Sensing phosphatase activity by using gold nanoparticles.
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Alkaline Phosphatase↗
Biomedical subjects
Ching-Hsuan Tung
Publications and source records attributed to Ching-Hsuan Tung.
Enzyme-targeted fluorescent imaging probes on a multiple antigenic peptide core.
Peptide dendrimers have a variety of applications in biology such as the vehicles for drug and gene delivery, molecular inhibitors, protein mimics, and synthetic vaccines. The multiple antigenic peptide (MAP) system is a well-known example of a discrete, dendrimeric scaffold. We explored a novel application of the MAP-based scaffold by designing molecular probes that fluoresce only after enzymatic treatment. The probes, which were synthesized on solid support, incorporate a cathepsin S dipeptide substrate (Leu-Arg), and a poly(ethylene glycol) (PEG) chain in their dendritic arms. The fluorescence emission of the near-infrared fluorochromes attached to the N-termini of the dendritic arms was quenched. Mechanistic studies revealed formation of H-type dye aggregates within the tetravalent MAP system. By varying the length of the PEG chain, three probes were synthesized, CyPEG-1, CyPEG-2, and CyPEG-3 with 4, 8, and 12 ethylene oxide units, respectively. CyPEG-2 showed optimum aqueous solubility and quenching efficiency for imaging applications. Upon proteolytic activation with cathepsin S (EC 3.4.22.27), CyPEG-2 showed greater than 70-fold increase and more than 95% recovery in fluorescence emission.
Cathepsins↗
Selective antitumor effect of novel protease-mediated photodynamic agent.
A new approach to selective photodynamic therapy (PDT) was developed by designing chlorin e6 (Ce6)-containing macromolecules, which are sensitive to tumor-associated proteases. The agents are nontoxic in their native state but become fluorescent and produce singlet oxygen on protease conversion. Coupled with optimized delivery systems, we show that (a) the agents efficiently accumulate in tumors due to the enhanced permeability and retention effect, (b) the agents are locally activated by proteases, (c) local drug concentrations can be measured by quantitative fluorescence tomography, and (d) light-treated tumors show reduced growth. A single low dose of PDT (0.125 mg Ce6 equivalent/kg) was sufficient to suppress tumor growth by >50%. Activatable singlet oxygen generation agents provide increased efficacy with reduced toxicity, and it could become a powerful PDT.
Animals↗
Inflammation in atherosclerosis: visualizing matrix metalloproteinase action in macrophages in vivo.
BACKGROUND: Matrix metalloproteinases (MMPs) in inflamed atherosclerotic plaques may contribute to extracellular matrix remodeling and the onset of acute thrombotic complications. METHODS AND RESULTS: To test the hypothesis that optical molecular imaging with the use of an activatable near-infrared fluorescence (NIRF) probe can detect enzymatic action of MMP in atherosclerotic plaques, we used a NIRF substrate for gelatinases (MMP-2/gelatinase-A and MMP-9/gelatinase-B) in apolipoprotein E-deficient (apoE-/-) mice that consumed a high-cholesterol diet for 12 weeks and age-matched apoE+/+ mice as control. The aortas of apoE-/- mice at 24 hours after probe yielded intense NIRF signals, as detected by NIRF reflectance ex vivo, compared with negligible signals in aortas of apoE+/+ mice with/without probe administration or atherosclerotic apoE-/- aortas without probe. Gelatinase inhibitor treatment abolished NIRF signals in apoE-/- mouse aortas ex vivo. Sites of gelatinase activity visualized by NIRF colocalized with macrophage accumulation, immunoreactive MMP-2 and MMP-9, and gelatinolytic activity detected by in situ zymography. Furthermore, fluorescence molecular tomography indicated in vivo that atherosclerotic aortas of apoE-/- mice produced NIRF signals for gelatinase action, whereas aortas of apoE+/+ mice injected with the probe or apoE-/- aortas with no probe exhibited negligible NIRF signals. CONCLUSIONS: These results suggest the feasibility of noninvasively imaging the enzymatic action of MMPs in vivo, an approach that may gauge inflammatory foci in atherosclerosis, assess cardiovascular risk, and evaluate the effects of therapeutic interventions.
Animals↗
Development of a dual fluorogenic and chromogenic dipeptidyl peptidase IV substrate.
