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Aim: This study focuses on biotinylated nanocarriers designed to encapsulate amphiphilic molecules with self-biodegradable properties for enhanced drug delivery.Methods: Biotin-zein conjugated nanoparticles were synthesized and tested in C6 cell lines to evaluate their viability and cellular uptake. Optimization was achieved using a a central composite design. The nanoparticles underwent thermogravimetric analysis, and their pharmacokinetics and biodistribution were also studied.Results: The optimized nanoparticles displayed 96.31% drug encapsulation efficiency, a particle size of 95.29 nm and a zeta potential of -17.7 mV. These nanoparticles showed increased cytotoxicity and improved cellular uptake compared with free drugs. Thermogravimetric analysis revealed that the drug-loaded nanocarriers provided better protection against drug degradation. Pharmacokinetic and biodistribution studies indicated that the formulation had an extended brain residence time, highlighting its effectiveness.Conclusion: The biotin-zein conjugated nanoparticles developed in this study offer a promising nano-vehicle for in vivo biodistribution and pharmacokinetic applications. Their high drug encapsulation efficiency, stability and extended brain residence time suggest they are effective for targeted drug delivery and therapeutic uses.

[Box: see text].

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A novel biothiols-sensitive near-infrared (NIR) fluorescent probe RhDN based on a rhodamine skeleton was developed for early detection of drug-induced hepatotoxicity in living mice. RhDN can be used not only as a conventional large stokes shift fluorescent (FL) probe, but also as a kind of anti-Stokes frequency upconversion luminescence (FUCL) molecular probe, which represents a long wavelength excitation (808 nm) to short wavelength emission (760 nm), and response to Cys/Hcy/GSH with high sensitivity. Compared with traditional FL methods, the FUCL method exhibited a lower detection limit of Cys, Hcy, and GSH in 75.1 nM, 101.8 nM, and 84.9 nM, respectively. We exemplify RhDN for tracking endogenously biothiols distribution in living cells and further realize real-time in vivo bioimaging of biothiols activity in mice with dual-mode luminescence system. Moreover, RhDN has been successfully applied to visualize the detection of drug-induced hepatotoxicity in living mice. Overall, this report presents a unique approach to the development of large stokes shift NIR FUCL molecular probes for in vitro and in vivo biothiols biosensing.

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The increasing demand for reliable near-infrared (NIR) probes exhibiting enduring fluorescence in living systems and facile compatibility with biomolecules such as peptides, antibodies or proteins is driven by the increasing use of NIR imaging in clinical diagnostics. To address this demand, a series of carboxy-functionalized unsymmetrical squaraine dyes (SQ-27, SQ-212, and SQ-215) along with non-carboxy-functionalized SQ-218 absorbing and emitting in the NIR wavelength range were designed and synthesized followed by photophysical characterization. This study focused on the impact of structural variations in the alkyl chain length, carboxy functionality positioning, and spacer chain length on dye aggregation and interaction with bovine serum albumin (BSA) as a model protein. In phosphate buffer (PB), the absorption intensity of the dyes markedly decreased accompanied by pronounced shoulders indicative of dye aggregation, and complete fluorescence quenching was seen in contrast to organic solvents. However, in the presence of BSA in PB, there was a enhancement in absorption intensity while regaining the fluorescence coupled with a remarkable increase in the intensity with increasing BSA concentrations, signifying the impact of dye-BSA interactions on preventing aggregation. Further analysis of Job's plot unveiled a 2:1 interaction ratio between BSA and all dyes, while the binding studies revealed a robust binding affinity (Ka) in the order of 107/mol. SQ-212 and SQ-215 were further tested for their in vitro and in vivo imaging capabilities. Notably, SQ-212 demonstrated nonpermeability to cells, while SQ-215 exhibited easy penetration and prominent cytoplasmic localization in in vitro studies. Injection of the dyes into laboratory mice showcased their efficacy in visualization, displaying stable and intense fluorescence in tissues without toxicity, organ damage, or behavioral changes. Thus, SQ-212 and SQ-215 are promising candidates for imaging applications, holding potential for noninvasive cellular and diagnostic imaging as well as biomarker detection when coupled with specific vectors in living systems.

