In her second publication, Josepha showed her ambitious strategies to load functional siRNA into milk-derived EVs. In her first approach, she tried to fuse siRNA loaded liposomes with EVs. She found out that the fusion success depends on the fusion technique and the lipid composition of the liposomes. Although fusion of EVs and liposomes was proven by FRET assays, siRNA loading remained low.
Following the concept that the ratio of the siRNA-rich outer phase to the volume inside the EVs is decisive for successful siRNA loading of the EVs, she concentrated the purified EVs via ultracentrifugation. Then, she complexed siRNA in neutrally charged Calciumphosphate nanoparticles (CaP-NP), concentrated them, and finally combined the siRNA-rich CaP-NP with the EV concentrate in a dual centrifugation step. With this procedure, Josepha could preserve the siRNA function and delivery via EVs to Caco-2 cells.
Nucleic acid delivery with extracellular vesicles
Advanced Drug Delivery Reviews 173 (2021) 89-111
Michaela Schulz-Siegmund, Achim Aigner
https://doi.org/10.1016/j.addr.2021.03.005
siRNA as active pharmaceutical ingredient (API)
Therapeutic application of siRNA is a fascinating way to positively influence regeneration. However, nucleic acid drugs in general do not meet the requirements for an active pharmaceutical ingredient: They have a molecular weight of more than 10 kDa, they are strongly negatively changed and they are prone to enzymatic degradation. By complex formation with cationic lipids, polymers or inorganic compounds, however, they can be packed as net positively charged complexes that are protected against nucleases, show interaction with the cell membrane and uptake by e.g. endocytosis.
Amphiphilic Anionic Oligomer-Stabilized Calcium Phosphate Nanoparticles with Prospects in siRNA Delivery via Convection-Enhanced Delivery
Franziska Mitrach, Max Schmid, Magali Toussaint, Sladjana Dukic-Stefanovic, Winnie Deuther-Conrad, Heike Franke, Alexander Ewe, Achim Aigner, Christian Wölk, Peter Brust, Michael C. Hacker, Michaela Schulz-Siegmund
Pharmaceutics. 2022;14(2):326. doi: 10.3390/pharmaceutics14020326
Graphical abstract as published:
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In a recent study, we used calcium phosphate nanoparticles (CaP-NP) as drug delivery systems for siRNA. CaP-NP are usually generated by precipitation and tend to rapidly grow in size upon storage. We developed a stabilizer for the CaP-NP containing maleic anhydride, a lipophilic component and polyethylene glycol in order to decrease particle growth. Survivin siRNA was used to transfect brain cancer cells and induce apoptosis in these cells. We extensively characterized the stabilized CaP-NP in vitro for their suitability for convection enhanced delivery in the brain.
How to control the release of siRNA from biomaterials?
Microtissues from mesenchymal stem cells and siRNA-loaded cross-linked gelatin microparticles for bone regeneration
Sandra Hinkelmann, Alexandra H. Springwald, Annett Starke, Hermann Kalwa, Christian Wölk, Michael C. Hacker, Michaela Schulz-Siegmund
Materials Today Bio 13 (2022) 100190: https://doi.org/10.1016/j.mtbio.2021.100190
Graphical abstract as published:
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Finding controlled release matrices for complexed siRNA is difficult, as RNA complexes might be decomplexed via electrostatic interactions with the matrix or may show rapid release [1]. In order to bring cells in close contact with the siRNA releasing matrices, we set up crosslinked gelatin microparticles (cGM) [2], loaded them with complexed siRNA and formed aggregate cultures with human mesenchymal stem cells (hMSC). These aggregate cultures were referred to as microtissues. Release of siRNA against chordin (an antagonist of bone morphogenetic protein-2 (BMP-2)) [3] from the microparticles led to remarkably improved osteogenic differentiation compared to controls. Moreover, co-culture of precultivated microtissues with freshly seeded hMSC showed improved differentiation of the co-culture.
[1] Katharina Schwabe, Alexander Ewe, Caroline Kohn, Tina Loth, Achim Aigner, Michael C. Hacker, and Michaela Schulz-Siegmund, Int J Pharm. 2017;526(1-2):178-187. doi: 10.1016/j.ijpharm.2017.04.065
[2] Tina Loth, Rudi Hötzel, Christian Kascholke, Ulf Anderegg, Michaela Schulz-Siegmund, Michael C. Hacker, Biomacromolecules 2014, 15: 2104-18, 2014. https://doi.org/10.1021/bm500241y
[3] Hellen Schneider, Bita Sedaghati, Andreas Naumann, Michael C. Hacker, Michaela Schulz-Siegmund, Tissue Engineering Part A 2014; 20 (1-2), 335-345. doi: 10.1089/ten.TEA.2012.0563
Mineralizing Gelatin Microparticles as Cell Carrier and Drug Delivery System for siRNA for Bone Tissue Engineering
Sandra Hinkelmann, Alexandra H. Springwald, Sabine Schulze, Ute Hempel, Franziska Mitrach, Christian Wölk, Michael C. Hacker, Michaela Schulz-Siegmund
Pharmaceutics 2022, 14, 548. https://doi.org/10.3390/pharmaceutics14030548
Graphical abstract as published:
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In a second paper on that subject, we employed stronger crosslinked self-mineralizing microparticles. With these microparticles, we could show a strong silencing effect on chordin as well as improved bone like mineral formation. The mineral phase in the microtissues that formed in the chordin siRNA group was different to the controls. In a supplement-free co-culture of hMSC and human peripheral blood monocytes we found improved osteoprotegerin amounts beside improved alkaline phosphatase in the co-culture system with chordin silenced microtissues. Our microtissues may provide positive effects on bone regeneration similar as autologous bone grafts.
