real‐time cell tracking in vivo. The design of this probe relies on a new phenomenon observed upon fluorobenzene‐conjugated fluorescent dyes, which can form complexes with cytosolic glutathione and actively translocates to lysosomes, exhibiting enhanced and stable cell labeling. Fluorobenzene‐coupled hemicyanine, a near‐infrared fluorophore manifests to efficiently staining tumor cells without affecting their invasive property and enables persistent monitoring of cell migration in metastatic tumor murine models at high resolution for one week. The method of fluorobenzene functionalization also provides a simple and universal “add‐on” strategy to render ordinary fluorescent probes suitable for long‐term live‐cell tracking, for which currently there is a deficit of suitable molecular tools.     

Nanoparticle‐Based Platform for Activatable Fluorescence Imaging and Photothermal Ablation of Endometriosis

Endometriosis is a painful disorder where endometrium‐like tissue forms lesions outside of the uterine cavity. Intraoperative identification and removal of these lesions are difficult. This study presents a nanoplatform that concurrently delineates and ablates endometriosis tissues using real‐time near‐infrared (NIR) fluorescence and photothermal therapy (PTT). The nanoplatform consists of a dye, silicon naphthalocyanine (SiNc), capable of both NIR fluorescence imaging and PTT, and a polymeric nanoparticle as a SiNc carrier to endometriosis tissue following systemic administration. To achieve high contrast during fluorescence imaging of endometriotic lesions, nanoparticles are constructed to be non‐fluorescent prior to internalization by endometriosis cells. In vitro studies confirm that these nanoparticles activate the fluorescence signal following internalization in macaque endometrial stromal cells and ablate them by increasing cellular temperature to 53 ^°C upon interaction with NIR light. To demonstrate in vivo efficiency of the nanoparticles, biopsies of endometrium and endometriosis from rhesus macaques are transplanted into immunodeficient mice. Imaging with the intraoperative Fluobeam 800 system reveals that 24 h following intravenous injection, nanoparticles efficiently accumulate in, and demarcate, endometriotic grafts with fluorescence. Finally, the nanoparticles increase the temperature of endometriotic grafts up to 47 °C upon exposure to NIR light, completely eradicating them after a single treatment.     

Prolonged Dye Release from Mesoporous Silica‐Based Imaging Probes Facilitates Long‐Term Optical Tracking of Cell Populations In Vivo

Nanomedicine is gaining ground worldwide in therapy and diagnostics. Novel nanoscopic imaging probes serve as imaging tools for studying dynamic biological processes in vitro and in vivo. To allow detectability in the physiological environment, the nanostructure‐based probes need to be either inherently detectable by biomedical imaging techniques, or serve as carriers for existing imaging agents. In this study, the potential of mesoporous silica nanoparticles carrying commercially available fluorochromes as self‐regenerating cell labels for long‐term cellular tracking is investigated. The particle surface is organically modified for enhanced cellular uptake, the fluorescence intensity of labeled cells is followed over time both in vitro and in vivo. The particles are not exocytosed and particles which escaped cells due to cell injury or death are degraded and no labeling of nontargeted cell populations are observed. The labeling efficiency is significantly improved as compared to that of quantum dots of similar emission wavelength. Labeled human breast cancer cells are xenotransplanted in nude mice, and the fluorescent cells can be detected in vivo for a period of 1 month. Moreover, ex vivo analysis reveals fluorescently labeled metastatic colonies in lymph node and rib, highlighting the capability of the developed probes for tracking of metastasis.     

一种用于电视跟踪的实时视频图像处理平台设计

在视频跟踪中,为了在有限的时间内实现大量的运算处理,需要采取一些提高运算速度的措施。介绍一种基于TMS320C6201高速DSP的实时视频图像处理硬件平台。实验证明,该平台在进行复杂算法和多目标提取方面比以前的DSP平台更容易满足实时性要求。     

One-pot synthesis of near-infrared fluorescent gold clusters for cellular fluorescence lifetime imaging

A facile strategy to synthesize water-soluble fluorescent gold nanoclusters (Au NCs) stabilized with the bidentate ligand dihydrolipoic acid (DHLA) is reported. The DHLA-capped Au NCs are characterized by UV-vis absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy. The Au NCs possess many attractive features including ultrasmall size, bright near-infrared luminescence, high colloidal stability, and good biocompatibility, making them promising imaging agents for biomedical and cellular imaging applications. Moreover, their long fluorescence lifetime (>100 ns) makes them attractive as labels in fluorescence lifetime imaging (FLIM) applications. As an example, the internalization of Au NCs by live HeLa cells is visualized using the FLIM technique.     

量子点在细胞以及体内生物中成像的研究进展

量子点是一种荧光半导体纳米材料,与生物分子结合成一种高亮度而稳定的荧光探针应用于生物成像。通过生物成像可观察量子点标记分子与其靶标的相互作用,实时观测其在活细胞及活体中的运行轨迹,实现对细胞水平及在活体层次的研究。利用这种生物成像技术还可以研究疾病的发生发展过程。介绍了量子点的光学特性,重点综述了量子点在细胞、体内生物成像中的应用,并展望了其发展前景。     

Eliminating Size‐Associated Diffusion Constraints for Rapid On‐Surface Bioassays with Nanoparticle Probes

Nanoparticle probes enable implementation of advanced on‐surface assay formats, but impose often underappreciated size‐associated constraints, in particular on assay kinetics and sensitivity. The present study highlights substantially slower diffusion‐limited assay kinetics due to the rapid development of a nanoprobe depletion layer next to the surface, which static incubation and mixing of bulk solution employed in conventional assay setups often fail to disrupt. In contrast, cyclic solution draining and replenishing yields reaction‐limited assay kinetics irrespective of the probe size. Using common surface bioassays, enzyme‐linked immunosorbent assays and immunofluorescence, this study shows that this conceptually distinct approach effectively “erases” size‐dependent diffusion constraints, providing a straightforward route to rapid on‐surface bioassays employing bulky probes and procedures involving multiple labeling cycles, such as multicycle single‐cell molecular profiling. For proof‐of‐concept, the study demonstrates that the assay time can be shortened from hours to minutes with the same probe concentration and, at a typical incubation time, comparable target labeling can be achieved with up to eight times lower nanoprobe concentration. The findings are expected to enable realization of novel assay formats and stimulate development of rapid on‐surface bioassays with nanoparticle probes.