In Situ Ratiometric Quantitative Tracing of Intracellular Leucine Aminopeptidase Activity via an Activatable Near-Infrared Fluorescent Probe

Leucine aminopeptidase (LAP), one of the important proteolytic enzymes, is intertwined with the progress of many pathological disorders as a well-defined biomarker. To explore fluorescent aminopeptidase probe for quantitative detection of LAP distribution and dynamic changes, herein we report a LAP-targeting near-infrared (NIR) fluorescent probe (DCM–Leu) for ratiometric quantitative trapping of LAP activity in different kinds of living cells. DCM–Leu is composed of a NIR-emitting fluorophore (DCM) as a reporter and l-leucine as a triggered moiety, which are linked together by an amide bond specific for LAP cleavage. High contrast on the ratiometric NIR fluorescence signal can be achieved in response to LAP activity, thus enabling quantification of endogenous LAP with “build-in calibration” as well as minimal background interference. Its ratiometric NIR signal can be blocked in a dose-dependent manner by bestatin, an LAP inhibitor, indicating that the alteration of endogenous LAP activity results in these obviously fluorescent signal responses. It is worth noting that DCM–Leu features striking characteristics such as a large Stokes shift (∼205 nm), superior selectivity, and strong photostability responding to LAP. Impressively, not only did we successfully exemplify DCM–Leu in situ ratiometric trapping and quantification of endogenous LAP activity in various types of living cells, but also, with the aid of three-dimensional confocal imaging, the intracellular LAP distribution is clearly observed from different perspectives for the first time, owing to the high signal-to-noise of ratiometric NIR fluorescent response. Collectively, these results demonstrate preclinical potential value of DCM–Leu serving as a useful NIR fluorescent probe for early detection of LAP-associated disease and screening inhibitor

GSH-Activated NIR Fluorescent Prodrug for Podophyllotoxin Delivery

Theranostic prodrug therapy enables the targeted delivery of anticancer drugs with minimized adverse effects and real-time <i>in situ</i> monitoring of activation of the prodrugs. In this work, we report the synthesis and biological assessment of the near-infrared (NIR) prodrug DCM-S-PPT and its amphiphilic copolymer (<i>m</i>PEG-DSPE)-encapsulated nanoparticles. DCM-S-PPT is composed of podophyllotoxin (PPT) as the anticancer moiety and a dicyanomethylene-4<i>H</i>-pyran (DCM) derivative as the NIR fluorescent reporter, which are linked by a thiol-specific cleavable disulfide bond. <i>In vitro</i> experiments indicated that DCM-S-PPT has low cytotoxicity and that glutathione (GSH) can activate DCM-S-PPT resulting in PPT release and a concomitant significant enhancement in NIR fluorescence at 665 nm. After being intravenously injected into tumor-bearing nude mice, DCM-S-PPT exhibited excellent tumor-activated performance. Furthermore, we have demonstrated that <i>m</i>PEG-DSPE as a nanocarrier loaded with DCM-S-PPT (<i>m</i>PEG-DSPE/DCM-S-PPT) showed even greater tumor-targeting performance than DCM-S-PPT on account of the enhanced permeability and retention effect. Its tumor-targeting ability and specific drug release in tumors make DCM-S-PPT a promising prodrug that could provide a significant strategy for theranostic drug delivery systems

S, N Codoped Graphene Quantum Dots Embedded in (BiO)₂CO₃: Incorporating Enzymatic-like Catalysis in Photocatalysis

In this study, S, N codoped graphene quantum dots/(BiO)₂CO₃ hollow microspheres have been fabricated by a facile electrostatic self-assembly method. The nanosized S, N:GQDs, which can be obtained by a bottom-up approach, are superior surface modification materials for photocatalytic applications due to their better electron transfer and peroxidase mimetic properties. The excellent oxidation property of the synthesized nanocomposite is confirmed by degradation of different model pollutants, such as rhodamine B, tetracycline, and bisphenol A under light irradiation or dark situation. Based on several experiments, the essential roles of S, N:GQDs can be described as (i) a photocarrier transport center strengthening photoinduced charge carriers (h⁺–e^–) separation and (ii) an enzymatic-like catalysis center to accelerate H₂O₂ decomposition to produce ·OH because the surface accumulation of H₂O₂ is harmful for photocatalytic processes. The present work may pave the way for integrating enzymatic-like cocatalysis into a photocatalytic process to generate more reactive oxygen species, thus advancing the field of environmental remediation and synthetic chemistry