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Real-time fluorescence sensing can provide insight into biodynamics. However, few fluorescent tools are available to overcome the tissue scattering and autofluorescence interference for high-contrast in vivo sensing with high spatiotemporal resolution. Here, we develop a molecular-based FRET nanosensor (MFN) capable of producing a dynamic ratiometric NIR-IIb (1500-1700 nm) fluorescence signal under a frequency-modulated dual-wavelength excitation bioimaging system. The MFN provides reliable signals in highly scattering tissues and enables in vivo real-time imaging at micrometer-scale spatial resolution and millisecond-scale temporal resolution. As a proof of concept, a physiological pH-responsive nanosensor (MFNpH) was designed as a nanoreporter for intravital real-time monitoring of the endocytosis dynamics of nanoparticles in the tumor microenvironment. We also show that MFNpH allows the accurate quantification of pH changes in a solid tumor through video-rate ratiometric imaging. Our study offers a powerful approach for noninvasive imaging and sensing of biodynamics with micrometer-scale spatial resolution and millisecond-scale temporal resolution.

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Visualizing biological tissues in vivo at a cellular or subcellular resolution to explore molecular signaling and cell behaviors is a crucial direction for research into biological processes. In vivo imaging can provide quantitative and dynamic visualization/mapping in biology and immunology. New microscopy techniques combined with near-infrared region fluorophores provide additional avenues for further progress in vivo bioimaging. Based on the development of chemical materials and physical optoelectronics, new NIR-II microscopy techniques are emerging, such as confocal and multiphoton microscopy, light-sheet fluorescence microscopy (LSFM), and wide-field microscopy. In this review, we introduce the characteristics of in vivo imaging using NIR-II fluorescence microscopy. We also cover the recent advances in NIR-II fluorescence microscopy techniques in bioimaging and the potential for overcoming current challenges.

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Optical imaging in the second near-infrared (NIR-II, 900-1700 nm) window has been extensively investigated for bioimaging. However, a strong autofluorescence background from real-time excitation light significantly reduces the images' quality of NIR-II fluorescence (FL) imaging. To resolve this issue, a NIR-II self-luminous small molecule (CLPD) based on bioluminescence (BL) resonance energy transfer (BRET) mechanism is first developed. The reactive oxygen species (ROS) can trigger NIR-II BL and reduce the NIR-II FL signals of the CLPD simultaneously, enabling ROS-correlated ratiometric BL/FL imaging. CLPD is used for high-contrast NIR-II BL imaging of osteoarthritis as well as guiding the treatment process by ratiometric BL/FL imaging. Moreover, CLPD is applied for NIR-II BL imaging of tumor triggered by the generated ROS during PDT. A correlation between the ratiometric NIR-II BL/FL signal and tumor size is constructed, providing a trustworthy tool for early assessment of PDT effect. Overall, this study presents a novel NIR-II self-luminous small molecular probe for in vivo imaging and provides a strategy for design a self-evaluation system of therapeutic effect.

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Systemic cancer therapy is always accompanied with toxicity to normal tissue, which has prompted concerted efforts to develop precise treatment strategies. Herein, we firstly develop an approach that enables spatiotemporally controlled formation and rotation of magnetic nanochains in vivo, allowing for precise mechanotherapy of tumor. The nanochain comprised nanocomposites of pheophorbide-A (PP) modified iron oxide nanoparticle (IONP) and lanthanide-doped down-conversion NP (DCNP). In a permanent magnetic field, the nanocomposites would be aligned to form nanochain. Next, MnO2 NPs were subsequently administered to accumulate in tumor as suppliers of Mn2+ , which coordinates with PP to immobilize the nanochain. In a rotating magnetic field, the nanochain would rapidly rotate, leading to apoptosis/necrosis of tumor cell. The nanochain showed high T2 -MR and NIR-II fluorescence imaging signals, which facilitated guided therapy. The strategy has great potential in practical applications.

