gms | German Medical Science

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2018)

23.10. - 26.10.2018, Berlin

Chitosan based drug delivery system for Virus-like Particle mediated RANKL knockdown

Meeting Abstract

  • presenting/speaker Kai O. Böker - Universitätsmedizin Göttingen, Unfallchirurgie, Orthopädie und Plastische Chirurgie, Göttingen, Germany
  • Jens Gruber - Deutsches Primatenzentrum Göttingen, Göttingen, Germany
  • Stefan Schneider - Deutsches Primatenzentrum Göttingen, Göttingen, Germany
  • Marina Komrakova - Universitätsmedizin Göttingen, Unfallchirurgie, Orthopädie und Plastische Chirurgie, Göttingen, Germany
  • Stephan Sehmisch - Universitätsmedizin Göttingen, Unfallchirurgie, Orthopädie und Plastische Chirurgie, Göttingen, Germany
  • Wolfgang Lehmann - Universitätsmedizin Göttingen, Unfallchirurgie, Orthopädie und Plastische Chirurgie, Göttingen, Germany
  • Arndt F. Schilling - Universitätsmedizin Göttingen, Unfallchirurgie, Orthopädie und Plastische Chirurgie, Göttingen, Germany
  • Daniel B. Hoffmann - Universitätsmedizin Göttingen, Unfallchirurgie, Orthopädie und Plastische Chirurgie, Göttingen, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2018). Berlin, 23.-26.10.2018. Düsseldorf: German Medical Science GMS Publishing House; 2018. DocPT20-508

doi: 10.3205/18dkou713, urn:nbn:de:0183-18dkou7130

Veröffentlicht: 6. November 2018

© 2018 Böker et al.
Dieser Artikel ist ein Open-Access-Artikel und steht unter den Lizenzbedingungen der Creative Commons Attribution 4.0 License (Namensnennung). Lizenz-Angaben siehe http://creativecommons.org/licenses/by/4.0/.


Gliederung

Text

Objectives: Rebalancing the RANKL/OPG-system seems to be an effective treatment-strategy for osteoporosis. Here we used virus-like particles (VLPs) as a gene delivery vehicle for siRNAs to inhibit RANKL expression. In our former studies we showed a significant short-term knockdown of RANKL by VLP mediated RNA-interference via weekly i.p. injection. Chitosan is a promising possibility for prolonged drug delivery in vivo. In the present study we investigated the effects of prelonged RANKL-knockdown by chitosan embedded siRNA-VLPs.

Methods: VLP production and loading: VLPs were produced in Hi5 insect cells and purified via size-exclusion chromatography. VLPs were loaded with desired target drug (e.g. expression cassette for GFP or RANKL siRNA) and incubated for 30 min at room temperature. Reassembly was performed in 5 L HEPES buffer containing CaCl2 for capsid formation at 4 °C overnight.

Chitosan gel production: For chitosan gel production 190 mg of Chitosan (95% deacetylated, Heppe Medical Chitosan GmbH, Germany) was dissolved in 10 mL acetic acid (0.1 M). The solution was cooled to 4°C and ß-Glycerophosphate (3g) was added slowly. The final liquid was clear and homogeneous and stored at 4°C until usage.

In vitro validation: For in vitro applications precooled chitosan gel was mixed with GFP-cDNA loaded VLPs and transferred into transwells (400 µm pore size, Corning, NY, US). Transwells were inserted into prepared 24well plates containing human cell lines like osteoblasts or embryonic kidney cells. GFP expression was documented daily up to 14 days. Furthermore, we tested VLP stability and release under physiological conditions daily by dot blot analysis.

In vivo application: All animal procedures were approved by the local Animal Care and Use Committee and performed according to the German animal protection laws. 3-month-old female Sprague-Dawley rats were used for VLP injection (Janvier Lab, France). Chitosan embedded VLPs (300 µg) were i.p. injected. At the end of the experiments, the rats were euthanized under Isofluran aneasthesia and decapitated. Tibia, lumbar vertebrae and blood samples were removed for analysis.

Results: VLP release was detected for 14 days through fluorescence microscopy and for 21 days by dot blot analysis. The in vivo experiments showed a significant and time depending knockdown of RANKL. 3 Days after VLP injection we detected a RANKL knockdown by 43 %. This knockdown was even increased after 14 days (67%). Knockdown was superior to former studies with VLP mediated RNA interference without chitosan. Toxic effects or inflammations were not observed in vivo.

Conclusion: We demonstrate here a successful transduction of chitosan embedded VLPs in vitro and in vivo. A significant knockdown of RANKL expression 3 and 14 days after injection in vivo indicates a long time function of VLP-encapsulated siRNA. This improved effect of chitosan embedded VLPs suggests it as a promising tool for specific siRNA therapy.