Design of Water-Soluble Rotaxane-Capped Superparamagnetic, Ultrasmall Fe3O4 Nanoparticles for Targeted NIR Fluorescence Imaging in Combination with Magnetic Resonance Imaging.

Integrating an NIR fluorescent probe with a magnetic resonance imaging (MRI) agent to harvest complementary imaging information is challenging. Here, we have designed water-soluble, biocompatible, noncytotoxic, bright-NIR-emitting, sugar-functionalized, mechanically interlocked molecules (MIMs)-capped superparamagnetic ultrasmall Fe3O4 NPs for targeted multimodal imaging. Dual-functional stoppers containing an unsymmetrical NIR squaraine dye interlocked within a macrocycle to construct multifunctional MIMs are developed with enhanced NIR fluorescence efficiency and durability. One of the stoppers of the axle is composed of a lipophilic cationic TPP+ functionality to target mitochondria, and the other stopper comprises a dopamine-containing catechol group to anchor at the surface of the synthesized Fe3O4 NPs. Fe3O4 NPs surface-coated with targeted NIR rotaxanes help to deliver ultrasmall magnetic NPs specifically inside the mitochondria. Two carbohydrate moieties are conjugated with the macrocycle of the rotaxane via click chemistry to improve the water solubility of MitoSQRot-(Carb-OH)2-DOPA-Fe3O4 NPs. Water-soluble, rotaxane-capped Fe3O4 NPs are used for live-cell mitochondria-targeted NIR fluorescence confocal imaging, 3D and multicolor imaging in combination with T2-weighted MRI on a 9.4 T MR scanner with a high relaxation rate (r2) of 180.7 mM-1 s-1. Biocompatible, noncytotoxic, ultrabright NIR rotaxane-capped superparamagnetic ultrasmall monodisperse Fe3O4 NPs could be a promising agent for targeted multimodal imaging applications.

Live-Cell Mitochondrial Targeted NIR Fluorescent Covalent Labeling of Specific Proteins Using a Dual Localization Effect.

Here, our designed water-soluble NIR fluorescent unsymmetrical Cy-5-Mal/TPP+ consists of a lipophilic cationic TPP+ subunit that can selectively target and accumulate in a live-cell inner mitochondrial matrix where a maleimide residue of the probe undergoes faster chemoselective and site-specific covalent attachment with the exposed Cys residue of mitochondrion-specific proteins. On the basis of this dual localization effect, Cy-5-Mal/TPP+ molecules remain for a longer time period even after membrane depolarization, enabling long-term live-cell mitochondrial imaging. Due to the adequate concentration of Cy-5-Mal/TPP+ reached in live-cell mitochondria, it facilitates site-selective NIR fluorescent covalent labeling with Cys-exposed proteins, which are identified by the in-gel fluorescence assay and LC-MS/MS-based proteomics and supported by a computational method. This dual targeting approach with admirable photostability, narrow NIR absorption/emission bands, bright emission, long fluorescence lifetime, and insignificant cytotoxicity has been shown to improve real-time live-cell mitochondrial tracking including dynamics and interorganelle crosstalk with multicolor imaging applications.

Acidic pH-Triggered Live-Cell Lysosome Specific Tracking, Ratiometric pH Sensing, and Multicolor Imaging by Visible to NIR Switchable Cy-7 Dyes.

We have demonstrated an efficient synthetic route with crystal structures for the construction of acidic pH-triggered visible-to-NIR interchangeable ratiometric fluorescent pH sensors. This bioresponsive probe exhibits pH-sensitive reversible absorption/emission features, low cytotoxicity, a huge 322 nm bathochromic spectral shift with augmented quantum yield from neutral to acidic pH, high sensitivity and selective targeting ability of live-cell lysosomes with ideal pKa , off-to-on narrow NIR absorption/fluorescence signals with high molar absorption coefficient at acidic lysosomal lumen, and in-situ live-cell pH-activated ratiometric imaging of lysosomal pH. Selective staining and ratiometric pH imaging in human carcinoma live-cell lysosomes were monitored by dual-channel confocal laser scanning microscope using a pH-activatable organic fluorescent dye comprising a morpholine moiety for lysosome targeting and an acidic pH openable oxazolidine ring. Moreover, real-time tracking of lysosomes, 3D, and multicolor live-cell imaging have been achieved using the synthesized pH-activatable probe.

Construction of Red Fluorescent Dual Targeting Mechanically Interlocked Molecules for Live Cancer Cell Specific Lysosomal Staining and Multicolor Cellular Imaging.

We have designed and synthesized red fluorescent mechanically interlocked molecules with dual targeting functionality for live cancer cell specific active targeting followed by selective internalization and imaging of malignant lysosomes along with real-time tracking, 3D, and multicolor cellular imaging applications.

Acidic pH-Activatable Visible to Near-Infrared Switchable Ratiometric Fluorescent Probe for Live-Cell Lysosome Targeted Imaging.

