2-Nitroimidazole-tricarbocyanine conjugate as a near-infrared fluorescent probe for in vivo imaging of tumor hypoxia

We developed a novel near-infrared (NIR) fluorescent probe, GPU-167, for in vivo imaging of tumor hypoxia. GPU-167 comprises a tricarbocyanine dye as an NIR fluorophore and two 2-nitroimidazole moieties as exogenous hypoxia markers that undergo bioreductive activation and then selective entrapment in hypoxic cells. After treatment with GPU-167, tumor cells contained significantly higher levels of fluorescence in hypoxia than in normoxia. In vivo fluorescence imaging specifically detected GPU-167 in tumors 24 h after administration. Ex vivo analysis revealed that fluorescence showed a strong correlation with hypoxia inducible factor (HIF)-1 active hypoxic regions. These data suggest that GPU-167 is a promising in vivo optical imaging probe for tumor hypoxia.     

A new fluorescent pH probe for imaging lysosomes in living cells

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5–4.1 with the pK_a value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no ‘alkalizing effect’ on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells.     

Synthesis and evaluation of a novel Tc-labeled bioreductive probe for tumor hypoxia imaging

Tumor hypoxia is closely associated with the malignant progression and/or the high metastatic ability of tumors and often induces resistance to chemo- and/or radiotherapy. Thus, the detection and evaluation of hypoxia is important for the optimization of cancer therapy. We designed a novel ^99mTc-labeled probe for tumor hypoxia imaging that utilizes bioreductive reactions in hypoxic cells. This probe, which contains a 4-nitrobenzyl ester group, is reduced in hypoxic cells to produce a corresponding carboxylate anion that cannot penetrate cell membranes because of its hydrophilicity and negative charge; therefore, it is expected to be trapped inside hypoxic cells. Based on this unique strategy, we synthesized the Technetium-99m (^99mTc)-labeled probe ^99mTc-SD32. The uptake of ^99mTc-SD32 in tumor cells was investigated under normoxic and hypoxic conditions. ^99mTc-SD32 showed sufficient accumulation and good retention in hypoxic cells. In addition, we demonstrated that ^99mTc-SD32 was subjected to bioreduction in hypoxic cells and was trapped as the corresponding carboxylate anion. These results indicated that ^99mTc-SD32 would be a promising agent for in vivo hypoxia imaging.     

Design strategy of optical probes for tumor hypoxia imaging

Science China Life Sciences - Clinical manifestations of tumors indicate that malignant phenotypes developing in the hypoxic microenvironment lead to resistance to cancer treatment, rendering...     

Dual probe with fluorescent and magnetic properties for imaging solid tumor xenografts

A dual probe with fluorescent and magnetic reporter groups was constructed by linkage of the near-infrared (NIR) fluorescent transferrin conjugate (Tf(NIR)) on the surface of contrast agent-encapsulated cationic liposome (Lip-CA). This probe was used for magnetic resonance imaging (MRI) and optical imaging of MDA-MB-231-luc breast cancer cells grown as a monolayer in vitro and as solid tumor xenografts in nude mice. Confocal microscopy, optical imaging, and MRI showed a dramatic increase of in vitro cellular uptake of the fluorescent and magnetic reporter groups from the probe compared with the uptake of contrast agent or Lip-CA alone. Pretreatment with transferrin (Tf) blocked uptake of the probe reporters, indicating the importance and specificity of the Tf moiety for targeting. Intravenous administration of the dual probe to nude mice significantly enhanced the tumor contrast in MRI, and preferential accumulation of the fluorescent signal was clearly seen in NIR-based optical images. More interestingly, the contrast enhancement in MRI showed a heterogeneous pattern within tumors, which reflected the tumor's morphologic heterogeneity. These results indicate that the newly developed dual probe enhances the tumor image contrast and is superior to contrast agent alone for identifying the tumor pathologic features on the basis of MRI but also is suitable for NIR-based optical imaging.     

Deep-red fluorescent imaging probe for bacteria

A versatile deep-red fluorescent imaging probe is described that is comprised of a bis(zinc(II)-dipicolylamine) targeting unit covalently attached to a pentamethine carbocyanine fluorophore with Cy5-like spectroscopic properties. A titration assay based on fluorescence resonance energy transfer is used to prove that the probe selectively associates with anionic vesicle membranes whose composition mimics bacterial cell membranes. Whole-body optical imaging experiments show that the probe associates with the surfaces of both Gram-positive and Gram-negative bacteria cells, and it can target the site of bacterial infection in a living mouse. In vivo accumulation at the infection site and subsequent clearance occurs more quickly than a structurally related near-infrared bis(zinc(II)-dipicolylamine) probe. The fact that the same deep-red probe molecule can be used for spectroscopic assays, cell microscopy, and in vivo imaging studies, is an important and attractive technical feature.     

