A novel apoptosis probe, cyclic ApoPep-1, for in vivo imaging with multimodal applications in chronic inflammatory arthritis.

Apoptosis plays an essential role in the pathophysiologic processes of rheumatoid arthritis. A molecular probe that allows spatiotemporal observation of apoptosis in vitro, in vivo, and ex vivo concomitantly would be useful to monitoring or predicting pathophysiologic stages. In this study we investigated whether cyclic apoptosis-targeting peptide-1 (CApoPep-1) can be used as an apoptosis imaging probe in inflammatory arthritis. We tested the utility of CApoPep-1 for detecting apoptotic immune cells in vitro and ex vivo using flow cytometry and immunofluorescence. The feasibility of visualizing and quantifying apoptosis using this probe was evaluated in a murine collagen-induced arthritis (CIA) model, especially after treatment. CApoPep-1 peptide may successfully replace Annexin V for in vitro and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for ex vivo in the measurement of apoptotic cells, thus function as a sensitive probe enough to be used clinically. In vivo imaging in CIA mice revealed that CApoPep-1 had 42.9 times higher fluorescence intensity than Annexin V for apoptosis quantification. Furthermore, it may be used as an imaging probe for early detection of apoptotic response in situ after treatment. The CApoPep-1 signal was mostly co-localized with the TUNEL signal (69.6% of TUNEL+ cells) in defined cell populations in joint tissues of CIA mice. These results demonstrate that CApoPep-1 is sufficiently sensitive to be used as an apoptosis imaging probe for multipurpose applications which could detect the same target across in vitro, in vivo, to ex vivo in inflammatory arthritis.

Intraarterial gene delivery in rabbit hepatic tumors: transfection with nonviral vector by using iodized oil emulsion.

PURPOSE: To evaluate the feasibility of an iodized oil emulsion that is used for the chemoembolization of hepatocellular carcinoma as a modifier of a nonviral gene transfer system for intraarterial gene delivery in experimentally induced hepatic tumors. MATERIALS AND METHODS: Experiments were performed in accordance with National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the animal research committee at Seoul National University Hospital. VX2 carcinoma was implanted into the liver of 26 rabbits. Four nonviral gene transfer systems were prepared by using pCMV-luc+ as a reporter gene. The first system consisted of a DNA and polyethylenimine (PEI) complex (n = 7); the second, of a DNA and PEI complex mixed with iopamidol and iodized oil (n = 7); the third, of a DNA and PEI complex mixed with iopamidol (n = 7); and the fourth, of a DNA and PEI complex mixed with iodized oil (n = 5). For the DNA and PEI complex that was mixed with iopamidol and iodized oil, iopamidol was used to stabilize the emulsion. Twenty days after tumor implantation, intraarterial gene delivery was performed by selective catheterization of the hepatic artery. Rabbits were euthanized 24 hours after gene delivery. Luciferase activity was assayed in the tumor, left hepatic lobe, right hepatic lobe, and other organs and was statistically analyzed for comparison between complexes by using the Kruskal-Wallis test. RESULTS: Luciferase activity in the tumor was significantly higher for the group that received DNA, PEI, iopamidol, and iodized oil than for any other group (Kruskal-Wallis test, P < .05). Luciferase activity in the left hepatic lobe, right hepatic lobe, and other organs was not significantly different between complexes. Selective gene expression in tumor cells was confirmed by means of immunohistochemical analysis for luciferase. CONCLUSION: It is feasible to use an iodized oil emulsion system for the intratumoral transfection of nonviral vectors in experimentally induced hypervascular hepatic tumors.

PEGylated polyethylenimine for in vivo local gene delivery based on lipiodolized emulsion system.

Polyethylenimine (PEI) is one of the most efficient vectors for non-viral gene delivery, whereas its poor transfection activity, compared to viral vectors, and cytotoxicity need to be improved for in vivo applications. In this study, we prepared two PEI conjugates with 6 and 10 wt.% of poly(ethylene glycol) (PEG) grafts (referred to PEI-PEG-6 and PEI-PEG-10, respectively) in order to investigate the effects of PEGylation on cytotoxicity and transfection activity in vitro. In addition, their suitability as vectors for local gene delivery in vivo was assessed by injecting lipiodolized emulsions containing polymer/DNA complexes into the femoral artery of Sprague-Dawley (SD) rats, occluded by a surgical suture to block inflow of the blood to the leg. Both PEGylated PEIs showed significantly lower cytotoxicity and higher transfection activity in COS-1 cells than PEI taken as a control; in particular, PEI-PEG-10 produced the most promising results. The stable water-in-oil emulsion, composed of aqueous domains containing the complexes and lipiodol as an oil phase, was formed in the presence of a hydrogenated castor oil. From in vivo experiments, it was found that all the complexes, dispersed in the lipiodolized emulsion, delivered effectively gene to muscle, surrounding the injection site, rather than other organs such as liver, spleen, kidney, heart and lung. The in vivo transfection activity of PEI-PEG-10 was 3-folds higher in muscle than that of PEI. Based on these results, it can be concluded that PEGylated PEIs (based on the lipiodolized emulsion system) hold a promising potential for local gene delivery in vivo.