Dual-Responsive Near-Infrared BODIPY-Based Fluorescent Probe for the Detection of F- and HClO in Organisms.

Fluoride anions (F-) play a crucial role in human physiological processes. However, excessive intake of F- would affect oxygen metabolism and promote the generation of oxygen-free radicals. Hence, it is essential to develop a precise and efficient fluorescent probe for visualizing F--induced oxidative stress. In this work, we developed the first bifunctional BODIPY-based fluorescent probe dfBDP with p-tert-butyldimethylsilanolate benzyl thioether as the sensing site for the detection of F- and HClO via two distinct reactions, the self-immolative removal and the thioether oxidation, which generate the sensing products with two nonoverlap fluorescence bands: 800-1200 and 500-750 nm, respectively. The probe dfBDP displays rapid response, high specificity, and sensitivity for the detection of F- (LOD, 316.2 nM) and HClO (LOD, 33.9 nM) in vitro. Cellular imaging reveals a correlation between F--induced oxidative stress and the upregulation of HClO. Finally, probe dfBDP was employed to detect F- and HClO in mice under the stimulation of F-. The experimental results display that the level of HClO elevates in the liver of mice.

Tumor-Targeted Fluorescent/Photoacoustic Imaging of Legumain Activity In Vivo.

Legumain has been identified as a target for diagnosis and treatment of associated cancers. Therefore, real-time imaging of legumain activity in vivo is helpful in diagnosing and evaluating therapeutic efficacy of associated cancers. Fluorescent/photoacoustic (FL/PA) dual-modal imaging developed rapidly because of its good sensitivity and spatial resolution. As far as we know, a tumor-targeted probe for FL/PA imaging of legumain activity in vivo has not been reported. Hence, we intended to develop a tumor-targeted hemicyanine (HCy) probe (HCy-AAN-Bio) for FL/PA imaging of legumain in vivo. The control probe HCy-AAN does not have tumor-targeting ability. Legumain can specifically cleave HCy-AAN-Bio or HCy-AAN with the generation of FL/PA signal while more HCy-AAN-Bio could be recognized by legumain than HCy-AAN with higher sensitivity in vitro. Due to the tumor-targeting ability, HCy-AAN-Bio could image 4T1 cells with an additional 1.3-fold FL enhancement and 1.9-fold PA enhancement than HCy-AAN. In addition, HCy-AAN-Bio could image legumain activity in vivo with an additional 1.5-fold FL enhancement and 1.9-fold PA enhancement than HCy-AAN. We expected that HCy-AAN-Bio will be a powerful tool for early diagnosis of associated cancer.

A BODIPY-based fluorescent probe for simultaneous detection of H2O2 and viscosity during the pyroptosis process.

A dual functional BODIPY fluorescent probe was developed for simultaneous detection of H2O2 and viscosity, by collecting fluorescence from 800-1100 nm and 550-750 nm, respectively. Bioimaging based on the probe shows that H2O2 accumulates and cytoplasmic viscosity increases during the palmitic acid (PA)-induced pyroptosis process.

ß-Galactosidase-Activatable Fluorescent and Photoacoustic Imaging of Tumor Senescence.

ß-Galactosidase (ß-gal) is the gold standard marker of cellular senescence, which is linked with various age-related diseases. Therefore, it is essential to develop more excellent probes that can real-time monitor ß-gal activity in cellular senescence in vivo. Fluorescent/photoacoustic (FL/PA) dual-modal imaging possesses excellent sensitivity and spatial resolution. To our knowledge, there has been no tumor-targeted FL/PA probe to image cellular senescence by monitoring the activity of ß-gal in vivo. Therefore, we developed a tumor-targeted FL/PA probe (Gal-HCy-Biotin) for ß-gal-activatable imaging of tumor senescence. Gal-HCy without tumor-targeted biotin is used as a control probe. Gal-HCy-Biotin is superior to Gal-HCy due to the higher kinetic parameter of Gal-HCy-Biotin than Gal-HCy in vitro. Moreover, biotin could help Gal-HCy-Biotin enter and accumulate in tumor cells with higher FL/PA signal. In detail, Gal-HCy-Biotin or Gal-HCy could image senescent tumor cells with 4.6-fold or 3.5-fold FL enhancement and 4.1-fold or 3.3-fold PA enhancement. Gal-HCy-Biotin or Gal-HCy could image tumor senescence with 2.9-fold or 1.7-fold FL enhancement and 3.8-fold or 1.3-fold PA enhancement. We envision that Gal-HCy-Biotin will be applied for FL/PA imaging of tumor senescence in clinic.