A new far-red dual fluorogenic and chromogenic substrate, 5-glycylprolylglycylprolyl-9-di-3-sulfonyl-propylaminobenza[a]phenoxazonium perchlorate (GPGP-2SBPO), was developed for dipeptidyl peptidase IV (DPP-IV) sensing. The glycylprolylglycylprolyl tetrapeptide was chosen as the recognition sequence due to its stability under physiological conditions. In contrast, the truncated substrate, GP-2SBPO, containing only a glycylprolyl peptide, is unstable. Proteolysis of GPGP-2SBPO was assayed by monitoring the absorbance and fluorescence signals from the released fluorochrome, 2SBPO, at 625 and 670nm, respectively.
Dipeptidyl Peptidase 4↗
Factor XIII deficiency causes cardiac rupture, impairs wound healing, and aggravates cardiac remodeling in mice with myocardial infarction.
BACKGROUND: Identification of key molecular players in myocardial healing could lead to improved therapies, reduction of scar formation, and heart failure after myocardial infarction (MI). We hypothesized that clotting factor XIII (FXIII), a transglutaminase involved in wound healing, may play an important role in MI given prior clinical and mouse model data. METHODS AND RESULTS: To determine whether a truly causative relationship existed between FXIII activity and myocardial healing, we prospectively studied myocardial repair in FXIII-deficient mice. All FXIII(-/-) and FXIII(-)(/+) (FXIII activity <5% and 70%) mice died within 5 days after MI from left ventricular rupture. In contradistinction, FXIII(-/-) mice that received 5 days of intravenous FXIII replacement therapy had normal survival rates; however, cardiac MRI demonstrated worse left ventricular remodeling in these reconstituted FXIII(-/-) mice. Using a FXIII-sensitive molecular imaging agent, we found significantly greater FXIII activity in wild-type mice and FXIII(-/-) mice receiving supplemental FXIII than in FXIII(-/-) mice (P<0.05). In FXIII(-/-) but not in reconstituted FXIII(-/-) mice, histology revealed diminished neutrophil migration into the MI. Reverse transcriptase-polymerase chain reaction studies suggested that the impaired inflammatory response in FXIII(-/-) mice was independent of intercellular adhesion molecule and lipopolysaccharide-induced CXC chemokine, both important for cell migration. After MI, expression of matrix metalloproteinase-9 was 650% higher and collagen-1 was 53% lower in FXIII(-/-) mice, establishing an imbalance in extracellular matrix turnover and providing a possible mechanism for the observed cardiac rupture in the FXIII(-/-) mice. CONCLUSIONS: These data suggest that FXIII has an important role in murine myocardial healing after infarction.
Animals↗
Conjugation of a photosensitizer to an oligoarginine-based cell-penetrating peptide increases the efficacy of photodynamic therapy.
To improve the efficiency of intracellular delivery of photosensitizers and the efficacy of photodynamic therapy, a membrane-penetrating arginine oligopeptide (R7) was conjugated to 5-[4-carboxyphenyl]-10,15,20-triphenyl-2,3-dihydroxychlorin (TPC). The resulting conjugate (R7-TPC) enhanced intracellular TPC uptake, which increased proportionally with the incubation time of the conjugate. The water solubility of the highly hydrophobic TPC photosensitizer was also improved after conjugation. Increased phototoxicity of R7-TPC was observed after an incubation time of only 30 min. Tumor cells mainly underwent apoptosis at lower concentrations of the photosensitizer-polyarginine conjugate, whereas necrotic cell damage became prevalent at higher concentrations.
Arginine↗
Protease-mediated phototoxicity of a polylysine-chlorin(E6) conjugate.
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Cell Line, Tumor↗
Construction of a novel chimera consisting of a chelator-containing Tat peptide conjugated to a morpholino antisense oligomer for technetium-99m labeling and accelerating cellular kinetics.
The attempt to target the limited copies of messenger RNA (mRNA) in vivo with radiolabeled nucleobase oligomers as antisense probes is challenging. Selecting an antisense molecule with superior properties, enhancing the cellular kinetics, and improving the radiolabeling chemistry would be the reasonable approach to accomplish this goal. The present study reports a method to construct a chimera of phosphorodiamidate morpholino nucleobase oligomer (MORF) covalently conjugated to a peptide containing a cell membrane transduction Tat peptide and an N(2)S(2) chelator for technetium-99m ((99m)Tc) radiolabeling (N(2)S(2)-Tat-MORF). The radiolabeling properties and cellular kinetics of (99m)Tc-N(2)S(2)-Tat-MORF were measured. As hypothesized, the preparation of (99m)Tc-N(2)S(2)-Tat-MORF could be achieved by an instant one-step method with labeling efficiency greater than 95%, and the (99m)Tc-N(2)S(2)-Tat-MORF showed distinct properties in cell culture from those of a control, the same MORF sequence without Tat but with mercaptoacetyltriglycine (MAG(3)) as chelator for (99m)Tc ((99m)Tc-MAG(3)-MORF). (99m)Tc-N(2)S(2)-Tat-MORF achieved maximum accumulation of about 35% within 2 h, while (99m)Tc-MAG(3)-MORF showed lower and steadily increasing accumulations but of less than 1% in 24 h. These preliminary results demonstrated that the proposed chimera has properties for easy labeling, and (99m)Tc-N(2)S(2)-Tat-MORF prepared by this method possesses enhanced cellular kinetics and merits further investigation for in vivo mRNA targeting.