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In this study, Eu3+/Gd3+ co-doped fluoroapatitååe (Eu/Gd:FAP) nanocrystals were synthesized by the hydrothermal method as a fluorescent bioimaging agent. The phase composition, morphology, fluorescence, and biosafety of the resulting samples were characterized. Moreover, the in vivo fluorescent bioimaging application of Eu/Gd:FAP nanocrystals was evaluated in mice with subcutaneously transplanted tumors. The results showed that the Eu/Gd:FAP nanocrystals were short rod-like particles with a size of 59.27 ± 13.34 nm × 18.69 ± 3.32 nm. With an increasing F substitution content, the Eu/Gd:FAP nanocrystals displayed a decreased size and enhanced fluorescence emission. Eu/Gd:FAP nanocrystals did not show hemolysis and cytotoxicity, indicating good biocompatibility. In vivo fluorescent bioimaging study demonstrated that Eu/Gd:FAP nanocrystals could be used as a bioimaging agent and displayed stable fluorescence emitting in tumors, indicating an accumulation in tumor tissue due to the passive targeting ability. In addition, any adverse effects of Eu/Gd:FAP nanocrystals on major organs were not observed. This study shows that biocompatible rare earth co-doped FAP nanocrystals have the potential to be used as a bioimaging agent in vivo.

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As a new type of "zero-dimensional" fluorescent carbon nanomaterials, carbon dots (CDs) have some unique optical and chemical properties, they are being explored for a variety of applications in bio-related fields, such as bioimaging, biosensors, and therapy. This review mainly summarizes the recent progress of CDs in bioimaging. The overview of this review can be roughly divided into two categories: (1) In vitro bioimaging based on CDs in different cells and important organelles. (2) The distribution, imaging and application of CDs in mice and zebrafish. In addition, this review also points out the potential advantages and future development directions of CDs for bioimaging, which may promote the development of CDs in the field of bioimaging.

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Eccentric contractions (ECC) facilitate cytosolic calcium ion (Ca2+) release from the sarcoplasmic reticulum (SR) and Ca2+ influx from the extracellular space. Ca2+ is a vital signaling messenger that regulates multiple cellular processes via its spatial and temporal concentration ([Ca2+]i) dynamics. We hypothesized that 1) a specific pattern of spatial/temporal intramyocyte Ca2+ dynamics portends muscle damage following ECC and 2) these dynamics would be regulated by the ryanodine receptor (RyR). [Ca2+]i in the tibialis anterior muscles of anesthetized adult Wistar rats was measured by ratiometric (i.e., ratio, R, 340/380 nm excitation) in vivo bioimaging with Fura-2 pre-ECC and at 5 and 24 h post-ECC (5 × 40 contractions). Separate groups of rats received RyR inhibitor dantrolene (DAN; 10 mg/kg ip) immediately post-ECC (+DAN). Muscle damage was evaluated by histological analysis on hematoxylin-eosin stained muscle sections. Compared with control (CONT, no ECC), [Ca2+]i distribution was heterogeneous with increased percent total area of high [Ca2+]i sites (operationally defined as R ≥ 1.39, i.e., ≥1 SD of mean control) 5 h post-ECC (CONT, 14.0 ± 8.0; ECC5h: 52.0 ± 7.4%, P < 0.01). DAN substantially reduced the high [Ca2+]i area 5 h post-ECC (ECC5h + DAN: 6.4 ± 3.1%, P < 0.01) and myocyte damage (ECC24h, 63.2 ± 1.0%; ECC24h + DAN: 29.1 ± 2.2%, P < 0.01). Temporal and spatially amplified [Ca2+]i fluctuations occurred regardless of DAN (ECC vs. ECC + DAN, P > 0.05). These results suggest that the RyR-mediated local high [Ca2+]i itself is related to the magnitude of muscle damage, whereas the [Ca2+]i fluctuation is an RyR-independent phenomenon.

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Sulfur is an element that is indispensable throughout the growth of plants. In plant cells, reactive sulfur species (RSS) play a vital role in maintaining cellular redox homeostasis and signal transduction. There is demand accordingly for a simple, highly selective, and sensitive method of RSS detection and imaging for monitoring dynamic changes and clarifying the biological functions of RSS in plant systems. Fluorescent analysis based on organic small-molecule fluorescent probes is an effective and specific approach to tracking plant RSS characteristics. This perspective summarizes the recent progress regarding organic small-molecule fluorescent probes for RSS monitoring, including small-molecule biological thiols, hydrogen sulfide, and sulfane sulfurs, in plants; it also discusses their response mechanism toward RSS and their imaging applications in plants across the agricultural chemistry field.