Facile Preparation of AIE-Active Fluorescent Nanoparticles through Flash Nanoprecipitation

Flash nanoprecipitation (FNP) is an easily scalable and fast processing method for the preparation of nanoparticles (NPs) with simple vortex equipment. By using the FNP method, fluorescent NPs are prepared in less than 1 s in a multi-inlet vortex mixer, in which hydrophobic aggregation-induced emission (AIE)-active dye of EDP is incorporated within the biocompatible block copolymer poly­(ethylene glycol)-<i>b</i>-poly­(ε-caprolactone) for EDP NP assembly. The formulation parameters of stream velocity, dyes, and loading and concentration in FNP are optimized. The sizes of the NPs ranged from 20 to 60 nm with a ratio change of mixed solvents. As a control, an aggregation-caused quenching (ACQ) molecule of BDP was also synthesized for BDP NPs. To gain insight into the effect of the polymer on the aggregation state of hydrophobic dyes, the preparation of EDP and BDP NPs without block copolymer was also investigated. Apparently, the sizes of the NPs display large distributions without an amphiphilic block copolymer as the engineering template, suggesting that the block of polymers plays a key role in tuning the aggregation state of encapsulated dyes in FNP processes. Moreover, the peak shifts of dye with different microenvironments also confirmed the successful encapsulation of fluorescent dye in the NP cores. Finally, by externally applied forces in the FNP method, the engineered assembly of AIE-active fluorescent NPs possessing a narrow size distribution with desirable fluorescence properties was obtained. These features provide the possibility of rapidly constructing controllable AIE-active fluorescent NPs as biomedical tracers

Facile Preparation of AIE-Active Fluorescent Nanoparticles through Flash Nanoprecipitation

Flash nanoprecipitation (FNP) is an easily scalable and fast processing method for the preparation of nanoparticles (NPs) with simple vortex equipment. By using the FNP method, fluorescent NPs are prepared in less than 1 s in a multi-inlet vortex mixer, in which hydrophobic aggregation-induced emission (AIE)-active dye of EDP is incorporated within the biocompatible block copolymer poly­(ethylene glycol)-<i>b</i>-poly­(ε-caprolactone) for EDP NP assembly. The formulation parameters of stream velocity, dyes, and loading and concentration in FNP are optimized. The sizes of the NPs ranged from 20 to 60 nm with a ratio change of mixed solvents. As a control, an aggregation-caused quenching (ACQ) molecule of BDP was also synthesized for BDP NPs. To gain insight into the effect of the polymer on the aggregation state of hydrophobic dyes, the preparation of EDP and BDP NPs without block copolymer was also investigated. Apparently, the sizes of the NPs display large distributions without an amphiphilic block copolymer as the engineering template, suggesting that the block of polymers plays a key role in tuning the aggregation state of encapsulated dyes in FNP processes. Moreover, the peak shifts of dye with different microenvironments also confirmed the successful encapsulation of fluorescent dye in the NP cores. Finally, by externally applied forces in the FNP method, the engineered assembly of AIE-active fluorescent NPs possessing a narrow size distribution with desirable fluorescence properties was obtained. These features provide the possibility of rapidly constructing controllable AIE-active fluorescent NPs as biomedical tracers

Morphology Tuning of Aggregation-Induced Emission Probes by Flash Nanoprecipitation: Shape and Size Effects on in Vivo Imaging

Aggregation-induced emission (AIE) imaging probes have recently received considerable attention because of their unique property of high performance in the aggregated state and their imaging capability. However, the tendency of AIE molecules to aggregate into micron long irregular shapes, which significantly limits their application in vivo, is becoming a serious issue that needs to be addressed. Here, we introduce a novel engineering strategy to tune the morphology and size of AIE nanoaggregates, based on flash nanoprecipitation (FNP). Quinolinemalononitrile (ED) is encapsulated inside properly selected amphiphilic block copolymers of varying concentration. This leads to a variety of ED particle morphologies with different sizes. The shape and size are found to have strong influences on tumor targeting both in vitro and in vivo. The current results therefore indicate that the FNP method together with optimal choice of an amphiphilic copolymer is a universal method to systematically control the aggregation state of AIE materials and hence tune the morphology and size of AIE nanoaggregates, which is potentially useful for precise imaging at specific tumor sites