5(6)-ROX (5(6)-Carboxy-X-rhodamine)

Dual-labeled prostate-specific membrane antigen (PSMA)-targeting agent for preoperative molecular imaging and fluorescence-guided surgery for prostate cancer

The objective of this study was to report the synthesis and characteristics of a dual modality imaging agent, Tc-99m GRFLTGGTGRLLRIS-GHEG-ECG-K(-5-carboxy-X-rhodamine)-NH2 (GRFLT-ECG-ROX), and to verify its feasibility as both molecular imaging and intraoperative guidance agent. GRFLT-ECG-ROX was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling of GRFLT-ECG-ROX with Tc-99m was accomplished using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed using LNCaP and PC-3 tumor-bearing murine models. Surgical removal of tumor nodules in murine models with peritoneal carcinomatosis was performed under a fluorescence imaging system. After radiolabeling procedures with Tc-99m, Tc-99m GRFLT-ECG-ROX complexes were prepared in high yield (>96%). The binding affinity value (Kd ) of Tc-99m GRFLT-ECG-ROX for LNCaP cells was estimated to be 9.5 ± 1.3 nM. In gamma camera imaging, the tumor to normal muscle uptake ratios of Tc-99m GRFLT-ECG-ROX increased with time (3.1 ± 0.2, 4.0 ± 0.4, and 6.3 ± 0.9 at 1, 2, and 3 h, respectively). Under real-time optical imaging, the removal of visible nodules was successfully performed. Thus, Tc-99m GRFLT-ECG-ROX could provide both preoperative molecular imaging and fluorescence imaging guidance for tumor removal.

A novel dual-modality imaging agent targeting folate receptor of tumor for molecular imaging and fluorescence-guided surgery

Objective: Folate receptor (FR) is an ideal target for cancer imaging because it is frequently overexpressed in major types of human tumor, whereas its expression in normal organs is highly limited. Combining nuclear and fluorescence-imaging techniques provides a novel approach for cancer imaging and monitoring the surgery. The objective of this study was to report the synthesis and characteristics of a dual-modality imaging agent, Tc-99m Folate-Gly-His-Glu-Gly-Glu-Cys-Gly-Lys(-5-carboxy-X-rhodamine)-NH₂ (Folate-ECG-ROX), and verify its feasibility as both molecular imaging agent and intra-operative guidance.
Methods: Folate-ECG-ROX was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling of Folate-ECG-ROX with Tc-99m was done using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution and ex vivo imaging studies were performed using KB and HT-1080 tumor-bearing murine models. Tumor tissue slides were prepared and analyzed with immunohistochemistry staining and confocal microscopy. Surgical removal of tumor nodules in murine models with peritoneal carcinomatosis was performed under the fluorescence-imaging system.
Results: After radiolabeling procedures with Tc-99m, Tc-99m Folate-ECG-ROX complexes were prepared in high yield (> 97%). The binding affinity value (K_d) of Tc-99m Folate-ECG-ROX for KB cells was estimated to be 6.9 ± 0.9 nM. In gamma camera imaging, tumor to normal muscle uptake ratio of Tc-99m Folate-ECG-ROX increased with time (3.4 ± 0.4, 4.4 ± 0.7, and 6.6 ± 0.8 at 1, 2, and 3 h, respectively). In biodistribution study, %IA/g for KB tumor was 2.50 ± 0.80 and 4.08 ± 1.16 at 1 and 3 h, respectively. Confocal microscopy with immunohistochemistry staining detected strong Tc-99m Folate-ECG-ROX fluorescence within KB tumor tissue which is correlating with the fluorescent activity of anti-FR antibody. Under real-time optical imaging, the removal of visible nodules was successfully performed.
Conclusions: In vivo and in vitro studies revealed substantial and specific uptake of Tc-99m Folate-ECG-ROX in FR-positive tumors. Thus, Tc-99m Folate-ECG-ROX could provide both pre-operative molecular imaging and fluorescence image-guidance for tumor.