Dicarboxy-dichlorofluorescein: a new fluorescent probe for measuring acidic intracellular pH

Derivatives of fluorescein sensitive to pH are extensively utilized for the determination of intracellular pH (pHi). Available dyes have pKa values of approximately 7.0, and are not well suited for measuring acidic pHi. We examined the fluorescein derivative, 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDCF) for its potential in the microspectrofluorometric measurement of pHi during acidic conditions. CDCF showed intense fluorescence and pH sensitivity near its "effective" pKa value of 4.2, using a 495/440 nm dual excitation wave-length ratio method. Protein interactions caused fluorescence ratio deviations which were most pronounced at the extremes of pH, whereas calcium and magnesium concentrations had little effect on the fluorescent ratio intensity. Intracellular calibration performed using nigericin in the presence of high potassium eliminated the need to correct for protein interactions, and the ratio method minimized any variations due to dye concentration differences or instrument fluctuation. Intracellular retention of the dye was high, and 95% of the initial signal remained after 1 h. Fluorescence bleaching was 14.5% after 1 h of continuous excitation and cell survival was not affected by dye loading. We conclude that CDCF is an excellent intracellular pH indicator in the pH range of 4-5.     

Synthesis and evaluation of a novel (99m)Tc-labeled bioreductive probe for tumor hypoxia imaging

Tumor hypoxia is closely associated with the malignant progression and/or the high metastatic ability of tumors and often induces resistance to chemo- and/or radiotherapy. Thus, the detection and evaluation of hypoxia is important for the optimization of cancer therapy. We designed a novel (99m)Tc-labeled probe for tumor hypoxia imaging that utilizes bioreductive reactions in hypoxic cells. This probe, which contains a 4-nitrobenzyl ester group, is reduced in hypoxic cells to produce a corresponding carboxylate anion that cannot penetrate cell membranes because of its hydrophilicity and negative charge; therefore, it is expected to be trapped inside hypoxic cells. Based on this unique strategy, we synthesized the Technetium-99m ((99m)Tc)-labeled probe (99m)Tc-SD32. The uptake of (99m)Tc-SD32 in tumor cells was investigated under normoxic and hypoxic conditions. (99m)Tc-SD32 showed sufficient accumulation and good retention in hypoxic cells. In addition, we demonstrated that (99m)Tc-SD32 was subjected to bioreduction in hypoxic cells and was trapped as the corresponding carboxylate anion. These results indicated that (99m)Tc-SD32 would be a promising agent for in vivo hypoxia imaging.     

Membrane permeable esterase-activated fluorescent imaging probe

An esterase-triggered probe 2 derived from a cyanine-based pH sensitive dye was developed for cell labeling. Permeation of probe 2 into cells and subsequent hydrolytic activation by cellular esterases result in a bright fluorescent intracellular signal.     

Estimation of matrix pH in isolated heart mitochondria using a fluorescent probe

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the pH-sensitive 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The resulting charged forms of the probes are retained in the mitochondrial matrix and appear well-suited for the estimation of pCa and pH in this compartment. The mitochondria esterase activity is stimulated by Ca2+, inhibited by butacaine and quinine, and shows an alkaline pH optimum. The esterase has a similar affinity for the two probes (Km about 1.5 microM) and a somewhat higher Vmax for BCECF. Intramitochondrial pH can be determined by recording the ratio of the fluorescence of matrix BCECF at its excitation maximum of 509 nm to that at 450 nm, an excitation wavelength that is unresponsive to pH. A calibration plot relating the fluorescence ratio to pH is constructed using detergent-lysed mitochondria and the excitation maximum of 500 nm for BCECF in aqueous solution. Estimates of matrix pH by BCECF fluorescence in its useful range (pH 6 to 8) agree well with values obtained using the distribution of 5,5-dimethyl-2,4-oxazolidenedione. In protocols in which the fluorescence with excitation at 450 nm does not vary, a direct recording of BCECF fluorescence with excitation at 509 nm can be used to follow the kinetics of matrix pH changes.     

Genetically encoded fluorescent indicators for live cell pH imaging

Background

Intracellular pH underlies most cellular processes. There is emerging evidence of a pH-signaling role in plant cells and microorganisms. Dysregulation of pH is associated with human diseases, such as cancer and Alzheimer's disease.

Detection of dysplastic intestinal adenomas using a fluorescent folate imaging probe

Macrophages have long been recognized as a prominent component of tumors. Activated macrophages overexpress folate receptors and we used this phenomenon to image inflammatory reactions in colon dysplasia using a fluorescent folate probe (FFP). APC(Delta468) mice injected with FFP showed fluorescent adenomas (target-to-background ratio, adenoma vs. adjacent normal mucosa, of 2.46 +/- 0.41), significantly higher (p < .001) than adenomas in animals injected with a non-folate-containing control probe. Fluorescence-activated cell-sorting analysis revealed a 3-fold higher content of Mac1-positive cells in colonic adenomas compared with normal adjacent mucosa (6.8% vs. 2.2%), and confirmed the source of FFP-positive cells to be primarily an F4/80-positive macrophage subpopulation. Taken together, these results indicate that probe potentially can be used to image dysplastic intestinal adenomas in vivo.     

