Fluorogenic, Two-Photon-Triggered Photoclick Chemistry in Live Mammalian Cells

The tetrazole-based photoclick chemistry has provided a powerful tool to image proteins in live cells. To extend photoclick chemistry to living organisms with improved spatiotemporal control, here we report the design of naphthalene-based tetrazoles that can be efficiently activated by two-photon excitation with a 700 nm femtosecond pulsed laser. A water-soluble, cell-permeable naphthalene-based tetrazole was identified that reacts with acrylamide with the effective two-photon cross-section for the cycloaddition reaction determined to be 3.8 GM. Furthermore, the use of this naphthalene-tetrazole for real-time, spatially controlled imaging of microtubules in live mammalian cells via the fluorogenic, two-photon-triggered photoclick chemistry was demonstrated

Subcellular Optogenetics Enacted by Targeted Nanotransformers of Near-Infrared Light

Light activation of photoswitchable molecules enables control over the course of physicochemical processes with high spatiotemporal precision, offering revolutionizing potential in multiple areas of contemporary biomedicine. Yet, application of this technology in live organisms remains severely limited due to the reliance on visible light that has poor penetration in biological tissues. Herein, we introduce highly efficient upconversion nanoparticles (UCNPs) as photon nanotransformers that intracellularly convert tissue-penetrating, near-infrared light into visible light required for photoactivation. The core/shell nanoparticles described here are determined to be about six times brighter in the blue range than the canonical hexagonal (NaYF₄:Yb³⁺30%/Tm³⁺0.5%)/NaYF₄ core/shell UCNPs with record efficiency. An application of such efficient photon nanotransformers can significantly advance optogenetics technology, wherein the signaling of genetically modified neurons is controlled through interaction of visible light with optogenetic proteins inserted into the cell membranes. We demonstrate that our photon nanotransformers, targeted to cultured cells, enable optogenetic activation with incident near-infrared light. The resulting membrane potential modulation by ion channel activity is probed by Ca ²⁺ sensitive dye. In contrast to conventional optogenetic approaches involving unselective activation of optogenetic proteins in the cellular volume with incident light irradiation, the upconverted light generated in situ by intracellular UCNPs, activates the optogenetic proteins in close vicinity to the nanoparticles, thus, providing a high subcellular precision of photoactivation

Tunable Narrow Band Emissions from Dye-Sensitized Core/Shell/Shell Nanocrystals in the Second Near-Infrared Biological Window

We introduce a hybrid organic–inorganic system consisting of epitaxial NaYF₄:Yb³⁺/X³⁺@NaYbF₄@NaYF₄:Nd³⁺ (X = null, Er, Ho, Tm, or Pr) core/shell/shell (CSS) nanocrystal with organic dye, indocyanine green (ICG) on the nanocrystal surface. This system is able to produce a set of narrow band emissions with a large Stokes-shift (>200 nm) in the second biological window of optical transparency (NIR-II, 1000–1700 nm), by directional energy transfer from light-harvesting surface ICG, via lanthanide ions in the shells, to the emitter X³⁺ in the core. Surface ICG not only increases the NIR-II emission intensity of inorganic CSS nanocrystals by ∼4-fold but also provides a broadly excitable spectral range (700–860 nm) that facilitates their use in bioapplications. We show that the NIR-II emission from ICG-sensitized Er³⁺-doped CSS nanocrystals allows clear observation of a sharp image through 9 mm thick chicken breast tissue, and emission signal detection through 22 mm thick tissue yielding a better imaging profile than from typically used Yb/Tm-codoped upconverting nanocrystals imaged in the NIR-I region (700–950 nm). Our result on in vivo imaging suggests that these ICG-sensitized CSS nanocrystals are suitable for deep optical imaging in the NIR-II region

Organotellurium Fluorescence Probes for Redox Reactions: 9‑Aryl-3,6-diamino­telluro­xanthylium Dyes and Their Telluroxides

Several 9-aryl-3,6-diamino­telluro­xanthylium dyes with phenyl, 2-methylphenyl, and 2,4,6-trimethylphenyl substituents at the 9-position were prepared. The characterization of these compounds included determination of ¹²⁵Te NMR spectra, fluorescence quantum yields (Φ_F), and quantum yields for the generation of singlet oxygen [Φ­(¹O₂)]. While these compounds were essentially nonfluorescent (Φ_F < 0.005), they produce ¹O₂ with Φ­(¹O₂) between 0.43 and 0.90. The tellurorosamines were oxidized with ¹O₂ via self-photosensitization to the corresponding telluroxides, which allowed their preparation free of excess oxidant. Telluroxides with a 9-(2-methylphenyl) or 9-(2,4,6-trimethylphenyl) substituent were fluorescent with quantum yields for fluorescence between 0.20 and 0.31. Steric bulk at the 9-position of the resulting telluroxides impacted rates of inter- and intramolecular attack of nucleophiles and stability of the telluroxide in aqueous media near physiological pH. The yield of reduction of the telluroxide with glutathione was also dependent on the steric bulk of the 9-aryl substituent. The structure of products from oxidation of the 9-(4-bromophenyl) tellurorosamine was determined by X-ray crystallography and indicated the addition of oxygen nucleophiles to the 9-position of the telluroxide oxidation state of the tellurorosamine