Caspase-3-Responsive Fluorescent/Photoacoustic Imaging of Tumor Apoptosis.

Caspase-3 is an essential executor in apoptosis, and its activation has been regarded as a biomarker of cell apoptosis. The development of Caspase-3-responsive multimodal probes is a promising research prospect. Fluorescent/photoacoustic (FL/PA) imaging has attracted considerable attention due to the high sensitivity of FL as well as the high spatial resolution and penetration depth of PA. To our knowledge, there has been no tumor-targeted FL/PA probe to monitor the activity of Caspase-3 in vivo. Therefore, we developed a tumor-targeted FL/PA probe (Bio-DEVD-HCy) for Caspase-3-responsive imaging of tumor apoptosis. Ac-DEVD-HCy without tumor-targeted biotin is used as a control probe. In vitro experiments indicated that Bio-DEVD-HCy is superior to Ac-DEVD-HCy because of the higher kinetic parameter of Bio-DEVD-HCy in comparison to Ac-DEVD-HCy. Cell and tumor imaging results suggested that Bio-DEVD-HCy could enter and accumulate in tumor cells with higher FL/PA signal with the help of tumor-targeted biotin. In detail, Bio-DEVD-HCy or Ac-DEVD-HCy could image apoptotic tumor cells with 4.3-fold or 3.5-fold FL enhancement and 3.4-fold or 1.5-fold PA enhancement. Bio-DEVD-HCy or Ac-DEVD-HCy could image tumor apoptosis with 2.5-fold or 1.6-fold FL enhancement and 4.1-fold or 1.9-fold PA enhancement. We envision that Bio-DEVD-HCy will be applied for FL/PA imaging of tumor apoptosis in clinical settings.

Intracellular Formation of Hemicyanine Nanoparticle Enhances Tumor-Targeting Photoacoustic Imaging and Photothermal Therapy.

Alkaline phosphatase (ALP) is a tumor marker for early diagnosis and treatment. Tumor targeting can recognize and fight tumor cells more accurately from healthy cells. Glycyrrhetinic acid (GA) is a targeting ligand of liver tumors. Photoacoustic imaging (PAI) and photothermal therapy (PTT) are promising techniques for tumor diagnosis and treatment. The outstanding characteristics of Hemicyanine (HCy) dye make it suitable for tumor diagnosis and treatment. However, using HCy nanoparticle (HCy NP) for liver tumor-targeting PAI and PTT has not been reported. Herein, Probe-1 is developed to enhance PAI and PTT of liver tumors due to GA targeting and intracellular ALP-instructed self-assembly of HCy NP. Compared to Probe-2 without self-assembly ability, Probe-1 displays a 4.6-fold higher PAI signal or 1.7-fold lower half inhibitory concentrations in HepG2 cells. Moreover, Probe-1 shows extended retention time (10 vs 6 h) and 2.1-fold higher PAI signal than Probe-2 in HepG2 tumors. The HepG2 tumors in Group Probe-1 obviously increase 18 °C (Tmax : 55 °C) with a 3.3-fold decreased volume while that in Group Probe-2 mildly increase 9.8 °C (Tmax : 46.8 °C) with a 4.3-fold increased volume. It is envisioned that this smart self-assembly strategy can be easily adjusted for PAI and PTT of more tumors.

Caspase-1-responsive fluorescence biosensors for monitoring endogenous inflammasome activation.

