Nanomagneto-assisted cell- and gene therapy in the heart for the treatment of post-infarct complications
Zusammenfassung der Projektergebnisse
Initially clinically relevant cell types were loaded with MNP and to test their magnet-based positioning in vitro and in vivo. SOMag5 were identified as most suitable MNP type, because of their good loading properties of embryonic cardiomyocytes (eCM, e14.5-15.5), embryonic stem cell derived cardiomyocytes (ES-CM) and adult bone marrow cells (BMC)) and their low toxicity. Next, their in vitro and in vivo behavior under magnetic force was first simulated and then tested. Based on these results, 200.000 MNP-loaded cells were injected intramyocardially into cryolesioned murine hearts with- and without superimposition (ca. 0.5mm distance) of a 1.3 T bar magnet on the injection site during and 10 minutes after the injection. Two weeks after this treatment approximately a 7-fold increase of engraftment rates was noticed for all three cell types following magnet assisted transplantation (M+). The left ventricular function of eCM- and ES-CM injected hearts was also investigated and strongly improved left ventricular function was detected in M+ hearts compared to controls. In eCM transplanted hearts also the long term fate of grafted cells was analyzed and 8 weeks postoperatively still long term survival of grafted cells (3.4 fold) and left ventricular function were significantly increased compared to controls. We found that several different mechanisms are responsible for improved integration and long-term survival of MNP loaded eCM following magnet assisted intramyocardial transplantation. Magnetorelaxometry revealed a relatively high cell retention rate of 58% in M+ hearts, while in M- hearts only 24% of injected MNP loaded eCM were found 10 minutes after intramyocardial injection. In the further course, the M+ injected cells were found to display strongly reduced rates of apoptosis (Caspase3) and strongly increased rates of proliferation (Ki-67, pHH3) thereby explain the increased engraftment rates. Ultrastructural analysis proved that engrafted and magnet-treated eCM were further differentiated (sarcomeric structure, mitochondria content) and displayed a better structural organization (presence of desmosomes, different collagen and glucose-amino-glycan content of the extracellular matrix) compared to controls. Beside the above described cell transplantation experiments we also investigated, if resident cells of the myocardial scar, mainly (myo)fibroblasts, could be modulated by a direct gene therapeutic approach. Due to the massive initial cell death and inflammatory response, direct MNP/magnet assisted lentiviral transduction of the infarct zone was carried out at the 3rd day after myocardial infarction, in order to obtain proliferating or immigrating cells like (myo)fibroblasts or endothelial cells. The magnet assisted injection of LV/MNP complexes resulted in prominent expression of the reporter gene compared to LV/MNP injection without magnet application. Additionally to the direct intramyocardial injection also the feasibility of a local magnet assisted MNP/LV gene therapy via the transvascular/intracoronary application route was investigated. Prominent recirculation of infused MNP/LV complexes within the coronary system was achieved but so far local magnet application resulted in intramyocardial but also intracoronary aggregation of MNP, causing malperfusion downstream of the target area and lack of gene expression in the established heterotopic heart transplantation model. To solve the problem, smaller MNP with less aggregation tendency and better biodistribution were designed and are tested at the moment in vitro.
Projektbezogene Publikationen (Auswahl)
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(2012). Identification of magnetic nanoparticles for combined positioning and lentiviral transduction of endothelial cells. Pharmaceutical research, 29(5), 1242-1254
Wenzel,D., Rieck,S., Vosen,S., Mykhaylyk,O., Trueck,C., Eberbeck,D., Trahms,L., Zimmermann,K., Pfeifer,A., Fleischmann,B.K.
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(2012). Local gene targeting and cell positioning using magnetic nanoparticles and magnetic tips: comparison of mathematical simulations with experiments. Pharm Res. 29, 1380-91
Kilgus,C., Heidsieck,A., Ottersbach,A., Roell,W., Trueck,C., Fleischmann,B.K., Gleich,B., Sasse,P.
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(2012). Optimization of magnetic nanoparticle-assisted lentiviral gene transfer. Pharmaceutical research, 29(5), 1255-1269
Trueck,C., Zimmermann,K., Mykhaylyk,O., Anton,M., Vosen,S., Wenzel,D., Fleischmann,B.K., Pfeifer, A.
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(2013). Embryonic cardiomyocyte, but not autologous stem cell transplantation, restricts infarct expansion, enhances ventricular function, and improves long-term survival. PLoS One. 8, e61510
Paulis,L.E., Klein,A.M., Ghanem,A., Geelen,T., Coolen,B.F., Breitbach,M., Zimmermann,K., Nicolay,K., Fleischmann,B.K., Roell,W., Strijkers,G.J.
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(2015). Systemic gene transfer enables optogenetic pacing of mouse hearts. Cardiovasc Res. 106, 338-43
Vogt,C.C., Bruegmann,T., Malan,D., Ottersbach,A., Roell,W., Fleischmann,B.K., Sasse,P.
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(2016). Vascular Repair by Circumferential Cell Therapy Using Magnetic Nanoparticles and Tailored Magnets. ACS Nano. 10, 369-76
Vosen,S., Rieck,S., Heidsieck,A., Mykhaylyk,O., Zimmermann,K., Bloch,W., Eberbeck,D., Plank,C., Gleich,B., Pfeifer,A., Fleischmann,B.K., Wenzel,D.