Chelating Agents↗
Peptide-based biomaterials for protease-enhanced drug delivery.
Controlled delivery of drugs in response to environments has the potential of targeting therapies and personalized treatments. Here, we described self-assembled peptide sequences that release therapeutic payloads upon specific interaction with disease-associated proteases. The core peptide sequence consists of a protease cleavable region flanked by two self-assembly motifs. In aqueous solution, the peptides self-assemble as a gel scaffold. With treatment of the model preparations with the appropriate protease, the matrix can be degraded in a controlled fashion, where the degradation rate is fine-tuned by varying the peptide compositions. Protease-mediated drug release was demonstrated by enzymatic treatment of a model therapeutic peptide incorporated into the optimized matrix. Our results suggest that this type of material may have far-reaching applications for functionally targeted drug delivery.
Biocompatible Materials↗
A fluorescent nanosensor for apoptotic cells.
A biocompatible surface-functionalized nanoparticle was designed to sense phosphatidylserine exposed on apoptotic cells. We conjugated synthetic artificial phosphatidylserine binding ligands in a multivalent fashion onto magnetofluorescent nanoparticles. Our results show that (1) the synthetic nanoparticles bind to apoptotic cells, (2) there is excellent correlation with annexin V staining by microscopy, and (3) FACS analysis with nanoparticles allows the measurement of therapeutic apoptosis induction. The described nanomaterials should be useful for a variety of biomedical applications including in vivo imaging of apoptosis.
Apoptosis↗
The Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 replicates in mature phagolysosomes within J774 macrophages.
Adherent-invasive Escherichia coli (AIEC) bacteria isolated from Crohn's disease patients are able to extensively replicate within macrophages in large vacuoles. The mechanism by which AIEC bacteria survive within phagocytic cells is unknown. This report describes the maturation of AIEC LF82-containing phagosomes within J774 macrophages. LF82-containing phagosomes traffic through the endocytic pathway as shown by the sequential acquisition and loss of EEA1 and Rab7 and by accumulation of Lamp-1, Lamp-2 and cathepsin D. We demonstrated that AIEC LF82-containing phagosomes mature into active phagolysosomes where bacteria are exposed to low pH and to the degradative activity of cathepsin D. Finally, we showed that an acidic environment is necessary for replication of AIEC LF82 bacteria within J774 macrophages. Thus, evidence is provided that AIEC LF82 bacteria do not escape from the endocytic pathway but undergo normal interaction with host endomembrane organelles and replicate within acidic and cathepsin D-positive vacuolar phagolysosomes.
Animals↗
A mitochondrial targeted fusion peptide exhibits remarkable cytotoxicity.
A potent cytotoxic peptide (r7-kla) was synthesized by incorporating a mitochondrial membrane disrupting peptide, kla (klaklakklaklak), with a cell-penetrating domain, r7 (rrrrrrr). The IC(50) of r7-kla (3.54 +/- 0.11 micromol/L) was more than two orders of magnitude lower than that of kla. r7-kla induced cell death in both in vitro and in vivo environments, and showed rapid kinetics. Within minutes, the morphologic changes in cells and mitochondrial leakage were apparent by microscopy and was consistent with rapid apoptosis. Our results suggested that r7-kla is an apoptosis inducer and can be potentially used as an antitumor agent, especially when combined with the appropriate systemic delivery systems.
Animals↗
Tissue inhibitor of metalloproteinase-3 expression from an oncolytic adenovirus inhibits matrix metalloproteinase activity in vivo without affecting antitumor efficacy in malignant glioma.
Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, AdDelta24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus AdDelta24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by AdDelta24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2-activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the AdDelta24TIMP-3-treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87deltaEGFR) tumors with AdDelta24TIMP-3 and AdDelta24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of AdDelta24 did not significantly affect the antitumor efficacy of this oncolytic agent.
Adenoviridae↗
Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction.