Here, we have designed and synthesized acidic pH-activatable visible to NIR switchable ratiometric pH-sensitive fluorescent dye. The design consists of a cell-permeable organic probe containing a lysosome targeting morpholine functionality and an acidic pH-activatable oxazolidine moiety. The visible closed oxazolidine form (λabs 418 nm) can be switched to the highly conjugated NIR Cy-7 form (λabs 780 nm) through ring opening of the oxazolidine moiety at acidic pH. This switching of the ratiometric fluorescent probe is highly reversible and can be controlled by pH. NMR, UV/vis, and fluorescence spectroscopies allowed monitoring of pH switching behavior of the probe. This bioresponsive in situ acidic organelle activatable fluorophore showed reversible pH-switchable ratiometric optical properties, high photostability, huge bathochromic emission shift of 320 nm from basic to acidic pH, off-to-on narrow NIR absorption and emission bands with enhanced molar extinction coefficient at lysosomal pH, good quantum yield, low cytotoxicity, and targeted imaging ability of live cell lysosomes with ideal pKa. The report demonstrated ratiometric imaging with improved specificity of the acidic lysosome while minimizing signals at the NIR region from nontargeted neutral or basic organelles in human carcinoma HeLa and A549 as well as rat healthy H9c2(2-1) live cells, which is monitored by confocal laser scanning microscopy.

Design and Synthesis of Near-Infrared Mechanically Interlocked Molecules for Specific Targeting of Mitochondria.

The entrapment of squaraine (SQ) within a molecular container to form rotaxane has been shown to improve the dye stability and the fluorescence proficiency inside the mitochondria. The macrocycle provides shelter and protects the near-infrared (NIR) SQ chromophore from nucleophilic attacks made by the exposed thiol of Cys-containing mitochondrial proteins and mitochondrial glutathione. Herein a microwave-assisted template-directed clipping reaction on low-loading 2-chlorotrityl chloride resin is used to develop an NIR unsymmetrical squaraine rotaxane in high quantum yield.

Supramolecular ß-Sheet Forming Peptide Conjugated with Near-Infrared Chromophore for Selective Targeting, Imaging, and Dysfunction of Mitochondria.

Herein, conjugation of the amyloid-ß (Aß) peptide fragment, Lys-Leu-Val-Phe-Phe (KLVFF, fragment of Aß16-20), with an unsymmetrical near-infrared (NIR) cyanine-5 (Cy-5) chromophore is achieved using microwave-assisted solid phase synthesis on 2-chlorotrityl chloride resin. Selective mitochondria tracking and staining in human carcinoma cells are accomplished by the KLVFF/Cy-5 conjugate containing triphenylphosphonium functionality, and this is compared to a control molecule KLVFF/Cy-5c. Mitochondrial target specificity of KLVFF/Cy-5 is established by the colocalization assay using mitochondria selective probe MitoTracker Red, which is monitored by confocal laser scanning microscope and shows a high Pearson's correlation coefficient. The KLVFF/Cy-5 conjugate has high photostability, NIR absorption/emission, high molar extinction coefficient, narrow absorption/emission band, high fluorescence lifetime, and high fluorescence quantum yield. Moreover, mitochondria targeting KLVFF/Cy-5 conjugate reaches the critical aggregation concentration inside the mitochondria of cancer cells due to the strong negative inner mitochondrial membrane potential [(ΔΨm)cancer -220 mV] and self-assembles to form amyloid fibrils at the target site, which is responsible for the mitochondrial dysfunction and cytotoxicity. Annexin V-FITC/PI apoptosis detection assay is used to determine the signal pathway of mitochondria targeted cellular dysfunction.

Targeting and Imaging of Mitochondria Using Near-Infrared Cyanine Dye and Its Application to Multicolor Imaging.

Herein, we report water-soluble mitochondria-selective molecules that consist of a target-specific moiety conjugated with a near-infrared (NIR) imaging agent through variable spacer length. The presented NIR fluorescent cyanine-5 (Cy-5) chromophore exhibits excellent photostability, narrow NIR absorption and emission bands, high molar extinction coefficient, high fluorescence quantum yield, and long fluorescence lifetime. The biological compatibility and negligible cytotoxicity further make the dye an attractive choice for biological applications. Confocal fluorescence microscopic studies in the fixed human lung carcinoma cell line (A549) stained with the targeting NIR Cy-5 dyes (Cy-5a and Cy-5b) at 700 nM concentration show their cellular uptake and localization, which is compared with the nontargeting Cy-5c. Mitochondrial target specificity is demonstrated by colocalization experiments using the mitochondrion-tracking probe, MitoTracker Red and lysosome-tracking probe, LysoTracker Green. Multicolor imaging of cellular organelles in A549 cells is achieved in combination with suitable target-specific dyes with distinct excitation and emission, such as green emitting FM 1-43FX to selectively image the plasma membrane, blue-fluorescent DAPI to stain the nucleus, and the synthesized NIR Cy-5 to image the mitochondria. Higher accumulation of the dye inside the cancer cell mitochondria compared to the noncancerous cell is also demonstrated.