NIR-II Dyad-Doped Ratiometric Nanosensor with Enhanced Spectral Fidelity in Biological Media for In Vivo Biosensing.

Ratiometric fluorescence nanosensors provide quantitative biological information. However, spectral shift and distortion of ratiometric nanosensors in biological media often compromise sensing accuracy, limiting in vivo applications. Here, we develop a fluorescent dyad (aBOP-IR1110) in the second near-infrared (NIR-II) window by covalently linking an asymmetric aza-BODIPY with a ONOO--responsive meso-thiocyanine. The dyad encapsulated in the PEGylated nanomicelle largely improves spectral fidelity in serum culture by >9.4 times compared to that of its noncovalent counterpart. The increased molecular weights (>1480 Da) and hydrophobicity (LogP of 7.87-12.36) lock dyads inside the micelles, which act as the shield against the external environment. ONOO--altered intramolecular Förster resonance energy transfer (FRET) generates linear ratiometric response with better serum tolerance, enabling us to monitor the dynamics of oxidative stress in traumatic brain injury and evaluate therapeutic efficiency. The results show high correlation with in vitro triphenyltetrazolium chloride staining, suggesting the potential of NIR-II dyad-doped nanosensor for in vivo high-fidelity sensing applications.

HClO-Activated Near-Infrared Fluorogenic Aza-BODIPY-Bisferrocene Triad with High Turn-on Ratio for In Vivo Biosensing.

Optically monitoring hypochlorous acid (HClO) in living body favors diagnosis and study of inflammatory diseases. However, this has been hampered by limited strategies to develop highly fluorogenic tools in the deep-penetration near-infrared spectrum. Herein, a near-infrared aza-BODIPY-bisferrocene triad Fc2 -CBDP that unexpectedly achieves an exceptionally sensitive and selective fluorescence turn-on (>220-fold) response toward HClO through single-ferrocene oxidation and boron-alkynyl hydrolysis cascade is reported. Mechanism insight shows that Fc2 -CBDP features "enhanced charge transfer"-caused quenching due to intramolecular bisferrocene electronic coupling, which is decoupled in the reaction with HClO. The utility of Fc2 -CBDP for intracellular HClO imaging is evaluated and, more importantly, in vivo high-contrast deep-tissue imaging of lymphatic inflammation and colitis is realized. This work provides new insights into both HClO and ferrocene chemistry, and extends the reach of fluorogenic strategies in the near-infrared biosensing.

A Bright, Renal-Clearable NIR-II Brush Macromolecular Probe with Long Blood Circulation Time for Kidney Disease Bioimaging.

Early detection of kidney disease is of vital importance due to its current prevalence worldwide. Fluorescence imaging, especially in the second near-infrared window (NIR-II) has been regarded as a promising technique for the early diagnosis of kidney disease due to the superior resolution and sensitivity. However, the reported NIR-II organic renal-clearable probes are hampered by their low brightness (ϵmax Φf>1000 nm <10 M-1 cm-1 ) and limited blood circulation time (t1/2 <2 h), which impede the targeted imaging performance. Herein, we develop the aza-boron-dipyrromethene (aza-BODIPY) brush macromolecular probes (Fudan BDIPY Probes (FBP 912)) with high brightness (ϵmax Φf>1000 nm ≈60 M-1 cm-1 ), which is about 10-fold higher than that of previously reported NIR-II renal-clearable organic probes. FBP 912 exhibits an average diameter of ≈4 nm and high renal clearance efficiency (≈65 % excretion through the kidney within 12 h), showing superior performance for non-invasively diagnosis of renal ischemia-reperfusion injury (RIR) earlier than clinical serum-based protocols. Additionally, the high molecular weight polymer brush enables FBP 912 with prolonged circulation time (t1/2 ≈6.1 h) and higher brightness than traditional PEGylated renal-clearable control fluorophores (t1/2 <2 h), facilitating for 4T1 tumor passive targeted imaging and renal cell carcinoma active targeted imaging with higher signal-to-noise ratio and extended retention time.

NIR-II cell endocytosis-activated fluorescent probes for in vivo high-contrast bioimaging diagnostics.