Oberflächenmodifikationen von Biomaterialien zur Knochenregeneration
Biodegradable macromers for implant bulk and surface engineering
Biological Chemistry, Band 402, Heft 11
Jan Krieghoff, Mathis Gronbach, Michaela Schulz-Siegmund, Michael C. Hackerhttps://doi.org/10.1515/hsz-2021-0161
This review summarizes our work in the SFB Transregio 67 that was funded from 2009 to the end of 2021 with a focus on the theses of Jan Krieghoff and Mathis Gronbach. Aim of the SFB TRR67 was to elucidate the mechanisms of sulfated glycosaminoglycans (GAGs) in bone regeneration and wound healing.
Our part within this project was to develop biomaterials to present the GAGs on and in biomaterials for in vitro and in vivo testing. In the 12 year funding period, we created a platform of macromer-based biodegradable materials for scaffold film and scaffold fabrication. Our materials allowed to control scaffold mechanics via crosslinking degree as well as degradation time over a period of up months to years. In contrast to pure poly-α-hydroxyesters (e.g. polylactids (PLA) and polyglycolids (PGA)), our biomaterials degraded more regularly avoiding the typical bulk degradation related burst release of acidic products well known from PLAs:
Composition-controlled degradation behavior of macroporous scaffolds from three-armed biodegradable macromers
Polymer Degradatio and Stability 195 (2022) 109775
Jan Krieghoff, Christian Kascholke, Rudi Loth, Annett Starke, Andreas Koenig, Michaela Schulz-Siegmund, Michael C. Hacker
https://doi.org/10.1016/j.polymdegradstab.2021.109775
Moreover, we processed the macromers into films as model surfaces for scaffolds. In contrast to 3D scaffolds, defined and smooth film surfaces allow for sophisticated characterization. For covalent binding of GAGs, we integrated a small anchor molecule, glycidylmethacrylate (GMA) in the macromer films, bound polyetheramines and decorated them with GAGs. These GAG decorated films were characterized for their interaction with wnt-antagonists, known to negatively impact bone regeneration e.g. in osteoporosis. We could show that highly sulfated hyaluronan (sHA3) was able to tightly bind and inactivate sclerostin and dickkopf-1 (Dkk-11). In the Acta Biomaterialia paper, Mathis Gronbach showed the effects of the GAG modifications on scavenging of Dkk-1 and osteogenic differentiation of mesenchymal stem cells, key players in bone regeneration.
Scavenging of Dickkopf-1 by macromer-based biomaterials covalently decorated with sulfated hyaluronan displays pro-osteogenic effects
Acta Biomaterialia 114 (2020) 76-89
M. Gronbach, F. Mitrach, V. Lidzba, B. Müller, S. Möller, S. Rother, J.Salbach-Hirsch, L.C. Hofbauer, M. Schnabelrauch, V. Hintze, M.C. Hacker, M. Schulz-Siegmund
https://doi.org/10.1016/j.actbio.2020.07.017
Effects of processing parameters for film production and polyetheramine length on the efficiency of surface modification were shown in a second paper by Mathis Gronbach. Here, we also showed that sclerostin was scavenged in addition to Dkk-1 which required an increased stability of the macromer films. In contrast to the wnt-antagonists, we showed that wnt-3a, a wnt-agonist remained unbound in presence of surface immobilized sHA3.
A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Ostegenic Scavenging of Wnt Antagonists
Pharmaceutics 2020 (12)11
Mathis Gronbach, Franziska Mitrach, Stephanie Möller, Sandra Rother, Sabrina Friebe, Stefan G. Mayr, Matthias Schnebelrauch, Vera Hintze, Michael C. Hacker, Michaela Schulz-Siegmund
https://doi.org/10.3390/pharmaceutics12111037
ACS Appl. Mater. Interfaces 2020, 12, 8, 8963–8977
Gene activated matrices for regenerative medicines
Contact-Triggered Lipofection from Multilayer Films Designed as Surfaces for in Situ Transfection Strategies in Tissue Engineering
Catharina Husterden, Falko Doberenz, Nathalie Goergen, Shashank Reddy Pinnapireddy, Christopher Janich, Andreas Langner, Frank Syrowakta, Alexandros Repanas, Frank Erdmann, Jarmila Jedelská, Udo Bakowsky, Thomas Groth, Christian Wölk
https://doi.org/10.1021/acsami.9b18968
We also try to develop gene activated matrices for regenerative medicines which can functionalize surfaces.