A dual-function fluorescent probe for the detection of pH values and formaldehyde

A dual-function fluorescent probe (Probe 1) was developed in this work for the separate detection of pH value and formaldehyde (HCHO). Probe 1 could recognize HCHO and the pH value from the amino group. The colour of the probe solution was changed from grey blue to light blue with the increase in the pH value, and luminous intensity became larger with the increase in formaldehyde concentration. The curve function relationship between fluorescence intensity and the pH value was also determined. A smartphone containing a colour detector for imaging was used to record the values of the three primary colours (R value, G value, and B value) for the probe solution in formaldehyde. Importantly, there was a linear functional relationship between the B*R/G value with HCHO concentration. Therefore, the probe could be used as a rapid tool for the detection of formaldehyde. More importantly, Probe 1 was successfully used to detect formaldehyde in an actual distilled liquor sample.     

Measurement of the intracellular pH of blood platelets using a photolabel fluorescent probe

The internal pH of blood platelets using the intracellular photolabel probe azidofluorescein diacetate was determined. No change of intracellular pH during thrombin activation of human platelets was observed. Platelets were found to adjust themselves very quickly to the external pH. Quantitative subcellular localization of the attachment sites of this probe reveals that most of it is bound to low molecular weight proteins or peptides.     

Dual‐function fluorescent probe for cancer imaging and therapy

To date, several fluorescent probes modified by a single targeting agent have been explored. However, studies on the preparation of dual‐function quantum dot (QD) fluorescent probes with dual‐targeting action and a therapeutic effect are rare. Here, a dual‐targeting CdTe/CdS QD fluorescent probe with a bovine serum albumin–glycyrrhetinic acid conjugate and arginine‐glycine‐aspartic acid was successfully prepared that could induce the apoptosis of liver cancer cells and showed enhanced targeting in in vitro cell imaging. Therefore, the as‐prepared fluorescent probe in this work is an efficient diagnostic tool for the simultaneous detection of liver cancer and breast cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Spectroscopically well-characterized RGD optical probe as a prerequisite for lifetime-gated tumor imaging

Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανβ3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανβ3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.     

A water‐soluble fluorescent pH probe based on perylene dyes and its application to cell imaging

A fluorescent pH probe, N,N′‐bi( l ‐phenylalanine amine)‐perylene‐3,4;9,10‐dicarboxylic diimide (PDCDA) was synthesized and used for pH sensing in living cells. A significant fluorescence intensity change was observed over a pH range from 7.0 to 4.0. Electrostatic potential maps (MEP) suggested that the electronic repulsion between PDCDAs was increased by the high negative electrostatic potential which resulted in a high water solubility of PDCDA. PDCDA was successfully applied as a high‐performance fluorochrome for living HeLa cell imaging. The results demonstrate that the probe PDCDA is a good candidate for monitoring pH fluctuations in living cells with good water solubility, low cytotoxicity, high fluorescence quantum yield and photostability. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis and characterization of a novel near-infrared fluorescent probe for applications in imaging A549 cells

Objective

To design and synthesize a novel near-infrared (NIR) fluorescent probe based on indocyanine Green (ICG), that can be applied in imaging living cells.

Results

A highly fluorescent novel NIR fluorescent probe (IR-793) was synthesized in two steps. IR-793 had better fluorescence and optical stability than ICG. In addition, no obvious cytotoxicity effect of IR-793 was observed and cell viability was above 75% at the maximum concentration (120 nM). IR-793 also exhibited good performance in imaging living A549 cells.

Conclusion

IR-793, a novel NIR fluorescent probe that is stable, low-cost, highly fluorescent and low cytotoxicity, has been designed and synthesized for imaging living cells.

     

Evaluation of 99mTc-CN5DG as a broad-spectrum SPECT probe for tumor imaging

Previously, we reported a [^99mTc(ǀ)]⁺ labeled d-glucoamine derivative (^99mTc-CN5DG) and evaluated it as a tumor imaging agent in mice bearing A549 tumor xenografts. In this paper, ^99mTc-CN5DG was further studied in U87 MG (human glioma cells), HCT-116 (human colon cancer cells), PANC-1 (human pancreatic cancer cells) and TE-1 (human esophageal cancer cells) tumor xenografts models to verify its potential application for imaging of different kinds of tumors. The biodistribution data showed that ^99mTc-CN5DG had a similar biodistribution pattern in four tumor models at 2 h post-injection with high accumulation in tumors and kidneys. The tumor/muscle ratios (from 4.08 ± 0.42 to 9.63 ± 3.53) and tumor/blood ratios (from 17.18 ± 7.40 to 53.17 ± 16.16) of ^99mTc-CN5DG in four tumor models were high. All four kinds of tumors could be clearly seen on their corresponding SPECT/CT images. Pharmacokinetic study in healthy CD-1 mice demonstrated that ^99mTc-CN5DG cleared fast from blood (2 min, 12.97 ± 0.88%ID/g; 60 min, 0.33 ± 0.06%ID/g) and the blood distribution, elimination half-life was 5.81 min and 21.16 min, respectively. No abnormality was observed through the abnormal toxicity study. All of the above results demonstrated that ^99mTc-CN5DG could be a broad-spectrum SPECT probe for tumor imaging and its further clinical application is warranted.