The activation of inflammasome leads to secretion of inflammatory factors and cell pyroptosis that are critical in the pathogenesis of various chronic and acute inflammatory diseases. Recruitment and activation of caspase-1 is a marker of inflammasome activation. However, there is still lack of real-time and efficient methods to detect the activation of inflammasome, especially in vivo. Herein, we developed two activatable caspase-1-responsive fluorescence biosensors, WEHD-HCy and YVAD-HCy, to specifically monitor the activation of inflammasome in vivo. Our in vitro study demonstrated that WEHD-HCy and YVAD-HCy can sensitively and specifically respond to caspase-1 activation. Moreover, these biosensors can efficiency and specifically activated in the common inflammatory disease model, including inflammatory bowel disease, Salmonella infection, and acute arthritis. In particular, WEHD-HCy is more advantageous than YVAD-HCy to specifically image of caspase-1 activity both in vitro and in vivo. These caspase-1-responsive fluorescence biosensors provide an efficient, rapid, and in situ tool for monitoring inflammasome activation, and have the potential to be suitable for clinical diagnosis of various inflammatory diseases associated with inflammasome activation.

An esterase-activatable curcumin prodrug for tumor-targeting therapy.

A tumor-targeting therapy strategy is urgently needed to increase the accumulation of drugs in tumors and reduce the side effects in normal tissues. Herein, we developed an esterase-activatable curcumin prodrug Cur-RGD for tumor-targeting therapy. Armed with the tumor-targeting RGD peptide and in situ esterase-triggered drug release, this prodrug Cur-RGD can efficiently improve the therapeutic effect of curcumin in tumors.

Sequential self-assembly and disassembly of curcumin hydrogel effectively alleviates inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal tract with unclear etiology and insufficient therapeutic efficacy. The development of specific, effective and safe IBD treatment drugs is of great clinical significance. Curcumin (Cur) is a good candidate to prevent and manage inflammatory diseases (such as IBD) due to its antioxidant and anti-inflammatory effects with safety profile. However, its poor aqueous solubility and instability under physiological conditions greatly limit its therapeutic efficacy. Herein, we exploited a Cur precursor Cur-FFEYp to locally deliver and slowly release Cur at inflamed regions for treatment of IBD by a sequential self-assembly and disassembly strategy. The much higher catalytic efficiency of alkaline phosphatase (ALP) than esterase towards Cur-FFEYp validated the sequential ALP-induced self-assembly with the formation of Cur hydrogel and esterase-guided disassembly with the slow release of Cur. In cell and animal experiments, Cur-FFEYp can effectively enhance the anti-inflammatory effect of Cur on inflammatory macrophages and significantly alleviate two types of IBD. We envision that by using other biomarkers to conduct the sequential self-assembly and disassembly processes and replacing other drugs, our smart strategy could be easily adjusted for the treatment of more diseases or cancers.

Simultaneous enhancement of T1 and T2 magnetic resonance imaging of liver tumor at respective low and high magnetic fields.

Background: Nowadays, magnetic resonance imaging (MRI) is routinely applied in clinical diagnosis. However, using one contrast agent (CA) to simultaneously enhance the T1 and T2 MR contrast at low and high magnetic fields respectively has not been reported. Methods: Herein, we investigated the MR property of a γ-glutamyl transpeptidase (GGT)-instructed, intracellular formed gadolinium nanoparticle (DOTA-Gd-CBT-NP) at low and high magnetic fields. Results: Experimental results showed that DOTA-Gd-CBT-NP possesses a low r2/r1 ratio 0.91 which enables it to enhance T1 MR imaging of liver tumor at 1.0 T, and a high r2/r1 ratio 11.8 which renders the nanoparticle to largely enhance T2 MR imaging of liver tumor at 9.4 T. Conclusion: We expect that our GGT-responsive Gd-nanoparticle could be applied for simultaneous T1 and T2 MRI diagnosis of early liver cancer in clinic at respective low and high magnetic fields when the 9.4 T MR machine is clinically available in the future.

Mitochondria-targeted photoacoustic probe for imaging of hydrogen peroxide in inflamed mouse model.