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Cell membrane-targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl-4H-chromen-4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

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Chemiluminescence imaging is imperative for diagnostics and imaging due to its intrinsically high sensitivity. To improve in vivo detection of biomarkers, chemiluminophores that simultaneously possess near-infrared (NIR) emission and modular structures amenable to construction of activatable probes are highly desired; however, these are rare. Herein, we report two chemiluminophores with record long NIR emission (>750 nm) via integration of dicyanomethylene-4H-benzothiopyran or dicyanomethylene-4H-benzoselenopyran with dioxetane unit. Caging of the chemiluminophores with different cleavable moieties produces NIR chemiluminescence probes (NCPs) that only produce signals upon reaction with reactive oxygen species or enzymes, for example, ß-galactosidase, with a tissue-penetration depth of up to 2 cm. Thus, this study provides NIR chemiluminescence molecular scaffolds applicable for in vivo turn-on imaging of versatile biomarkers in deep tissues.

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Hyaluronidase 1 (Hyal-1) is an important enzyme involved in intracellular hyaluronic acid (HA) catabolism for performing various physiological functions, and its aberrant level is closely associated with many malignant diseases. Bioluminescence imaging is advantageous for monitoring Hyal-1 activity in vivo, but it remains challenging to design an available probe for differentiating Hyal-1 from other isoforms by a traditional strategy that covalently masks the firefly luciferase substrate. Herein, we, for the first time, present a noncovalently caging approach to construct a Hyal-1-specific bioluminogenic nanosensor by entrapping d-luciferin (d-Luc) inside the cholesterylamine-modified HA (CHA) nanoassembly to inhibit the bioluminescence production. When encountered with intracellular Hyal-1, CHA could be fully dissembled to liberate multiple copies of the loaded d-Luc, thereby emitting light by the luciferase-catalyzed bioluminescence reaction. Because of its cascade signal amplification feature, d-Luc@CHA displayed a remarkable "turn-on" response (248-fold) to 5 µg/mL Hyal-1 with a detection limit of 0.07 ng/mL. Importantly, bioluminescence imaging results validated that d-Luc@CHA could be competent for dynamically visualizing endogenous Hyal-1 changes in living cells and animals and possessed the capability of discriminating between normal and cancer cells, thus offering a promising toolbox to evaluate Hyal-1 roles in biological processes as well as to diagnose Hyal-1-related diseases.

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Using near-infrared fluorophore Alexa Fluor 680 labeled peptide nucleic acids (PNAs) the biodistribution of such antisense agents can be analyzed in real time in live mice using in vivo imaging. Using the fluorescence intensity emitted from the mouse at different time points following administration, the systemic distribution and organ accumulation of PNA can be tracked. In addition, an estimation of the body half-life of the compound can be obtained by the change in fluorescence intensity over time. With this technique, the distribution of compounds can be monitored real time, while reducing the number of animals and amount of compounds required.

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Bioactive molecules play a vital role in the process of regulating the redox balance in the intracellular environment, especially in maintaining the function of organelles. To explore the association and function of bioactive molecules in organelles, it is essential to develop a chemosensor tool that uses multiresponse fluorescence signals to distinguish between and track two related bioactive molecules in organelles. However, the development of sensors with multiresponse functions is still a challenging task. Herein, we present a unique and practical single chemosensor (Mito-CTC) that can monitor HClO (as an oxidative substance) and H2S (as a reductive substance) in mitochondria (organelle targeting) with multiresponse fluorescence signals. The response of the sensor to HClO and H2S changes from red to green and blue channel emission simultaneously, respectively, thereby providing a specific signal response to reductive/oxidative substances in the mitochondria. Using a single chemosensor, we have realized multichannel bioimaging of the exogenous and endogenous HClO and H2S in cellular mitochondria. Additionally, the excellent properties of the sensor Mito-CTC can be used to reveal the relationship between HClO and H2S in mitochondria. Meanwhile, Mito-CTC has been endowed with the ability to image in bacteria and zebrafish attributed to the good permeability and low cytotoxicity. Expectantly, drug-induced liver injury (DILI) caused by fluoxetine (an antidepressant drug) and the degree of drug-induced toxicity to the liver were evaluated using Mito-CTC through discriminating and imaging HClO, indicating that Mito-CTC has the potential function of evaluating the toxicity of the drug to the liver.

