A biocompatible two-photon absorbing fluorescent mitochondrial probe for deep in vivo bioimaging

Mitochondria, key organelles which keep in tune with energy demands for eukaryotic cells, are firmly associated with neurological conditions and post-traumatic rehabilitation. In vivo fluorescence imaging of mitochondria, especially with...

Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis

Hepatic apoptosis is involved in a variety of pathophysiologic conditions in the liver, including hepatitis, steatosis, and drug-induced liver injury. The development of easy-to-perform and reliable in vivo assays would thus greatly enhance the efforts to understand liver diseases and identify associated genes and potential drugs. In this study, we developed a transgenic zebrafish line that was suitable for the assessment of caspase 3 activity in the liver by using in vivo fluorescence imaging. The larvae of transgenic zebrafish dominantly expressed Casper3GR in the liver under control of the promoter of the phosphoenolpyruvate carboxykinase 1 gene. Casper3GR is composed of two fluorescent proteins, tagGFP and tagRFP, which are connected via a peptide linker that can be cleaved by activated caspase 3. Under tagGFP excitation conditions in zebrafish that were exposed to the well-characterized hepatotoxicant isoniazid, we detected increased and decreased fluorescence associated with tagGFP and tagRFP, respectively. This result suggests that isoniazid activates caspase 3 in the zebrafish liver, which digests the linker between tagGFP and tagRFP, resulting in a reduction in the Förster resonance energy transfer to tagRFP upon tagGFP excitation. We also detected isoniazid-induced inhibition of caspase 3 activity in zebrafish that were treated with the hepatoprotectants ursodeoxycholic acid and obeticholic acid. The transgenic zebrafish that were developed in this study could be a powerful tool for identifying both hepatotoxic and hepatoprotective drugs, as well as for analyzing the effects of the genes of interest to hepatic apoptosis.

Perfecting and extending the near-infrared imaging window

In vivo fluorescence imaging in the second near-infrared window (NIR-II) has been considered as a promising technique for visualizing mammals. However, the definition of the NIR-II region and the mechanism accounting for the excellent performance still need to be perfected. Herein, we simulate the photon propagation in the NIR region (to 2340 nm), confirm the positive contribution of moderate light absorption by water in intravital imaging and perfect the NIR-II window as 900–1880 nm, where 1400–1500 and 1700–1880 nm are defined as NIR-IIx and NIR-IIc regions, respectively. Moreover, 2080–2340 nm is newly proposed as the third near-infrared (NIR-III) window, which is believed to provide the best imaging quality. The wide-field fluorescence microscopy in the brain is performed around the NIR-IIx region, with excellent optical sectioning strength and the largest imaging depth of intravital NIR-II fluorescence microscopy to date. We also propose 1400 nm long-pass detection in off-peak NIR-II imaging whose performance exceeds that of NIR-IIb imaging, using bright fluorophores with short emission wavelength.

MMP-14-targeted Nanoprobe for in vivo Fluorescence Imaging of Rupture-prone Carotid Plaques

Background: The identification of rupture-prone carotid plaques for preventing stroke remains a clinical challenge. Macrophage matrix metalloproteinase (MMP)-14, which contributes to plaque progression and destabilisation, could be a promising biomarker for plaque imaging. This study aimed to design and synthesise an MMP-14-targeted nanoprobe to noninvasively visualise the behaviour of M1 macrophages in atherosclerotic plaques.Methods: A fluorescence molecular imaging probe ([email protected]) was constructed by covalently attaching the fluorescent dye cyanine (Cy) 5.5, an MMP-14 substrate, and polyethylene glycol (PEG) 5000-wrapped gold nanoparticles (AuNPs), and then administered via tail vein injection to carotid atherosclerosis models for in vivo fluorescence imaging. Additionally, carotid tissues and cultured macrophages were analysed for nanoprobe binding, and MMP-14 and inflammation-related marker expression was evaluated by polymerase chain reaction, western blotting, and immunohistochemistry.Results: MMP-14 expression significantly increased with plaque progression, along with the upregulation of MMP-2 and inflammatory M1 markers, CD68 and F4/80, and significant downregulation of the M2 marker CD206. All of cell, tissue and in vivo fluorescence imaging exhibited a favourable targeting efficacy of [email protected] for MMP-14.Conclusions: MMP-14, a cell membrane-anchoring enzyme, can serve as a biomarker of vulnerable plaques, and MMP-14 substrate-based [email protected], with an intense fluorescence signal after activation and good biocompatibility, can be applied to screen for and monitor plaque progression in vivo.

Perfecting and extending the near-infrared biological window

In vivo fluorescence imaging in the second near-infrared window (NIR-II) has been considered as a promising technique for visualizing the mammals. However, the definition of the NIR-II region and the mechanism accounting for the excellent performance still need to be perfected. Herein, we simulated bioimaging in the NIR spectral range (to 2340 nm), confirmed the positive contribution of moderate light absorption by water in intravital imaging and perfected the NIR-II window as 900-1880 nm, where the 1400-1500 nm was defined as NIR-IIx region and the 1700-1880 nm was defined as NIR-IIc region, respectively. Moreover, the 2080-2340 nm was newly proposed as the third near-infrared (NIR-III) window, which was believed to provide the best imaging quality. The wide-field fluorescence microscopy in brain, in addition, was performed around NIR-IIx region with excellent optical sectioning strength and the largest imaging depth of in vivo NIR-II fluorescence microscopy to date. We also proposed 1400 nm long-pass detection in off-peak NIR-II imaging whose profits exceeded those of NIR-IIb imaging, using bright fluorophores with short peak emission wavelength.

Towards the Development of Long Circulating Phosphatidylserine (PS)- and Phosphatidylglycerol (PG)-Enriched Anti-Inflammatory Liposomes: Is PEGylation Effective?

The anionic phospholipids (PLs) phosphatidylserine (PS) and phosphatidylglycerol (PG) are endogenous phospholipids with anti-inflammatory and immunomodulatory activity. A potential clinical use requires well-defined systems and for several applications, a long circulation time is desirable. Therefore, we aimed the development of long circulating liposomes with intrinsic anti-inflammatory activity. Hence, PS- and PG-enriched liposomes were produced, whilst phosphatidylcholine (PC) liposomes served as control. Liposomes were either formulated as conventional or PEGylated formulations. They had diameters below 150 nm, narrow size distributions and composition-dependent surface charges. Pharmacokinetics were assessed non-invasively via in vivo fluorescence imaging (FI) and ex vivo in excised organs over 2 days. PC liposomes, conventionally formulated, were rapidly cleared from the circulation, while PEGylation resulted in prolongation of liposome circulation robustly distributing among most organs. In contrast, PS and PG liposomes, both as conventional or PEGylated formulations, were rapidly cleared. Non-PEGylated PS and PG liposomes did accumulate almost exclusively in the liver. In contrast, PEGylated PS and PG liposomes were observed mainly in liver and spleen. In summary, PEGylation of PS and PG liposomes was not effective to prolong the circulation time but caused a higher uptake in the spleen.

Ultra-low background signaling cascade amplifier for in vivo fluorescence imaging of hydroxyl radical production induced by testosterone

The designing of cascade assay strategies for sensing ultralow analyte concentration is of crucial importance. However, the conventional strategies are not fit for in vivo detection due to the need...