Synthesis and Properties of Heavy Chalcogen Analogues of the Texas Reds and Related Rhodamines

Analogues of Texas red incorporating the heavy chalcogens S, Se, and Te atoms in the xanthylium core were prepared from the addition of aryl Grignard reagents to appropriate chalcogenoxanthone precursors. The xanthones were prepared via directed metalation of amide precursors, addition of dichalcogenide electrophiles, and electrophilic cyclization of the resulting chalcogenides with phosphorus oxychloride and triethylamine. The Texas red analogues incorporate two fused julolidine rings containing the rhodamine nitrogen atoms. Analogues containing two “half-julolidine” groups (a trimethyltetrahydroquinoline) and one julolidine and one “half-julolidine” were also prepared. The photophysics of the Texas red analogues were examined. The S-analogues were highly fluorescent, the Se-analogues generated single oxygen (¹O₂) efficiently upon irradiation, and the Te-analogues were easily oxidized to rhodamines with the telluroxide oxidation state. The tellurorhodamine telluroxides absorb at wavelengths ≥690 nm and emit with fluorescence maxima >720 nm. A mesityl-substituted tellurorhodamine derivative localized in the mitochondria of Colo-26 cells (a murine colon carcinoma cell line) and was oxidized <i>in vitro</i> to the fluorescent telluroxide

(α-NaYbF₄:Tm³⁺)/CaF₂ Core/Shell Nanoparticles with Efficient Near-Infrared to Near-Infrared Upconversion for High-Contrast Deep Tissue Bioimaging

We describe the development of novel and biocompatible core/shell (α-NaYbF₄:Tm³⁺)/CaF₂ nanoparticles that exhibit highly efficient NIR_in–NIR_out upconversion (UC) for high contrast and deep bioimaging. When excited at ∼980 nm, these nanoparticles emit photoluminescence (PL) peaked at ∼800 nm. The quantum yield of this UC PL under low power density excitation (∼0.3 W/cm²) is 0.6 ± 0.1%. This high UC PL efficiency is realized by suppressing surface quenching effects <i>via</i> heteroepitaxial growth of a biocompatible CaF₂ shell, which results in a 35-fold increase in the intensity of UC PL from the core. Small-animal whole-body UC PL imaging with exceptional contrast (signal-to-background ratio of 310) is shown using BALB/c mice intravenously injected with aqueously dispersed nanoparticles (700 pmol/kg). High-contrast UC PL imaging of deep tissues is also demonstrated, using a nanoparticle-loaded synthetic fibrous mesh wrapped around rat femoral bone and a cuvette with nanoparticle aqueous dispersion covered with a 3.2 cm thick animal tissue (pork)

Structural and Epimeric Isomers of HPPH [3-Devinyl 3‑{1-(1-hexyloxy) ethyl}pyropheophorbide-a]: Effects on Uptake and Photodynamic Therapy of Cancer

The tetrapyrrole structure of porphyrins used as photosentizing agents is thought to determine uptake and retention by malignant epithelial cancer cells. To assess the contribution of the oxidized state of individual rings to these cellular processes, bacteriochlorophyll <i>a</i> was converted into the ring “D” reduced 3-devinyl-3-[1-(1-hexyloxy)­ethyl]­pyropheophorbide-a (HPPH) and the corresponding ring “B” reduced isomer (iso-HPPH). The carboxylic acid analogs of both ring “B” and ring “D” reduced isomers showed several-fold higher accumulation into the mitochondria and endoplasmic reticulum by primary culture of human lung and head and neck cancer cells than the corresponding methyl ester analogs that localize primarily to granular vesicles and to a lesser extent to mitochondria. However, long-term cellular retention of these compounds exhibited an inverse relationship with tumor cells generally retaining better the methyl-ester derivatives. <i>In vivo</i> distribution and tumor uptake was evaluated in the isogenic model of BALB/c mice bearing Colon26 tumors using the respective ¹⁴C-labeled analogs. Both carboxylic acid derivatives demonstrated similar intracellular localization and long-term tumor cure with no significant skin phototoxicity. PDT-mediated tumor action involved vascular damage, which was confirmed by a reduction in blood flow and immunohistochemical assessment of damage to the vascular endothelium. The HPPH stereoisomers (epimers) showed identical uptake (in vitro & in vivo), intracellular retention and photoreaction