2013 - CTS 2013 Congress

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Oral Communications 3

8.1 - Loco-regional detection and stimulation of transplanted liver cells by particle-based miRNA depletion

Presenter: Nathanael, Raschzok, Berlin, Germany
Authors: Nathanael Raschzok^1,2, Annekatrin Leder^1,2, Antje Butter^1,2, Susanne Kolano^1,2, Duygu Arabacioglu^1,2, Luisa De Sousa Lisboa^1,2, Wiebke Werner^1,2, Steffen Lippert^1,2, Christian Schmidt^1,2, Igor M. Sauer^1,2

Loco-regional detection and stimulation of transplanted liver cells by particle-based miRNA depletion

Nathanael Raschzok^1,2, Annekatrin Leder^1,2, Antje Butter^1,2, Susanne Kolano^1,2, Duygu Arabacioglu^1,2, Luisa De Sousa Lisboa^1,2, Wiebke Werner^1,2, Steffen Lippert^1,2, Christian Schmidt^1,2, Igor M. Sauer^1,2

¹General, Visceral, and Transplant Surgery, Experimental Surgery and Regenerative Medicine, CharitÃ© - UniversitÃ¤tsmedizin Berlin, Berlin, Germany; ²microparticles GmbH, Berlin, Germany

Introduction:

Liver cell transplantation (LCT) is a promising treatment option for children with inborn metabolic liver disease. However, both detection and proliferation stimulation of transplanted liver cells is challenging. Aim of this study was the development of a functionalized iron oxide particle for detection of transplanted liver cells by MRI and simultaneous loco-regional stimulation by targeting of specific microRNAs (miRNAs).

Methods:

Silica-based micron-sized iron oxide particles (sMPIOs) were used as carrier particles. Locked nucleic acid (LNA) antisense oligonucleotides specific for hsa-let-7g or rno-let-7g were covalently bound on the surface of the particles. For in vitro studies, primary human hepatocytes were labeled with LNA-sMPIO and miRNA depletion was investigated using qRT-PCR and Northern Blot. Protein expression of Cyclin D1 and c-Myc was investigated by western blot. Light and laser scanning microscopy were used to verify the intracellular localization of the particles. For in vivo proof of concept, male rat hepatocytes labeled with LNA-sMPIOs were transplanted to female Wistar rats via intrasplenic injection. 3.0 Tesla MRI was performed 7 days following transplantation. Cell engraftment was analyzed by Prussian Blue staining and Fish-Typing for the Y-chromosome.

Results:

In vitro incubation with LNA-sMPIOs enabled hepatocyte labeling within 4 hours. Particles were located near the cell nucleus, while not appearing to be encapsulated from cellular membranes. Labeling of human hepatocytes with LNA-sMPIOs lead to a decrease of endogenous let-7g levels by more than 85%. Knockdown efficiency was similar to controls with transfected cells.Cyclin D1 and c-Myc, which are both targets of let-7g, were significantly upregulated upon LNA-sMPIO internalization. In vivo, LNA-sMPIO labeled liver cells were detectable as punctual signal decrease of the liver and could be correlated with the presence of Prussian blue positive donor cells.

Discussion:

Our results demonstrate the feasibility of particle-based miRNA depletion. Moreover, we show functional effects on the protein expression of labeled cells. In vivo, the micron-sized iron core enabled non-invasive detection of transplanted cells. Our concept could be therefore suitable for LCT as well as for further cellular therapies where visualization and therapeutic modification is necessary.

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