ERA NanoSci - Magnetic nanoparticles for nerve regeneration

• Antragstellerin: Professorin Dr. Gerburg Keilhoff
• Fachliche Zuordnung: Molekulare Biologie und Physiologie von Nerven- und Gliazellen ; Theoretische Chemie: Elektronenstruktur, Dynamik, Simulation
• Förderung: Förderung von 2009 bis 2014
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 119418337

• Erstellungsjahr: 2015

Zusammenfassung der Projektergebnisse

The project was part of the ERA-Net Project (Magnetic nanoparticles for nerve regeneration). Magnetic nanoparticles (NP) should be generated, functionalized and used in vitro and in vivo to support the target-orientated regeneration of out-sprouting nerve fibers. The PNP were prepared at the INA (Saragossa). Functionalization with NGF and BDNF was the job of the SSSA (Pisa). At the IMSAT (Dundee) the particles were characterized by Atomic-force-Microscopy. Our group was responsible for the cell-specific testing of the NP and the connected with that establishment of an organotypic spinal cord culture model with the possibility to reconstruct the ventral root which is normally sheared off by preparation. The results performed by our group can be summarized as followed: (i) NP were vesicular (per endocytosis) up-taken. Thereby we found a cellular ranking of uptake capacity: Microglia ≫ Astroglia, Schwann cells > Oligodendroglia, Neurons. (ii) NP induced a (expected) Microglia proliferation. (iii) NP were toxic with ongoing concentration, whereby the Glia-toxicity was higher than the neurotoxicity. That was unexpected; endocytosis is a main function of microglia. (iv) A neuronal (receptor-mediated) uptake requests functionalization. Covalent bounded NGF or GDNF was, however, only partly bioactive. Non-covalent bounded both factors were able to develop good neuroprotective potency. The non-covalent binding of GDNF was, unfortunately, not stable enough for allowing its application in vivo. (v) The organotypic SC cultures (without and with reconstructed ventral horn) were very well suitable models for testing of neuro-protective/regeneration supporting potencies, also and especially under the aspects of animal welfare. (vi) NP were well tolerated from SC cultures. An improvement of the target-orientated neurites sprouting with the assistance of an external magnetic field was, however, not reached. One main reason was the insufficient NP up-take capacity of neurons, which, in turn, could be attributed to the instable functionalization of the NP. (vii) Minocycline, applied to SC cultures in a short-term and low-dosed manner, was neuro-protective. Supporting effects on number and length of out-sprouting neurites was more stable. Unexpected were the astroglia-inhibiting, Cx 43-mediated effects of minocycline. (viii) Cerebrolysin was neuro-protective also only when it was applied short-termed and low-dosed. With longer application time and/or higher dosage it developed neuro-toxic as well as gliainhibitory potencies. That should be carefully noted as Cerebrolysin is already in clinical use. (ix) The In-vivo-Testing of the project-relevant, functionalized NP in our well established in vivo rat/mice model of sciatic nerve injury and reconstruction was not possible to carry out. Therefore the number and quality of functionalized NP were too low.

Projektbezogene Publikationen (Auswahl)

• Magnetic nanoparticles in primary neural cell cultures are mainly taken up by microglia. BMC Neurosci. 2012;22;13:32
  Pinkernelle J, Calatayud MP, Goya GF, Fansa H, Keilhoff G
  
• Poly-l-lysine-coated magnetic nanoparticles as intracellular actuators for neural guidance. Int J Nanomedicine. 2012;7:3155-66
  Riggio C, Calatayud MP, Hoskins C, Pinkernelle J, Sanz B, Torres TE, Ibarra MR, Wang L, Keilhoff G, Goya GF, Raffa V, Cuschieri A
  
• Prolonged minocycline treatment impairs motor neuronal survival and glial function in organotypic rat spinal cord cultures. PLoS One. 2013 Aug 13;8(8):e73422
  Pinkernelle J, Fansa H, Ebmeyer U, Keilhoff G
  
• Effects of cerebrolysin on motor-neuron-like NSC-34 cells. Exp Cell Res. 2014;327(2):234-55
  Keilhoff G, Lucas B, Pinkernelle J, Steiner M, Fansa H
  
• Effects of cerebrolysin on rat Schwann cells in vitro. Acta Histochem. 2014;116(5):820-30
  Lucas B, Pinkernelle J, Fansa H, Keilhoff G
  
• Evaluation of In-Situ magnetic signals from iron oxide nanoparticle-labeled PC12 cells by atomic force microscopy. J Biomed Nanotechnol 2015;11(3):457-468
  Wang L, Min Y, Wang Z, Riggio C, Calatayud MP, Pinkernelle J, Raffa V, Goya GF, Keilhoff G, Cuschieri A
  
• Growth factor choice is critical for successful functionalization of nanoparticles. Front Neurosci, 2015, 9:305
  Pinkernelle J, Raffa V, Calatayud MP, Goya GF, Riggio C, Keilhoff G