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.

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.

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.

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.

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.

Alkaline phosphatase-triggered assembly of etoposide enhances its anticancer effect.

Etoposide is a cancer-targeting drug but an overdose of etoposide leads to immunosuppression in patients. Therefore, the development of a new strategy to enhance its anticancer effect, while in the meantime alleviating its adverse effects, is important but challenging. In this work, with the assistance of a hydrogelator precursor Nap-Phe-Phe-Tyr(H2PO3)-OH (1P), etoposide phosphate (EP) was subjected to alkaline phosphatase (ALP)-triggered assembly, which obviously enhanced its anticancer efficacy in vitro and in vivo. In vitro tests indicated that the assembly of EP with 1P resulted in a slow release of etoposide and long-term inhibitory effects on HeLa cells. In vivo experiments indicated that, compared with those of EP-treated mice, the tumor growth of EP + 1P-treated mice was further inhibited while their body weight loss was alleviated. We envision that our hydrogelator-assisted assembly strategy could be applied to enhance the therapeutic effects of more drugs, while in the meantime alleviating their adverse effects in the future.

Intracellular Proteolytic Disassembly of Self-Quenched Near-Infrared Nanoparticles Turning Fluorescence on for Tumor-Targeted Imaging.

The design of tumor-targeting, intracellular protease-activatable near-infrared fluorescence (NIRF) nanoprobes is broadly interesting but remains challenging. In this work, we report the rational design of a NIR probe Cys(StBu)-Lys(Biotin)-Lys-Lys(Cy5.5)-CBT (1) to facilely prepare the self-quenched nanoparticles 1-NPs for tumor-targeted imaging in vitro and in vivo. The biotinylated 1-NPs could be actively uptaken by biotin receptor-overexpressing tumor cells via receptor-mediated endocytosis. Upon intracellular proteolytic cleavage, 1-NPs were disassembled to yield the small molecular probe Lys(Cy5.5)-Luciferin-Lys(Biotin)-Lys-OH (1-D-cleaved), accompanied by fluorescence "Turn-On". With this NIRF "Turn-On" property, 1-NPs were successfully applied for tumor-targeted imaging. We envision that our nanoparticles could be applied for fluorescence-guided tumor surgery in the near future.

Bioluminescence Sensing of γ-Glutamyltranspeptidase Activity In Vitro and In Vivo.

γ-Glutamyltranspeptidase (GGT) is an important tumor biomarker but using a bioluminescence (BL) probe to real time monitor its activity has not been reported. Herein, we rationally designed two GGT-cleavable BL probes Glu-AmLH2 (1) and Glu-p-aminobenzyloxycarbonyl-AmLH2 (2), and successfully applied them for sensing GGT activity with high sensitivity and excellent selectivity both in vitro and in vivo. The results indicated that, although 2 had lower background BL signal than 1, GGT had higher catalytic efficiency for 1 than 2, and 1 was superior to 2 for sensing GGT activity in living cells and tumors. We envision that our probe 1 could be widely applied for the diagnosis of important GGT-related diseases in animal models in the near future.

Alkaline Phosphatase-Instructed Self-Assembly of Gadolinium Nanofibers for Enhanced T2-Weighted Magnetic Resonance Imaging of Tumor.

Alkaline phosphatase (ALP) is an important enzyme but using ALP-instructed self-assembly of gadolinium nanofibers for enhanced T2-weighted magnetic resonance imaging (MRI) of tumor has not been reported. In this work, we rationally designed a hydrogelator Nap-FFFYp-EDA-DOTA(Gd) (1P) which, under the catalysis of ALP, was able to self-assemble into gadolinium nanofibers to form hydrogel Gel I for enhanced T2-weighted MR imaging of ALP activity in vitro and in tumor. T2 phantom MR imaging indicated that the transverse relaxivity (r2) value of Gel I was 33.9% higher than that of 1P and both of them were 1 order of magnitude higher than that of Gd-DTPA. In vivo T2-weighted MR imaging showed that, at 9.4 T, ALP-overexpressing HeLa tumors of 1P-injected mice showed obviously enhanced T2 contrast. We anticipate that, by replacing ALP with other enzymes, our approach could be applied for MR diagnosis of other diseases in the future.

Discriminative fluorescence sensing of biothiols in vitro and in living cells.

Simultaneous discriminative sensing of biothiols in vitro and in living cells has remained challenging. Herein, we report a new sulfonamide-based self-quenched fluorescent probe 1 for this purpose with high sensitivity and good selectivity. Treatment of 1 with cysteine (Cys), homocysteine (Hcy), or glutathione (GSH) yields aminoluciferin, 2-cyano-6-aminobenzothiazole homocysteine (CBTHcy), or 2-cyano-6-aminobenzothiazole (CBT), turning "on" the fluorescence at wavelengths of 522, 517, or 490 nm, respectively. Kinetic study indicated that 1 reacts with Cys faster than with Hcy or GSH. With these unique properties of 1, we applied 1 for highly sensitive sensing of Cys, Hcy, and GSH among other 19 natural amino acids (AAs) with good selectivity. Confocal fluorescence microscopic imaging of 1-treated HepG2 cells at two channels (522 ± 8 and 490 ± 8 nm), together with quantitative analysis, indicated that the "turn-on" fluorescence was induced by intracellular Cys-dominating condensation and reduction of 1 but not by intracellular GSH-dominating reduction of 1. This suggests that 1 could be applied for discriminative sensing of intracellular Cys from the abundant GSH. Further development of 1 might bring about an efficient tool for probing cellular functions that relate to biothiols.

Pyridine-biquinoline-metal complexes for sensing pyrophosphate and hydrogen sulfide in aqueous buffer and in cells.

Herein, we report a new pyridine-biquinoline-derivative fluorophore L for effectively sensing pyrophosphate (PPi) and monohydrogen sulfide (HS(-)) in aqueous buffer and in living cells. L could selectively coordinate with metal ions (M(n+)) in Groups IB and IIB to form L-M(n+) complexes with 1:1 stoichiometry, resulting in fluorescence quenching via photoinduced electron transfer (PET) mechanism. L-Zn(2+) complex was applied to competitively coordinate with PPi to form a new "ate"-type complex, turning on the fluorescence by a 21-fold-increase. The limit of detection (LOD) of this assay for PPi detection in aqueous buffer is 0.85 µM. L-Cu(2+) complex was applied for highly selective detection of HS(-) with an excellent sensitivity by 25-fold decomplexation-induced fluorescence increase. LOD of L-Cu(2+) complex for HS(-) detection in aqueous buffer is 2.24 µM. With the in vitro data obtained, we successfully applied these two complexes for sequential imaging Zn(2+) and PPi, Cu(2+) and HS(-) in living cells, respectively. Since PPi and HS(-) occur in vascular calcification in positive correlation, our multifunctional probe L might help doctors to more precisely diagnose this disease in vivo in the future. For example, we could use radioactive tracer L-(64)Cu for qualitative and quantitative positron emission tomography/computed tomography (PET/CT) imaging of HS(-) in vivo.