In this chapter, we gave a brief introduction of hydrogen peroxide (H2O2) and its existing analytical methods and described the need of mitochondria-targeted photoacoustic (PA) probe for H2O2 detection in vivo. Then we provided the detailed protocols for the design and characterization of a mitochondria-targeted PA probe (TPP-HCy-BOH) to visualize H2O2in vivo, which was developed in our previous work. Compared to control probe without mitochondria-targeted ability (HCy-BOH), TPP-HCy-BOH could efficiently accumulate in mitochondria and activate its PA signals toward overproduced H2O2 in inflamed mouse model with higher PA sensitivity.

Cathepsin B-Activated Fluorescent and Photoacoustic Imaging of Tumor.

Early diagnosis is crucial to the treatment of cancer. Cathepsin B (CTB) plays an important role in numerous cancers, which is a promising biomarker for early diagnosis of cancer. It is necessary to exploit new probes for visualization of CTB in vivo. Fluorescent/photoacoustic (FL/PA) imaging is a powerful tool for in vivo study which possesses both excellent sensitivity and spatial resolution. To our knowledge, there has been no FL/PA probe to image CTB in vitro or in vivo. Therefore, we developed two CTB-activated FL/PA probes HCy-Cit-Val and HCy-Gly-Leu-Phe-Gly, which could successfully monitor CTB activity in vivo. Both two probes had excellent sensitivity and selectivity in vitro. Cell imaging showed that HCy-Cit-Val or HCy-Gly-Leu-Phe-Gly could image endogenous CTB in lysosome with 6.8-fold or 5.1-fold enhancement of the FL signal and 5.8-fold or 3.4-fold enhancement of the PA signal compared to their inhibitor contrast groups. Tumor imaging in vivo further confirmed the good applicability of these two probes to monitor CTB activity with high sensitivity and spatial resolution. Moreover, the property of HCy-Cit-Val is superior to HCy-Gly-Leu-Phe-Gly due to the higher catalytic efficiency of CTB toward HCy-Cit-Val than HCy-Gly-Leu-Phe-Gly. We envision that our FL/PA probe HCy-Cit-Val will be suitable for clinical early diagnosis of CTB-related cancer in the near future.

A Golgi-Targeting and Dual-Color "Turn-On" Probe for Spatially Precise Imaging of Furin.

Development of fluorescence probes for highly accurate detection of cancer-related enzyme activity is important in early cancer diagnosis. Herein, we report a Golgi-targeting and dual-color "Turn-On" probe Q-RVRR-DCM for imaging furin with high spatial precision. By integrating the principles of Förster resonance energy transfer and intramolecular charge transfer, the probe was designed to be non-fluorescent. Upon furin cleavage, Q-RVRR-DCM was converted into Q-RVRR and DCM-NH2, turning the dual fluorescence color "On" at 420 and 640 nm without spectral cross-talk. In furin-overexpressing HCT116 cells, Q-RVRR-DCM showed not only furin-specific, dual-color "Turn-On" fluorescence but also superior colocalization with a Golgi tracker than the single-color "Turn-On" probe RVRR-DCM. We envision that, with the excellent properties of Golgi-targeting and dual fluorescence color "Turn-On", our furin probe Q-RVRR-DCM could be applied for accurate early diagnosis of cancer in the near future.

Mitochondria-Targeted Fluorescent and Photoacoustic Imaging of Hydrogen Peroxide in Inflammation.