BACKGROUND: We used a molecular probe activated by protease cleavage to image expression of matrix metalloproteinases (MMPs) in the heart after myocardial infarction. METHODS AND RESULTS: We synthesized and characterized a near-infrared fluorescent (NIRF) probe that is activated by proteolytic cleavage by MMP2 and MMP9. The NIRF probe was injected into mice at various time points up to 4 weeks after myocardial infarction induced by ligation of the left anterior descending coronary artery. NIRF imaging of MMP activity increased in the infarct region, with maximal expression at 1 to 2 weeks, persisting to 4 weeks. Zymography and real-time polymerase chain reaction analysis showed that MMP9 expression is increased at 2 to 4 days, and MMP2 expression is increased at 1 to 2 weeks. Dual-label confocal microscopy showed colocalization of NIRF imaging with neutrophils on day 2, and flow cytometric analysis confirmed that NIRF signal is associated with leukocytes in the infarct zone. CONCLUSIONS: This study demonstrates that the activity of MMPs in the myocardium may be imaged by use of specific activity-dependent molecular probes.
Animals↗
Optical zymography for specific detection of urokinase plasminogen activator activity in biological samples.
Zymography techniques are routinely used to quantify proteolytic activity. In the current study, we describe an optical zymographic procedure that specifically detects urokinase-type plasminogen activator (uPA) activity in biological samples. The method employs a synthetic polymeric uPA fluorescent probe, which is copolymerized in sodium dodecyl sulfate (SDS)-polyacrylamide gel. Following electrophoresis and renaturation, enzymatic digestions of the substrate in 50 mM of Tris buffer at pH 7.4 generates fluorescence emission at 695 nm. The enzymatic activities can be analyzed directly by conventional gel imaging systems with a detection limit of 40 pg. This protocol is fast (hours) and does not require staining and destaining steps. The procedure is independent of plasminogen and, therefore, can efficiently distinguish the active two-chain uPA from its proenzyme. Densitometry analysis demonstrated a highly correlative relationship (r2=0.999) between the amount of uPA (over the range of 0.1-8.0 ng) and the average intensity of the fluorescent band. We were able to directly measure uPA activities in different cancer cell lines. This newly developed technique could be expanded to nearly all proteases, including the ones that cannot be analyzed by traditional zymography.
Caseins↗
Arthritis imaging using a near-infrared fluorescence folate-targeted probe.
A recently developed near-infrared fluorescence-labeled folate probe (NIR2-folate) was tested for in vivo imaging of arthritis using a lipopolysaccharide intra-articular injection model and a KRN transgenic mice serum induction mouse model. In the lipopolysaccharide injection model, the fluorescence signal intensity of NIR2-folate (n = 12) and of free NIR2 (n = 5) was compared between lipopolysaccharide-treated and control joints. The fluorescence signal intensity of the NIR2-folate probe at the inflammatory joints was found to be significantly higher than the control normal joints (up to 2.3-fold, P < 0.001). The NIR2-free dye injection group showed a persistent lower enhancement ratio than the NIR2-folate probe injection group. Excessive folic acid was also given to demonstrate a competitive effect with the NIR2-folate. In the KRN serum transfer model (n = 4), NIR2-folate was applied at different time points after serum transfer, and the inflamed joints could be detected as early as 30 hours after arthritogenic antibody transfer (1.8-fold increase in signal intensity). Fluorescence microscopy, histology, and immunohistochemistry validated the optical imaging results. We conclude that in vivo arthritis detection was feasible using a folate-targeted near-infrared fluorescence probe. This receptor-targeted imaging method may facilitate improved arthritis diagnosis and early assessment of the disease progress by providing an in vivo characterization of active macrophage status in inflammatory joint diseases.
Animals↗
Mechanism-based fluorescent reporter for protein kinase A detection.
A novel mechanism-based fluorescent reporter was designed for the detection of protein kinase A (PKA), which is known to mediate a variety of cellular responses in most eukaryotic cells. The probe consists of a specific binding peptide sequence, LRRRRFAFC, conjugated with 2'-thioethyl-5-(or -6)-carboxyfluoresceinamide (FAMS; 2) and 5-(or 6-)carboxytetramethylrhodamine (TAMRA) at the cysteine and leucine residues, respectively. In the absence of PKA, the two fluorophores associate by hydrophobic interactions, forming an intramolecular ground-state dimer; this results in fluorescein quenching (>93 %). Upon PKA addition, the reporter reacts with the sulfhydryl functionality at Cys199 through a disulfide-exchange mechanism. FAMS is subsequently released, resulting in significant fluorescence amplification. The remaining peptide sequence, which acts as an inhibitor, is attached covalently to the enzyme. Our results suggest that this type of sensors could have far-reaching applications in the molecular sensing of enzymes.
Amino Acid Sequence↗