Comparison of fluorescence energy transfer primers with different donor-acceptor dye combinations

Fluorescence energy transfer (ET) primers are far superior to single dye-labeled primers as labels for DNA sequencing and polymerase chain reaction amplification. We compare here ET primers with different donor and acceptor dye combinations with respect to the relative acceptor fluorescence emission intensity and the amount of residual donor fluorescence emission. Primers with the following donor/acceptor pairs were synthesized: 6-carboxyfluorescein/6-carboxy-X-rhodamine (FAM-ROX), 3-(epsilon-carboxypentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine/ 6-carboxy-X-rhodamine (CYA-ROX), and the 4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene-3-propionic acid (BODIPY) derivatives, 5,7-dimethyl-BODIPY/5-(4-phenyl-1,3-butadienyl) BODIPY (BODIPY503/512-BODIPY581/591). Variables examined included the length of the 5'-amino linker arm, the number of base pairs between the donor and acceptor, and the excitation wavelength (488 or 514 nm). Of the primers examined, CYA-ROX primers offer the best combination of acceptor fluorescence emission intensity and spectral purity.

High-resolution liquid chromatography of fluorescent dye-labeled nucleic acids

Using 100 mM of triethylammonium acetate as ion-pairing reagent, phosphodiester oligonucleotides labeled fluorescently at their 5' terminus could be separated successfully on alkylated nonporous 2.3-microns poly(styrene-divinylbenzene) particles by means of high-resolution liquid chromatography. Applying excitation wavelengths of 490, 520, 550, and 575 nm, respectively, optimum sensitivity was achieved for the fluorophores 5-carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein, N,N,N',N'-tetramethyl-6-carboxyrhodamine, and 6-carboxy-X-rhodamine (FAM, JOE, TAMRA, and ROX, respectively) at emission wavelengths of 520, 550, 580, and 605 nm, respectively. With calibration curves being linear over at least three orders of magnitude, the lower detection limits were 0.5, 2, 2, and 3 fmol, respectively. Depending on the type of fluorescent dye attached, retention times increased in the order JOE < FAM < TAMRA < ROX. Subsequently, fluorescent oligonucleotides were employed to prime polymerase chain reactions (PCR). Again the fluorophores were found to increase the retention times of double-stranded nucleic acids, but to a lesser degree than those of single-stranded oligonucleotides. Using a single FAM label attached to one of the two PCR primers, the sensitivity of fluorescence detection was found to be approximately 1 fmol or 30-70 times higher than that of uv absorbance detection depending on the length of the PCR product. Since the technique allows the separation of PCR products differing only 4 to 8 base pairs in length within a size range of 50 to 200 base pairs, it may be employed for the quantitative assessment of competitive PCR.

A simple QD-FRET bioprobe for sensitive and specific detection of hepatitis B virus DNA

We report here a simple quantum dot-FRET (QD-FRET) bioprobe based on fluorescence resonance energy transfer (FRET) for the sensitive and specific detection of hepatitis B virus DNA (HBV DNA). The proposed one-pot HBV DNA detection method is very simple, rapid and convenient due to the elimination of the washing and separation steps. In this study, the water-soluble CdSe/ZnS QDs were prepared by replacing the trioctylphosphine oxide on the surface of QDs with mercaptoacetic acid (MAA). Subsequently, DNA was attached to QDs surface to form the functional QD-DNA bioconjugates by simple surface ligand exchange. After adding 6-carboxy-X-rhodamine (ROX)-modified HBV DNA (ROX-DNA) into the QD-DNA bioconjugates solution, DNA hybridization between QD-DNA bioconjugates and ROX-DNA was formed. The resulting hybridization brought the ROX fluorophore, the acceptor, and the QDs, the donor, into proximity, leading to energy transfer from QDs to ROX. When ROX-DNA was displaced by the unlabeled HBV DNA, the efficiency of FRET was dramatically decreased. Based on the changes of both fluorescence intensities of QDs and ROX, HBV DNA could be detected with high sensitivity and specificity. Under the optimized conditions, the linear range of HBV DNA determination was 2.5 - 30 nmol L(-1), with a correlation coefficient (R) of 0.9929 and a limit of detection (3σ black) of 1.5 nmol L(-1). The relative standard deviation (R.S.D.) for 12 nmol L(-1) HBV DNA was 0.9% (n = 5). There was no interference to non-complementary DNA. Time-resolved fluorescence spectra and fluorescence images were performed to verify the validity of this method and the results were satisfying.