Fluorescence probes have great potential to empower bioimaging, precision clinical diagnostics and surgery. However, current probes are limited to in vivo high-contrast diagnostics, due to the substantial background interference from tissue scattering and nonspecific activation in blood and normal tissues. Here, we developed a kind of cell endocytosis-activated fluorescence (CEAF) probe, which consists of a hydrophilic polymer unit and an acid pH-sensitive small-molecule fluorescent moiety that operates in the "tissue-transparent" second near-infrared (NIR-II) window. The CEAF probe stably presents in the form of quenched nanoaggregates in water and blood, and can be selectively activated and retained in lysosomes through cell endocytosis, driven by a synergetic mechanism of disaggregation and protonation. In vivo imaging of tumor and inflammation with a passive-targeting and affinity-tagged CEAF probe, respectively, yields highly specific signals with target-to-background ratios over 15 and prolonged observation time up to 35 hours, enabling positive implications for surgical, diagnostic and fundamental biomedical studies.

High-Fidelity End-Functionalization of Poly(ethylene glycol) Using Stable and Potent Carbamate Linkages.

Commercial PEG-amine is of unreliable quality, and conventional PEG functionalization relies on esterification and etherification steps, suffering from incomplete conversion, harsh reaction conditions, and functional-group incompatibility. To solve these challenges, we propose an efficient strategy for PEG functionalization with carbamate linkages. By fine-tuning terminal amine basicity, stable and high-fidelity PEG-amine with carbamate linkage was obtained, as seen from the clean MALDI-TOF MS pattern. The carbamate strategy was further applied to the synthesis of high-fidelity multi-functionalized PEG with varying reactive groups. Compared to with an ester linkage, amphiphilic PEG-PS block copolymers bearing carbamate junction linkage exhibits preferential self-assembly tendency into vesicles. Moreover, nanoparticles of the latter demonstrate higher drug loading efficiency, encapsulation stability against enzymatic hydrolysis, and improved in vivo retention at the tumor region.

Regulating vesicle bilayer permeability and selectivity via stimuli-triggered polymersome-to-PICsome transition.

Compared to liposomes, polymersomes of block copolymers (BCPs) possess enhanced stability, along with compromised bilayer permeability. Though polyion complex vesicles (PICsomes) from oppositely charged block polyelectrolytes possess semipermeable bilayers, they are unstable towards physiologically relevant ionic strength and temperature; moreover, permselectivity tuning of PICsomes has remained a challenge. Starting from a single component diblock or triblock precursor, we solve this dilemma by stimuli-triggered chemical reactions within pre-organized BCP vesicles, actuating in situ polymersome-to-PICsome transition and achieving molecular size-selective cargo release at tunable rates. UV light and reductive milieu were utilized to trigger carboxyl decaging and generate ion pairs within hydrophobic polymersome bilayers containing tertiary amines. Contrary to conventional PICsomes, in situ generated ones are highly stable towards extreme pH range (pH 2-12), ionic strength (~3 M NaCl), and elevated temperature (70 °C) due to multivalent ion-pair interactions at high local concentration and cooperative hydrogen bonding interactions of pre-organized carbamate linkages.

Cytosolic NQO1 Enzyme-Activated Near-Infrared Fluorescence Imaging and Photodynamic Therapy with Polymeric Vesicles.

The utilization of enzymes as a triggering module could endow responsive polymeric nanostructures with selectivity in a site-specific manner. On the basis of the fact that endogenous NAD(P)H:quinone oxidoreductase isozyme 1 (NQO1) is overexpressed in many types of tumors, we report on the fabrication of photosensitizer-conjugated polymeric vesicles, exhibiting synergistic NQO1-triggered turn-on of both near-infrared (NIR) fluorescence emission and a photodynamic therapy (PDT) module. For vesicles self-assembled from amphiphilic block copolymers containing quinone trimethyl lock-capped self-immolative side linkages and quinone-bridged photosensitizers (coumarin and Nile blue) in the hydrophobic block, both fluorescence emission and PDT potency are initially in the "off" state due to "double quenching" effects, that is, dye-aggregation-caused quenching and quinone-rendered PET (photoinduced electron transfer) quenching. After internalization into NQO1-positive vesicles, the cytosolic NQO1 enzyme triggers self-immolative cleavage of quinone linkages and fluorogenic release of conjugated photosensitizers, leading to NIR fluorescence emission turn-on and activated PDT. This process is accompanied by the transformation of vesicles into cross-linked micelles with hydrophilic cores and smaller sizes and triggered dual drug release, which could be directly monitored by enhanced magnetic resonance (MR) imaging for vesicles conjugated with a DOTA(Gd) complex in the hydrophobic bilayer. We further demonstrate that the above strategy could be successfully applied for activated NIR fluorescence imaging and tissue-specific PDT under both cellular and in vivo conditions.