Hydrogen peroxide (H2O2) is a prominent reactive oxygen species with relative stability, which makes it a potential diagnostic marker for pathological states. Excessive H2O2 in mitochondria leads to oxidative stress and inflammation. However, precisely monitoring the level of H2O2 at specific organelles (e.g., mitochondria) in vivo is still of urgent necessity. Therefore, we rationally designed a mitochondria-targeted near-infrared probe TPP-HCy-BOH for fluorescent/photoacoustic (FL/PA) dual-modal imaging of overproduced H2O2 in an inflamed mouse model. TPP-HCy-BOH had a low LOD (0.348 µM), which is comparable to those of recently reported probes for H2O2 detection. The high kinetic rate constant (kobs = 4.72 × 10-3 s-1) of TPP-HCy-BOH toward H2O2 is superior to recently reported H2O2 probes. Compared to control probe HCy-BOH without the mitochondrial targeting moiety, TPP-HCy-BOH successfully images exogenous or endogenous H2O2 in mitochondria with an additional 2.4-fold FL increase and 4.7-fold PA increase in HeLa cells or additional 2.1-fold FL increase and 3.3-fold PA increase in RAW 264.7 cells. In LPS-induced acute inflammation in vivo, TPP-HCy-BOH is more competent to image overproduced H2O2 with additional 1.6-fold higher sensitivity of FL in abdomen and 2.0-fold higher sensitivity of PA in liver and longer retention time of 0.5 h than HCy-BOH. We anticipate that TPP-HCy-BOH could be employed for the FL/PA dual-modal diagnosis of pathological inflammation in clinic in near future.

Multi-Stimuli-Triggered Shape Transformation of Polymeric Filaments Derived from Dynamic Covalent Block Copolymers.

Using dynamic polymers to achieve the morphology transformation of polymeric assemblies under different conditions is challenging. Herein, we reported diversiform shape transformation of multi-responsive polymer filaments, which were self-assembled by a new kind of amphiphilic block copolymer (PVEG-PVEA) possessing dynamic and reversible acylhydrazone bonds through reacting benzaldehyde-containing block copolymers poly(vinylbenzaldehyde)-b-poly(N-(4-vinylbenzyl)-N,N-diethylamine) (PVBA-PVEA) with acylhydrazine-modified oligoethylene glycol. It was found that the resulting amphiphilic and dynamic PVEG-PVEA was capable of hierarchically self-assembling into intriguing core-branched filaments in aqueous solution. Notably, the features of acylhydrazone bonds and PVEA block endow the filaments with multi-responsiveness including acid, base, and temperature, leading to the multiple morphological transformations under such stimuli. Moreover, the core-branched filaments would further transform into polymeric braided bundles driven by hydrogen-bonding interactions of amide bonds. It is noteworthy that both core-branched filaments and braided bundles made from polymers are quite rare. These diversiform polymeric assemblies and their morphological evolution were characterized by TEM, Cryo-TEM, SEM, and DLS. Finally, we used PVBA-PVEA as a platform to facilely prepare functional polymers, such as glycopolymers via the reaction of amino-containing sugars and aldehyde groups. The obtained glycopolymers self-assembled into glycofibers for the biomimicry of glycans via binding with lectins. These findings not only are conducive to understanding of the stimulated shape change process of dynamic polymeric assemblies in water but also provide a new method for the facile fabrication of smart and functional polymeric assemblies for different potential applications, such as biomimicry and targeted drug nanocarriers or delivery vehicles.

Cathepsin B Turning Bioluminescence "On" for Tumor Imaging.

Cathepsin B (CTSB) is a lysosomal protease, and several human cancers are reported highly expressing CTSB. Many optical methods have been developed for CTSB detection but not a bioluminescence (BL) probe. Herein, a CTSB-specific bioluminescence probe Val-Cit-AL was rationally designed for selectively sensing CTSB activity in vitro with a 67-fold "Turn-On" of BL intensity and an excellent limit of detection. Inhibitory experiments indicated that Val-Cit-AL is capable of sensing CTSB activity in living cells and tumors. We anticipate that Val-Cit-AL might be applied to diagnose CTSB-related diseases in rodent models or evaluate CTSB roles in more biological processes in the near future.

Sustained Release of Two Bioactive Factors from Supramolecular Hydrogel Promotes Periodontal Bone Regeneration.

