Highly Sensitive Detection of Caspase-3/7 Activity in Living Mice Using Enzyme-Responsive ¹⁹F MRI Nanoprobes

Highly sensitive imaging of enzymatic activities in the deep tissues of living mammals provides useful information about their biological functions and for developing new drugs; however, such imaging is challenging. ¹⁹F magnetic resonance imaging (MRI) is suitable for noninvasive visualization of enzymatic activities without endogenous background signals. Although various enzyme-responsive ¹⁹F MRI probes have been developed, most cannot be used for in vivo imaging because of their low sensitivity. Recently, we developed unique nanoparticles, called FLAMEs, that are composed of a liquid perfluorocarbon core and a robust silica shell, and demonstrated their outstanding sensitivity in vivo. Here, we report a highly functionalized nanoprobe, FLAME-DEVD 2, with an OFF/ON ¹⁹F MRI switch for detecting caspase-3/7 activity based on the paramagnetic relaxation enhancement effect. To improve the cleavage efficiency of peptides by caspase-3, we designed a novel Gd³⁺ complex-conjugated peptide, DEVD <i>X</i> (<i>X</i> = 1, 2), which is a substrate peptide sequence tandemly repeated <i>X</i> times, and demonstrated that DEVD 2 showed faster cleavage kinetics than DEVD 1. By incorporating this novel concept into a signal activation strategy, FLAME-DEVD 2 showed a high ¹⁹F MRI signal enhancement rate in response to caspase-3 activity. After intravenous injection of FLAME-DEVD 2 and an apoptosis-inducing reagent, caspase-3/7 activity in the spleen of a living mouse was successfully imaged by ¹⁹F MRI. This imaging platform shows great potential for highly sensitive detection of enzymatic activities in vivo

Real-Time Background-Free Selective Imaging of Fluorescent Nanodiamonds in Vivo

Recent developments of imaging techniques have enabled fluorescence microscopy to investigate the localization and dynamics of intracellular substances of interest even at the single-molecule level. However, such sensitive detection is often hampered by autofluorescence arising from endogenous molecules. Those unwanted signals are generally reduced by utilizing differences in either wavelength or fluorescence lifetime; nevertheless, extraction of the signal of interest is often insufficient, particularly for in vivo imaging. Here, we describe a potential method for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds. This method is based on the property of NVCs that the fluorescence intensity sensitively depends on the ground state spin configuration which can be regulated by electron spin magnetic resonance. Because the NVC fluorescence exhibits neither photobleaching nor photoblinking, this protocol allowed us to conduct long-term tracking of a single nanodiamond in both <i>Caenorhabditis elegans</i> and mice, with excellent imaging contrast even in the presence of strong background autofluorescence

Real-Time Background-Free Selective Imaging of Fluorescent Nanodiamonds in Vivo

Recent developments of imaging techniques have enabled fluorescence microscopy to investigate the localization and dynamics of intracellular substances of interest even at the single-molecule level. However, such sensitive detection is often hampered by autofluorescence arising from endogenous molecules. Those unwanted signals are generally reduced by utilizing differences in either wavelength or fluorescence lifetime; nevertheless, extraction of the signal of interest is often insufficient, particularly for in vivo imaging. Here, we describe a potential method for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds. This method is based on the property of NVCs that the fluorescence intensity sensitively depends on the ground state spin configuration which can be regulated by electron spin magnetic resonance. Because the NVC fluorescence exhibits neither photobleaching nor photoblinking, this protocol allowed us to conduct long-term tracking of a single nanodiamond in both <i>Caenorhabditis elegans</i> and mice, with excellent imaging contrast even in the presence of strong background autofluorescence

Magnetic Resonance Imaging of Tumor with a Self-Traceable Phosphorylcholine Polymer

Polymers are concentration-amplified with respect to the monomeric units. We show here that a phosphorylcholine polymer enriched with ¹³C/¹⁵N at the methyl groups is self-traceable by multiple-resonance (heteronuclear-correlation) NMR in tumor-bearing mice inoculated with the mouse rectal cancer cell line (colon 26). Preliminary measurements indicated that the present polymeric nanoprobe was satisfactorily distinguished from lipids and detectable with far sub-micromolar spectroscopic and far sub-millimolar imaging sensitivities. Detailed ex vivo and in vivo studies for the tumor-bearing mice administered the probe with a mean molecular weight of 63 000 and a mean size of 13 nm, revealed the following: (1) this probe accumulates in the tumor highly selectively (besides renal excretion) and efficiently (up to 30% of the injected dose), (2) the tumor can thus be clearly in vivo imaged, the lowest clearly imageable dose of the probe being 100 mg/kg or 2.0 mg/20-g mouse, and (3) the competition between renal excretion and tumor accumulation is size-controlled; that is, the larger (higher molecular-weight) and smaller (lower molecular-weight) portions of the probe undergo tumor accumulation and renal excretion, respectively. The observed size dependence suggests that the efficient tumor-targeting of the present probe is stimulated primarily by the so-called enhanced permeability and retention (EPR) effect, that is, size-allowed invasion of the probe into the tumor tissue via defective vascular wall. Self-traceable polymers thus open an important area of magnetic resonance imaging (MRI) of tumors and may provide a highly potential tool to visualize various delivery/localization processes using synthetic polymers