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The difficulty of near-infrared (NIR) ratiometric detection imaging lies in the lack of high-efficiency NIR probes and the overlapping interference between two emission peaks. To achieve more accurate detection in living organisms, dual NIR-emissive luminescent nanoprobes were designed under the same excitation at 808 nm. The Er3+ ion-doped nanoparticles were employed as a reference with their fluorescence emission at 1525 nm. Meanwhile, a cyanine dye molecule (Cy925) was combined on the surface of nanoparticles as the ClO- recognition site with its NIR emission at 925 nm. The ratiometric nanoprobe relied on the ratio of aforementioned two separated NIR peaks ( I925nm/ I1525nm), featuring deeper imaging penetration depth and low autofluorescence. This nanoprobe was verified to be sensitive and highly selective to ClO- through photoluminescence titration. The in vitro detection experiment developed reasonable work curves, guaranteeing that we can detect the change in concentration of ClO- in mice limbs with arthritis through in vivo imaging experiments.

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Mild acidity matrix, rich blood vessels and special biomarkers constitute the primary tumor microenvironment. Nanoparticles could change their physicochemical characteristics by functionalizing a series of moieties which is responsive towards pH or specific markers. So precise regulation of nanocarrier-based drug delivery systems by the tumor microenvironment has showed great potential for theranostics. Herein, we developed a smart nano delivery system STD-NM, showing tumor microenvironment responsive targeting, efficient drug delivery and precise evaluation of therapeutic effect in vivo. STD-NM kept in 'stealth' state in normal environment while 'activated' in the tumor acidic environment, which could show stability in blood circulation while deeply penetrate into tumor tissues. Additionally, STD-NM was designed with aggregation-induced emission (AIE) characteristic, of which the fluorescence 'switch on' when apoptosis taken place. Gene analysis by RNA-seq also confirmed a superior therapeutic effect of drug loaded STD-NM treatment. We envisioned the well-designed smart nano materials for drug delivery could open a new avenue in precisely tumor imaging and specific cancer therapeutics.

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Two important challenges in the field of 19F magnetic resonance imaging (MRI) are the maintenance of high fluorine content without compromising imaging performance, and effective targeting of small particles to diseased tissue. To address these challenges, we have developed a series of perfluoropolyether (PFPE)-based hyperbranched (HBPFPE) nanoparticles with attached peptide aptamer as targeting ligands for specific in vivo detection of breast cancer with high 19F MRI sensitivity. A detailed comparison of the HBPFPE nanoparticles (NPs) with the previously reported trifluoroethyl acrylate (TFEA)-based polymers demonstrates that the mobility of fluorinated segments of the HBPFPE nanoparticles is significantly enhanced (19F T2 > 80 ms vs 31 ms), resulting in superior MR imaging sensitivity. Selective targeting was confirmed by auto- and pair correlation analysis of fluorescence microscopy data, in vitro immunofluorescence, in vivo 19F MRI, ex vivo fluorescence and 19F NMR. The results highlight the high efficiency of aptamers for targeting and the excellent sensitivity of the PFPE moieties for 19F MRI. Of relevance to in vivo applications, the PFPE-based polymers exhibit much faster clearance from the body than the previously introduced perfluorocarbon emulsions ( t1/2 ∼ 20 h vs up to months). Moreover, the aptamer-conjugated NPs show significantly higher tumor-penetration, demonstrating the potential of these imaging agents for therapeutic applications. This report of the synthesis of polymeric aptamer-conjugated PFPE-based 19F MRI CAs with high fluorine content (∼10 wt %) demonstrates that these NPs are exciting candidates for detecting diseases with high imaging sensitivity.

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The first example of the synthesis of mono-N,O-B-chelated dipyrromethene (BODIPY) derivatives through an unexpected intramolecular nucleophilic displacement of the fluorine by alkenols in the presence of boron trifluoride as Lewis acid is reported. The chlorine in the indacene core allowed for further structural modifications through nucleophilic substitutions or palladium-catalysed coupling reactions to afford new fluorophores with tuneable photophysical properties. Their expanded conjugation structure resulted in distinct red-shifted absorption and emission spectra in organic solutions. Furthermore, the twisted steric hindrance of the benzene substitution patterns suppressed aggregation-induced quenching, leading to an enhanced NIR emission in the aggregate/solid state, which was rarely observed for BODIPY dyes. Nanoparticles of the fluorophores formed by the assembly with the polymeric surfactant F127 were successfully used for bioimaging of living cells and for tumour-targeted imaging in a tumour-bearing mouse model.