Intact and stable bone reconstruction is ideal for the treatment of periodontal bone destruction but remains challenging. In research, biomaterials are used to encapsulate stem cells or bioactive factors for periodontal bone regeneration, but, to the best of our knowledge, using a supramolecular hydrogel to encapsulate bioactive factors for their sustained release in bone defect areas to promote periodontal bone regeneration has not been reported. Herein, we used a well-studied hydrogelator, NapFFY, to coassemble with SDF-1 and BMP-2 to prepare a supramolecular hydrogel, SDF-1/BMP-2/NapFFY. In vitro and in vivo results indicated that these two bioactive factors were ideally, synchronously, and continuously released from the hydrogel to effectively promote the regeneration and reconstruction of periodontal bone tissues. Specifically, after the bone defect areas were treated with our SDF-1/BMP-2/NapFFY hydrogel for 8 weeks using maxillary critical-sized periodontal bone defect model rats, a superior bone regeneration rate of 56.7% bone volume fraction was achieved in these rats. We anticipate that our SDF-1/BMP-2/NapFFY hydrogel could replace bone transplantation in the clinic for the repair of periodontal bone defects and periodontally accelerated osteogenic orthodontics in the near future.

γ-Glutamyltranspeptidase-Triggered Intracellular Gadolinium Nanoparticle Formation Enhances the T2-Weighted MR Contrast of Tumor.

Magnetic resonance imaging (MRI) is advantageous in the diagnosis of deep internal cancers, but contrast agents (CAs) are always needed to improve MRI sensitivity. Gadolinium (Gd)-based agents are routinely used as T1-dominated CAs in clinic but using intracellularly formed Gd nanoparticles to enhance the T2-weighted MRI of tumor in vivo at high magnetic field has not been reported. Herein, we rationally designed a "smart" Gd-based probe Glu-Cys(StBu)-Lys(DOTA-Gd)-CBT (1), which was subjected to γ-glutamyltranspeptidase (GGT) cleavage and an intracellular CBT-Cys condensation reaction to form Gd nanoparticles (i.e., 1-NPs) to enhance the T2-weighted MR contrast of tumor in vivo at 9.4 T. Living cell experiments indicated that the 1-treated HeLa cells had an r2 value of 27.8 mM-1 s-1 and an r2/r1 ratio of 10.6. MR imaging of HeLa tumor-bearing mice indicated that the T2 MR contrast of the tumor enhanced 28.6% at 2.5 h post intravenous injection of 1. We anticipate that our probe 1 could be employed for T2-weighted MRI diagnosis of GGT-related cancers in the future when high magnetic field is available in clinic.

Legumain-Specific Near-Infrared Fluorescence "Turn On" for Tumor-Targeted Imaging.

Legumain is one of the cysteine proteases which can serve as an essential indicator for cancer diagnosis. Near-infrared (NIR) nanoprobes with fluorescence "Turn On" property are advantageous in cancer diagnosis. However, to the best of our knowledge, using a completely organic NIR nanoprobe to image legumain activity either in vitro or in vivo has not been reported. Herein, employing a CBT-Cys click condensation reaction, we used a rationally designed NIR probe Cys(StBu)-Ala-Ala-Asn-Lys(Cy5.5)-CBT (1) to synthesize its nanoprobes 1-NPs with self-quenched fluorescence. Cell and animal experiments indicated that our nanoprobes were able to specifically image legumain activity in living cells and tumors with a NIR fluorescence "Turn On" manner. We envision that the nanoprobes could be applied for the diagnosis of legumain-related diseases in the near future.

Intracellular self-assembly of Ru(bpy)32+ nanoparticles enables persistent phosphorescence imaging of tumors.

Nanoprobes are advantageous over small molecular probes in sensitivity but most luminescence molecules used to construct nanoprobes often suffer from an aggregation-caused quenching effect. Herein, we rationally designed a small molecular probe Cys(StBu)-Lys(Ru(bpy)32+)-CBT (1) which "smartly" self-assembled into nanoparticles 1-NPs inside cells with non-quenched, persistent phosphorescence. Employing this property, we successfully applied 1 for long-term sensing of biothiol activity in living HepG2 cells and tumors. We envision that, by modifying the amino group with an enzyme substrate, our probe 1 could be further developed for sensing intracellular enzyme activity with non-quenched, persistent phosphorescence.